MicroRNA-182对浆液性卵巢癌生物学行为的影响及其分子机制研究
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摘要
第一部分MiR-182对瘦素诱导的卵巢癌细胞生长的影响及机制研究
研究背景
卵巢癌是妇科常见的恶性肿瘤,严重威胁妇女健康,死亡率居妇科肿瘤之首。早期诊断困难及复发转移导致治疗手段缺乏,是卵巢癌高死亡率的主要原因。因此及早捕捉预警信号并阐明卵巢癌发生发展的相关分子机制,对卵巢癌的防治至关重要,但目前相关机制尚未阐明。流行病学数据表明肥胖增加激素依赖性肿瘤(如卵巢癌)的发生风险,瘦素(Leptin)作为重要的脂肪因子类激素之一,参与调控机体的脂肪代谢,并与多种肿瘤的发生有关。研究发现卵巢癌时瘦素水平升高,且与患者无病生存期密切相关,但目前瘦素与卵巢癌增殖、凋亡的关系及其在卵巢癌起源中的作用尚不明确。
MicroRNA (miRNA)是一类非编码小RNA,可在转录后水平通过抑制翻译或者诱导降解抑制靶mRNA的功能,调节细胞分化、增殖和凋亡过程。研究表明多种肿瘤组织存在miRNAs的异常表达或者突变,并影响肿瘤的生长、侵袭和转移。MiR-182是重要的miRNAs分子,其过表达于输卵管内上皮性浆液性卵巢癌和高级别浆液性卵巢癌,并促进良性和恶性肿瘤细胞的侵袭和转化,提示miR-182在卵巢癌的早期发生中具有重要意义,而对其进行有效调节将有助于卵巢癌的防治。研究表明瘦素可通过影响miRNAs的表达调节骨骼肌细胞的生长,但关于卵巢癌发生和发展过程中瘦素影响miRNAs表达及机制未见报道。
综上所述,基于相关领域国内外的研究进展,本课题体外研究了瘦素通路下游信号分子STAT5介导的miR-182上调对卵巢癌细胞生长和转化的影响及机制。本研究肯定了瘦素和miR-182在卵巢癌中的作用,进一步阐明了卵巢癌发生发展的机制,为卵巢癌的早期诊断和治疗提供新的线索。
研究目的
1.瘦素对卵巢癌细胞生长、增殖及转化的作用。
2.探索介导瘦素调节卵巢癌细胞生长、增殖及转化的miRNAs及miR-182和miR-96在瘦素作用中的影响。
3.观察瘦素下游信号通路对miR-182和miR-96的作用。
研究方法
1.卵巢癌细胞系瘦素及瘦素受体的表达
卵巢癌细胞(SKOV3和A2780)37℃C培养24小时,RT-PCR检测瘦素和瘦素长型受体OB-Rb和短型受体OB-Ra表达,Western blot检测瘦素及其受体的蛋白水平。培养72小时后收集上清,ELISA法检测内源性瘦素表达。
2.瘦素对卵巢癌细胞增殖、凋亡和转化的影响
分别给予SKOV3和A2780细胞不同剂量瘦素(0、10、50、100和500ng/mL),每隔24小时收集细胞,WST-1法检测细胞增殖并绘制生长曲线。卵巢癌细胞SKOV3和A2780分为Control、Leptin、Anti-leptin、Leptin+Anti-leptin组,在0.3%的软琼脂中37℃C培养3周后,结晶紫染色观察不同组别软琼脂中形成的单细胞克隆数目。卵巢癌细胞分为Control、Leptin、Anti-leptin、Leptin+Anti-leptin组37℃培养48小时后,流式细胞术检测各组细胞的凋亡率,瘦素处理的结肠癌细胞-HCT-116用于阳性对照。
3.瘦素作用后卵巢癌细胞转录因子FOXO3表达的变化,并观察其在瘦素调节卵母巢癌细胞增殖中的作用
卵巢癌细胞(SKOV3和A2780)分为Control、Leptin、Anti-leptin、 Leptin+Anti-leptin组,37℃C培养48小时后,Western blot检测FOXO1和FOXO3蛋白水平变化,筛选表达变化的FOXO分子,然后Western Blot检测其下游靶分子蛋白水平表达。
siRNA干扰相应FOXO分子的表达,RT-PCR和Western Blot分别从基因水平和蛋白水平检测干扰效率,卵巢癌细胞分为Control、Leptin、Con-siRNA和FOXO-siRNA组,37℃培养48小时后,WST-1检测各组细胞的增殖变化。
4.瘦素对靶向作用于FOXO分子3'-UTR的miRNAs的影响,miRNAs在卵巢癌细胞增殖中的作用
PCR克隆扩增FOXO分子的3'-UTR并构建psiCHECK2荧光载体,用该载体及对照空载体分别转染卵巢癌细胞SKOV3和A2780。37℃培养24小时后,细胞分为Vector、Vector+Leptin、FOXO-3'-UTR, FOXO-3'-UTR+Leptin组并继续培养48小时,双荧光报告系统检测不同组别的荧光信号强度。
卵巢癌细胞SKOV3和A2780分为Control和Leptin组,培养48小时后,RT-PCR检测miRNAs分子的基因水平表达变化。用所测miRNAs的mimics转染卵巢癌细胞,37℃培养48小时,RT-PCR检测各组过表达效率,Western blot检测不同mimics转染细胞的FOXO分子蛋白水平的变化,WST-1检测细胞的增殖变化。用miRNAs抑制剂转染卵巢癌细胞,37℃培养24小时后RT-PCR检测各组抑制效率,细胞分为Control、Leptin、Leptin+miRNAs inhibitor并继续培养48小时,Western blot检测卵巢癌细胞的FOXO分子蛋白水平的变化,WST-1检测细胞的增殖变化。
5.瘦素受体下游信号传导分子STAT3、STAT5在瘦素调节的miRNAs表达上调中的作用
瘦素作用卵巢癌SKOV3和A2780细胞48小时后,Western blot检测瘦素长型受体OB-Rb表达水平的变化。
瘦素处理卵巢癌SKOV3和A2780细胞不同时间(0,15,30,60分钟)后,Western blot检测不同STAT信号通路蛋白(STAT3、STAT5)的表达变化。
卵巢癌SKOV3和A278048细胞分为Control、Leptin、STAT5-siRNA、 Cucurbitacin Ⅰ (p-STAT3抑制剂)、Leptin+STAT5-siRNA、Leptin+Cucurbitacin I组,RT-PCR检测目的miRNAs分子基因表达水平变化。
研究结果
1.卵巢癌细胞表达瘦素受体
实验表明,卵巢癌SKOV3和A2780细胞在蛋白水平和mRNA水平均可检测到瘦素、瘦素长型受体OB-Rb和短型受体OB-Ra的表达,两种细胞培养上清中也存在极低量的瘦素(分别为38.39±3.72pg/mL和35.40±2.44pg/mL),因远低于生理剂量,后续实验均给予外源性瘦素。
2.瘦素促进卵巢癌细胞的增殖和转化能力
瘦素以剂量依赖和时间依赖方式促进卵巢癌细胞SKOV3和A2780的增殖能力(P<0.05)。同时增加软琼脂中两种卵巢癌细胞克隆的数目和大小(SKOV3细胞：2.72--±0.34倍,P<0.05；A2780细胞：1.97±0.15倍,P<0.05),瘦素抗体可阻断瘦素的促细胞转化能力。流式细胞术显示瘦素不影响卵巢癌细胞SKOV3和A2780的凋亡率(P>0.05),但显著抑制结肠癌细胞HCT-116的凋亡(P=0.0169)。
3.瘦素抑制卵巢癌细胞FOXO3的表达,从而促进细胞增殖
Western blot结果表明瘦素(100ng/ml)显著抑制卵巢癌细胞SKOV3和A2780内FOXO3的表达(SKOV3细胞：55.1±1.8%,P<0.01；A2780细胞：48.4±10.1%,P<0.01), FOXO1的表达无显著变化(P>0.05)。进一步检测发现瘦素(100ng/ml)显著抑制细胞内FOXO3的两个下游靶基因Bim (SKOV3细胞：45.8±1.50%,P<0.01；A2780细胞：46.7±3.6%,P<0.01)和p27(SKOV3细胞：41.4±6.3%,P<0.01；A2780细胞：36.7±9.0%,P<0.01)的表达。利用FOXO3-siRNA干扰掉细胞内FOXO3基因和蛋白表达后,发现两种细胞的增殖能力均显著增强(SKOV3细胞：1.63±0.10倍,P<0.01；A2780细胞：1.59±0.16倍,P<0.05)。
4.瘦素通过上调靶向作用于FOXO33'端非翻译区(3'-UTR)的miRNAs抑制FOXO3表达,从而促进卵巢癌细胞增殖
利用包含FOXO33'-UTR的psiCHECK2荧光载体转染细胞后,双荧光报告系统显示瘦素(100ng/ml)显著降低卵巢癌细胞SKOV3和A2780内的荧光素酶强度(SKOV3细胞：53.4±2.5%,P=0.001；A2780细胞：41.6±8.5%,P=0.016)。RT-PCR结果显示瘦素(100ng/ml)显著上调SKOV3细胞中靶向作用于FOXO3的肿瘤相关miRNAs,包括miR-182(2.26±0.23倍,P<0.01)、miR-106(1.52±0.16倍,P=0.03)、miR-96(1.91±0.03倍,P<0.01)和miR-155(1.53±0.10倍,P=0.01),对A2780细胞中的上述miRNAs具有相似的作用。但两种细胞miR-93和miR-153的表达均不受瘦素影响(P>0.05)。
上述六种miRNAs的]mimics分别转染卵巢癌细胞SKOV3和A2780, Westernblot显示miR-182(SKOV3细胞：64.8%±11.4%,P<0.05；A2780细胞：51.8%±0.5%,P<0.05)和miR-96(SKOV3细胞：58.3%±2.5%,P<0.05；A2780细胞：42.2%±4.1%,P<0.05)两种mimics可以有效抑制两种细胞内FOX03的表达,miR-155和miR-106a的mimics则不具备此功能(P>0.05)。利用抑制剂干扰阻断两种细胞内miR-182和miR-96的表达后,Leptin对FOX03表达的抑制作用亦消失。WST-1检测两种细胞的增殖发现,miR-182(SKOV3细胞：2.65±0.05倍,P<0.01；A2780细胞：2.52±0.06倍,P<0.01)和miR-96(SKOV3细胞：2.03±0.08倍,P<0.01；A2780细胞：1.85±0.13倍,P<0.05)的mimics可促进两种细胞的增殖,而miR-182(SKOV3细胞：37.1%±3.3%,P<0.01；A2780细胞：37.4%±5.0%,P<0.01)和miR-96(SKOV3细胞：28.9%±6.2%,P<0.01；A2780细胞：29.9%±3.5%,P<0.01)抑制剂则可有效抑制瘦素对两种细胞的促增殖效应。
5.瘦素通过STAT5通路上调细胞中miR-182和miR-96的表达
瘦素(100ng/ml)能够促进卵巢癌细胞SKOV3和A2780瘦素长型受体OB-Rb的表达。同时瘦素可活化瘦素受体下游信号通路STAT3和STAT5,分别在60、30分钟活化达到高峰,特异性阻断两个通路发现cucurbitacin Ⅰ (STAT3抑制剂)不影响瘦素诱导的miR-182和miR-96表达上调(P>0.05),而STAT5-siRNA能够降低瘦素导致的miR-182(SKOV3细胞：43.5%±2.5%,P<0.01；A2780细胞：32.0%±9.4%,P<0.01)和miR-96(SKOV3细胞：34.1%±0.8%,P<0.01；A2780细胞：22.7%±4.2%,P<0.05)表达上调,表明STAT5通路参与miR-182和miR-96依赖的FOXO3抑制和细胞增殖。
结论
1.MiR-182和miR-96在瘦素抑制FOXO3表达、进而促进抑卵巢癌细胞增殖这一过程中发挥重要的介导作用。
2.瘦素通过下游STAT5信号途径上调卵巢癌细胞miR-182和miR-96的表达。
第二部分抗mi R-182化合物对卵巢癌体内生长和转移的影响及机制研究
研究背景
卵巢癌是女性死亡率最高的生殖系统肿瘤,高级别浆液性卵巢癌是卵巢癌中侵袭程度最高的类型,多数患者确诊时已属晚期,肿瘤已出现广泛转移。现有的手术治疗和化疗可在一定程度上减少肿瘤负荷,但对患者远期生存率并无显著影响。近年来抗microRNA治疗(anti-microRN A)成为癌症治疗的一种潜在有效的途径。miRNAs为一种小分子非编码RNA,某些miRNAs与肿瘤的生长、侵袭和远处转移密切相关。其中miR-182是一种原癌miRNAs,可负性调节多种与肿瘤生长、侵袭、转移调节有关的抑癌基因,从而促进多种肿瘤的发生发展。
研究表明miR-182过表达与高级别浆液性卵巢癌,与肿瘤的生长及侵袭密切相关,且与患者的不良预后有关。本课题组前期发现miR-182在瘦素介导的促卵巢癌细胞增殖和转化过程中发挥关键作用,因此在miR-182过表达的卵巢癌中,anti-miR-182治疗可能会减少肿瘤负荷、抑制侵袭转移。综上所述,基于本课题国内外的研究进展及课题组的前期工作基础,本课题组首次尝试应用anti-miR-182进行卵巢癌治疗。我们将人卵巢癌细胞固定植入小鼠卵巢包膜内,成功构建了小鼠原位卵巢癌模型,并给予小鼠anti-miR-182系统治疗,应用在体生物光学成像技术动态监测肿瘤的生长转移,治疗结束后用组织病理学手段确认了肿瘤在小鼠各脏器的侵袭转移情况及药物的作用,并进一步对肿瘤组织中miR-182靶基因的表达情况进行了分子生物学分析。本研究为侵袭性高级别浆液性卵巢癌提供了一种潜在的新的治疗手段。
研究目的
1.模拟人卵巢癌构建小鼠原位卵巢癌模型。
2.给与小鼠anti-miR-182治疗,动态观察药物对肿瘤生长和侵袭的影响。
3.组织学观察药物对肿瘤生长和侵袭转移的作用。
4.分析anti-miR-182影响肿瘤侵袭转移的分子机制。
研究方法
1.体外检测anti-miR-182对卵巢癌细胞的影响
体外实验利用人卵巢癌细胞系OVCAR3、HEY和SKOV3细胞,应用慢病毒包装转染系统构建miR-182稳定过表达的HEY和SKOV3细胞系。利用脂质体转染技术将anti-miR-182瞬时导入上述卵巢癌细胞系,细胞分为Control、 anti-miR-182组,37℃C培养48小时后RT-PCR检测细胞内miR-182的基因水平,Western blot检测miR-182靶基因的蛋白水平。
将anti-miR-182利用脂质体转入卵巢癌细胞OVCAR3、HEY和SKOV3,37℃培养24小时后,1)Transwell侵袭实验检测卵巢癌细胞侵袭能力的变化；2)将Control细胞和anti-miR-182分别种在5个96孔板上(2×103每孔,每种细胞每板5个复孔),利用WST-1连续五天每天检测细胞的增殖并绘制生长曲线；3)软琼脂克隆形成实验检测卵巢癌细胞非停泊性生长能力的变化。
2.构建裸鼠原位人卵巢癌模型
利用慢病毒包装转染系统将荧光素酶基因导入稳定过表达miR-182的SKOV3卵巢癌细胞(SKOV3-miR-182-luc),该荧光素酶与底物作用后可被在体生物光学成像系统检测(IVIS)。
SKOV3-miR-182-luc消化计数后,利用Ⅳ型胶原将肿瘤细胞(106个细胞)固定成直径约0.2厘米的球状小团,显微镜下手术将细胞团块植入8周龄裸鼠左侧卵巢包膜内。
3. Anti-miR-182治疗
术后小鼠随机分成对照组(20只)和anti-miR-182实验组(20只),并于术后第三天分别腹腔注射安慰剂或anti-miR-182(25mg/kg),每周两次,整个治疗周期为8周(其中实验组4只动物仅治疗1周)。
4. Anti-miR-182对卵巢原发肿瘤生长的影响
治疗期间每周一次应用IVIS技术动态监测对照组和实验组肿瘤的生长和侵袭,利用软件定量分析两组动物肿瘤信号强度。
8周后处死小鼠,测量对照组和实验组肿瘤实际长短径线,收集冰冻所有小鼠血清和部分肿瘤、肝脏组织,切除小鼠所有器官(肝、肾、脾、胰腺、肠道组织、子宫输卵管、卵巢肿瘤及对侧卵巢、肺、心脏)并固定备用。
ELISA检测对照组和实验组血清中CA125的水平。提取对照组和实验组肿瘤组织总RNA, RT-PCR检测两组肿瘤组织内与细胞周期调节或细胞死亡有关(CDKN1A、CDKN1B、FOXO1、CHEK2)的基因表达水平。
5.Anti-miR-182对卵巢肿瘤侵袭远处转移的影响
IVIS技术动态监测对照组和实验组原发瘤的转移信号。对两组动物所有器官进行组织切片和HE染色,显微镜下观察个脏器转移瘤的情况并进行统计分析。
6.肿瘤组织的分子生物学水平分析
提取对照组和实验组动物肿瘤及血清中的总RNA, RT和实时定量PCR检测肿瘤即血清中miR-182及其同一基因簇miR-183和miR-96的基因表达。提取两组动物肿瘤组织内的总蛋白,Western blot检测两组肿瘤组织内miR-182靶基因(BRCA1、MTSS1、FOX03)和诱导基因HMGA2的蛋白水平变化。
7. Anti-miR-182治疗对肿瘤微环境的影响
对对照组和实验组肿瘤组织切片和HE染色,镜下观察肿瘤与卵巢、输卵管和卵巢周围脂肪组织的关系,免疫组织化学染色确定肿瘤周围微环境中的细胞成分。炎症基因芯片技术观察两组肿瘤组织内炎症基因谱的变化。RT-PCR检测对照组和实验组(各8例)肿瘤内趋化因子和细胞因子(CCL2、IL-6、IL-8、TNF-α、 IFN-γ)的基因水平变化。
人卵巢癌细胞系SKOV3分为Control组、anti-miR-182组、miR-182过表达组、miR-182过表达+anti-miR-182组,37℃培养48小时后RT-PCR检测细胞内CCL2基因水平的变化。
8.药物毒性分析
8周治疗结束后,收集对照组和anti-miR-182实验动物的全部器官(肝脏、胰腺、胃肠道、肾、脾及子宫)并进行组织切片和HE染色,镜下观察两组组织内细胞的形态学变化,判断药物的毒性作用。
研究结果
1. Anti-miR-182抑制体外卵巢癌细胞的增殖、基质胶侵袭和转化能力
RT-PCR结果显示anti-miR-182显著抑制OVCAR3、HEY-miR-182和SKOV3-miR-182细胞内miR-182的基因水平(P<0.001)。Western blot结果显示,anti-miR-182显著上调SKOV3-miR-182细胞内miR-182靶基因FOXO3(1.47±0.18倍,P<0.05)、BRCAl (3.09±0.99倍,P<0.05)、和MTSS1(1.39±0.18倍,P<0.05)的蛋白水平,同时下调miR-182诱导的HMGA2(62.6%±17.2%,P<0.01)的蛋白表达,对OVCAR3和HEY-miR-182细胞具有相似效应。
基质胶侵袭实验结果显示anti-miR-182可显著抑制SKOV3-miR-182细胞的侵袭能力(抑制率57.1%±7.9%,P<0.001)。软琼脂克隆形成实验表明anti-miR-182可显著减少单细胞克隆形成数目和大小(抑制率54.8%±16.3%,P<0.01)。WST-1细胞增殖实验结果显示anti-miR-182抑制SKOV3-miR-182细胞的体外增殖(第3天,P<0.05；第四天P<0.05；第五天P<0.01)。
2.构建裸鼠原位人卵巢癌模型
将SKOV3-miR-182-luc细胞包埋入Ⅳ型胶原并植入裸鼠左侧卵巢包膜内,术后第二天,IVIS检测结果表明所有小鼠荧光信号相同(相对荧光强度3-3.5x109),表明卵巢癌细胞植入量在所有动物体内基本一致,无腹腔泄漏。
3. Anti-miR-182治疗抑制卵巢癌肿瘤的体内生长
术后第3天,小鼠被随机分为对照组和anti-miR-182实验组并分别给予安慰剂或anti-miR-182腹腔注射治疗,治疗剂量25mg/kg,一周两次,整个疗程为8周。IVIS图像显示从治疗第5周开始,实验组与对照组肿瘤大小之间开始出现差异(对照组信号强度为(17.73±7.72)×109,实验组信号强度为(5.20±6.63)×109,P<0.01),且该差异随着治疗时间延长而增大。
处死小鼠测量两组小鼠卵巢肿瘤实际大小发现表明anti-miR-182治疗组的肿瘤(5.42±1.00mm,90.59±37.31mm3)显著小于对照组(8.07±1.74mm,310.89±187.12mm3),其最长径线较对照组减少38%,体积较对照组减少70.9%(P<0.01). ELISA结果显示对照组小鼠血清CA125水平高于实验组小鼠(P=0.06)。
RT-实时定量PCR结果显示,mti-miR-182显著上调肿瘤组织内与细胞生长和死亡有关的miR-182假定靶基因CDKN1A(P=0.0199)、CDKN1B(P=0.0156)、 FOXO1(P<0.0001)、CHEK2(P<0.0001)。
4. Anti-miR-182治疗抑制小鼠体内卵巢癌细胞的侵袭和远处转移
治疗第8周IVIS图像显示,对照组肿瘤的远处转移率为28%(5/8),实验组的转移率为7%(1/15)。组织学分析结果显示对照组77.8%(14/18)的动物存在至少一处转移癌灶,实验组33.3%(5/15)的动物存在至少一处转移灶；对照组共发现31个转移瘤,实验组仅发现6个转移瘤。对照组和实验组肿瘤远处转移率具有显著的统计学意义(P<0.05)。转移瘤数目由多到少的部位是依次是脾脏(对照组阳性率为44%,8/18；实验组阳性率为20%,3/15)、大网膜(对照组阳性率为28%,5/18；实验组阳性率为13%,2/15)、胰腺(对照组阳性率为22%,4/18；实验组阳性率为0%)、和肝脏(对照组阳性率为11%,2/18；实验组阳性率为0%)。与其他部位的转移瘤相比,胰腺转移瘤更大、侵袭性更强。对照组有一只小鼠发生了对侧卵巢转移。虽然肾实质内未发现转移瘤,对照组50%(9/18)的动物的卵巢肿瘤与同侧发生肾粘连,而anti-miR-182治疗组动物未发现此现象(0/15)。对照组和实验组动物均未发现肺转移灶。
5. Anti-miR-182治疗后肿瘤组织的分子生物学分析
实时定量PCR结果显示与对照组肿瘤相比,anti-miR-182治疗组的肿瘤组织内miR-182表达水平极低(降低94.9%±13.3%)或低于测量范围(P<0.001),同时显著降低miR-183的表达(降低92.5%0±9.7%,P<0.001),但对miR-96的表达无影响(P>0.05)。小鼠血清内miR-182(降低53.2%±10.4%,P<0.01)和miR-183(降低84.3%±20.6%,P<0.01)的水平也显著降低。
Western blot结果显示经anti-miR-182治疗后肿瘤内miR-182的靶基因BRCA1(2.63±0.35倍,P<0.01)、FOXO3(1.4±0.18倍,P<0.01)和MTSS1(2.63±1.29倍,P<0.01)与对照组相比显著上调,而miR-182诱导的基因HMGA2表达下调(降低71.5%±4.0%,P<0.01)。
6. Anti-miR-182治疗诱导的炎症反应抑制肿瘤的侵袭
组织学观察发现anti-miR-182治疗组73.3%(11/15)的小鼠肿瘤细胞植入部位(卵巢旁软组织)有大量单核细胞浸润,对照组虽有16.7%(3/18)的动物存在单核细胞浸润,但单核细胞数量明显少于实验组。免疫组织化学染色证实了浸润细胞为KP1阳性小鼠单核细胞。单核细胞浸润少的肿瘤表现出浸润生长模式,并穿透卵巢包膜侵入周围脂肪组织,而周围有大量单核细胞浸润的肿瘤则局限于卵巢包膜内。
炎症基因谱分析显示对照组肿瘤与anti-miR-182治疗组肿瘤组织炎症基因谱发生改变。实时定量PCR结果表明与对照组相比,实验组肿瘤组织内CCL2(P<0.0001). IL-6(P=0.0019)、IL-8(P=0.0002)、TNF-α (P=0.0088)、IFN-γ(P<0.0001)的表达显著上调。体外实验表明,无论在初始SKOV3细胞还是SKOV3-miR-182细胞,anti-miR-182均可显著上调细胞内CCL2的基因表达(SKOV3细胞：3.6±0.73倍,P<0.001；SKOV3-miR-182细胞：11.39±0.21倍,P<0.001)。
7.Anti-miR-182治疗对小鼠无明显不良组织毒性作用
组织病理学观察发现经8周anti-miR-182治疗后,小鼠的肝脏、胰腺、胃肠道、肾、脾及子宫均无显著的组织学和细胞学变化。
结论
1.建立的裸鼠原位卵巢癌模型可成功模拟人原发性卵巢癌的生长和侵袭模式,适用于卵巢癌的体内研究。
2.Anti-miR-182体内、体外均可通过下调miR-182、恢复其靶分子的表达有效抑制人卵巢癌细胞的增殖、侵袭和远处转移；同时anti-miR-182可诱导肿瘤局部炎症反应抑制肿瘤侵袭转移。
3. Anti-miR-182体内应用无不良组织学毒性作用。
Part I THE EFFECT AND UNDERLYING MECHANISM OF MICRORNA-182ON LEPTIN INDUCED PROLIFERATION OF OVARIAN CANCER CELLS
Background
Ovarian cancer is the fifth leading cause of cancer-related death worldwide and is still the most lethal gynecologic malignancy among women. One of the main reasons for such a high mortality is that the majority of cases are diagnosed at an advanced stage. Therefore, it is critical to elucidate the mechanism of the tumorigenesis. The converging studies indicate that obesity is a risk factor for hormone-dependent cancers including ovarian cancer, and leptin, the product of obesity gene (Ob), is implicated in the tumorigenesis and development of many cancers. Leptin is elevated and is correlated with the poor progress-free survival of patient. But its exact effect and the underlying mechanism in ovarian cancer remains pooly understood.
microRNAs (miRNAs) are small non-coding RNAs that negatively regulate gene expression at post-transcriptional level by blocking of translation and/or degradation of target mRNAs.Tumor-related miRNAs may play a role in tumor cell differentiation, proliferation and apoptosis. Aberrant expression or mutation of miRNAs has been detected in several tumors. Of these miRNAs, miR-182is overexpressed in the high-grade type (HG-SOC) and can promote the invasion and tansformation of both benign and ovarian cancer cell and metastasis in vivo, suggesting the role of miR-182in early tumorigenesis of HG-SOC is of great important and it may be very helpful for the clinical therapy to find a way to manipulate the miR-182level. Some early carcinogenic factors, such as DNA damage and inflammation, may influence the expression of some miRNA. Leptin can regulate miRNAs expression profile in skeletal muscle cells. However, the exact effect and mechanism of leptin on the expression of miRNAs during ovarian cancer remain unknown.
Based on the domestic and overseas progress in this field and our preliminary research data, in this study, we elucidated the effect and underlying mechanism of leptin on proliferation and transformation of ovarian cancer in vitro, explored the role of STAT5signaling pathway induced miR-182expression in this progress. Our results support that leptin and the upregulated-miR-182contribute to the ovarian cancer, which helps us to further know the mechanism of tumorigenesis of this specific cancer and provides molecular foundation for the early detection and therapy of ovarian cancer.
Objective
1. Observation of the effect of leptin on ovarian cancer cells proliferation and transformation.
2. Exploration of miRNAs regulated by leptin in ovarian cancer cells and their effect on ovarian cancer cells proliferation.
3. Elucidation the role of the downstream signaling pathway of leptin receptor in the expression of miR-182and miR-96.
Methods
1. The expression of leptin and leptin receptor in ovarian cancer cell. Ovarian cancer cells (SKOV3and A2780) were cultured for24hours at37℃. RT-PCR was employed to detect the mRNA expression of leptin, the long isoform leptin receptor OB-Rb and the short isoform leptin receptor OB-Ra. Western blot was employed to detect the protein level of leptin and leptin receptor. Ovarian cancer cells (SKOV3and A2780) were cultured for72hours and the supernatant was collected. ELISA was employed to detect the secreted leptin by ovarian cancer cells.
2. The effect of leptin on the proliferation, apoptosis and anchorage-independent growth of ovarian cancer cells.
Ovarian cancer cells (SKOV3and A2780) were treated with leptin (0,50,100,250,500ng/ml), and the proliferation of cells was measured using WST-1reagent for5consecutive days and the growth curve was traced. Ovarian cancer cells were divided into control、leptin、anti-leptin、leptin+anti-leptin groups and cultured in0.3%soft agar for3weeks at37℃. The single cell colonies were stained by crystal violet and counted. Ovarian cancer cells were divided into control、leptin、anti-leptin、 leptin+anti-leptin groups and cultured for48hours at37℃,flow cytometry was employed to detect the apoptosis and the leptin-treated colon carcinoma cell HCT-116was used as the positive control.
3. Detection the expression of the transcriptor FOXO family in ovarian cancer cells with leptin stimulation and observing their role in leptin-induced proliferation of ovarian cancer cells.
Ovarian cancer cells (SKOV3and A2780) were divided into control, leptin, anti-leptin and leptin+anti-leptin groups and cutured for48hours at37℃. Western blot was employed to measure the FOXO1and FOXO3expression at the protein level to screen the changed molecules. Then change of its or their downstream target molecules was measured using Western blot assay.
SiRNA was used to silence the function of the FOXO molecule(s) and RT-PCR and Western blot were used to detect the interference efficacy at mRNA and protein level, respectively. Ovarian cancer cells (SKOV3and A2780) were divided into control, leptin, Leptin+Con-siRNA and Leptin+FOXO-siRNA groups and cutured for48hours at37℃. The proliferation of cells was measured using the WST-1reagent.
4. The effect of leptin on miRNAs that target the3'-UTR of FOXO and the role of mIRNAs in the leptin induced ovarian cancer cell proliferation.
The3'-UTR reagion of human FOXO was amplified by PCR and cloned into the psiCHECK2luciferase vector. The constructed vector and the negative control was tranfected into ovarian cancer cells (SKOV3and A2780). After24hours at37℃, the cells were divided into Vector, Vector+Leptin, FOXO-3'-UTR and FOXO-3'-UTR+Leptin groups and further cultured for48hours at37℃. The relative luciferase activity was measured using the dual-luciferase reporter system.
Ovarian cancer cells were divided into Control and Leptin groups and cultured for48hours. RT-PCR was used to detect the mature miRNAs. The mimics of the deteced miRNAs was transfected into ovarian cancer cells and cultured for48hours. RT-PCR was employed to confirm the transfection efficacy. Western blot was used to detect the protein expression of FOXO. MiRNA inhibitors were used to knock out the expression of miRNAs and RT-PCR was used to confirm the interference efficacy. Ovarian cancer cells were divided into Control, Leptin, Leptin+miRNAs inhibitor groups and cultured for48hours. Western blot was employed to measure the expression of FOXO and WST-1was used to assess the cell proliferation.
5. The effect of Leptin receptor downstream signaling molecules STAT3and STAT5in leptin induced miRNAs expression.
Ovarian cancer cells (SKOV3and A2780) were treated with Leptin for48hours and Western blot was employed to measure the protein level of Leptin long isoform receptor OB-Rb.
Ovarian cancer cells were treated with leptin (100ng/ml) and cells were collected at different time point (0,15,30,60minutes). Western blot was employed to detect the level of the STAT3and STAT5signaling pathway.
Ovarian cancer cells were divide into Control, Leptin, Cucurbitacin I, STAT5-siRNA, Leptin+Cucurbitacin I and Leptin+STAT5-siRNA groups, RT-PCR was employed to measure the level of the target miRNAs.
Results
1. The Leptin receptors are constitutely expressed in ovarian cancer cells.
RT-PCR results indicated that both OB-Ra and OB-Rb were expressed on ovarian cancer cells SKOV3and A2780at mRNA level. Western blot results showed that these two were expressed at protein levels. In addition, Leptin mRNA and protein were detected at varied levels. Moreover, we detected secrete leptin in cultured supernatants of SKOV3(38.39±3.72pg/ml) and A2780(35.40±2.44pg/ml).
2. Leptin promoted the proliferation and anchorage-independent growth ability of ovarian cancer cells.
WST-1assay indicated that leptin enhanced the proliferation of SKOV3and A2780cells in a time-and dose-dependent manner (P<0.05). Leptin (100ng/mL) significantly promoted the anchorage-independent growth ability of A2780and SKOV3cells (SKOV3:2.72±0.34-fold, P<0.05; A2780:1.97±0.15-fold, P<0.05) as reflected by the significant increase in the number and size of colonies formed in soft agar,, and leptin neutralizing mAb attenuated this process. However, the apoptosis of the two cells were not affected by leptin (P>0.05), and the apoptosis of HCT-116(positive control) was inhibited by leptin (P=0.0169).
3. Leptin promoted ovarian cancer cell proliferation by inhibiting expression of FOXO3.
Western blot data indicated that leptin (100ng/mL) suppressed the expression of FOXO3significantly in both SKOV3cells (55.1±1.8%, P<0.01) and A2780cells (48.4±10.1%, P<0.01). However, the expression of FOXO1was not affected by leptin in both cell lines (P>0.05). Further data showed that leptin also inhibited the expression of Bim (45.8%±1.5%for SKOV3, P<0.01;46.7%±3.6%for A2780, P<0.01) and p27(41.4%±6.3%for SKOV3, P<0.01;36.7%±9.0%for A2780, P<0.01), respectively. When the mRNA and protein expression was knocked down by FOXO3-siRNA, the proliferation of both SKOV3(1.63±0.10-fold, P<0.01) and A2780cells (1.59±0.16-fold, P<0.05) was enhanced remarkedly.
4. Leptin promoted ovarian cell proliferation by upregulating miRNAs targeting the3'-UTR of FOX03.
Leptin (100ng/mL) can significantly reduced the normalized luciferase activity of SKOV3cells (53.4±2.5%, P=0.001) and A2780cells (41.6±8.5%, P=0.016) transfected with vector containing the3'-UTR of FOXO3, compared with the mock group. RT-PCR data indicated that leptin (100ng/ml) significantly increased the expression of miR-182(2.26±0.23-fold, P<0.01), miR-106a (1.52±0.16-fold, P=0.03), miR-96(1.91±0.03-fold, P<0.01) and miR-155(1.53±0.10-fold, P=0.01) in SKOV3 cells. Similar trend was observed in A2780cells. No difference was found on the expression of miR-93and miR-153(P>0.05).
Only miR-182(64.8%±11.4%for SKOV3, P<0.05;51.8%±0.5%for A2780, P<0.05) and miR-96(58.3%±2.5%for SKOV3, P<0.05;42.2%±4.1%for A2780, P<0.05) mimics inhibited FOXO3expression both cell lines. MiR-155and miR-106a mimics failed to downregulate FOXO3in two cells (P>0.05). MiR-182and miR-96inhibitors could rescue leptin-mediated downregulation of FOXO3. WST-1assay indicated that miR-182(2.65±0.05-fold SKOV3, P<0.01;2.52±0.06-fold for A2780, P<0.01) and miR-96mimics (2.03±0.08-fold SKOV3, P<0.01;1.85±0.13-fold for A2780, P<0.05) promoted the proliferation of both cell lines, and miR-182(37.1%±3.3%for SKOV3, P<0.01;37.4%±5.0%for A2780, P<0.01) and miR-96(28.9%±6.2%for SKOV3, P<0.01;29.9%±3.5%for A2780, P<0.01) inhibitors prevented the effect of leptin.
5. Leptin increased the expression of miR-182and miR-96through STAT5pathway.
The expression of the OB-Rb was enhanced by leptin (100ng/mL). Leptin induced the phosphorylation of STAT3and STAT5, whose activation reached a peak at60min and30min, respectively. The specific inhibition of STAT5by STAT5-siRNA prevented upregulation of precursor and mature miR-182(43.5%±2.5%for SKOV3, P<0.01;32.0%±9.4%for A2780, P<0.01) as well as miR-96(34.1.8%±0.8%for SKOV3, P<0.01;22.7%±4.2%for A2780, P<0.05) induced by leptin treatment. However, the specific STAT3inhibitor cucurbitacin I did not affect the miR-182and miR-96expression (P>0.05).
Conclusion
1. Leptin promotes the proliferation of ovarian cancer cells by suppressing the expression of FOXO3, but the apoptosis is not affeccted.
2. Leptin promotes the proliferation of ovarian cancer cells by upregulating miR-182and miR-96that targeting FOXO3. promotion of the gastric cancer cell MT1-MMP membrane expression is a KIF1B dependent process, involved in gastric cancer cell invasion process.
3. Elucidation that the role of the leptin receptor downstream signaling pathway molecules STAT5in the leptin induced miR-182and miR-96, which provides new target and experimental data for the early detection and therapy of ovarian cancer.
Significance
In his study, we elucidates the effect and underlying mechanism of leptin on FOXO3, explores the role of this process in the proliferation of ovarian cancer cells, and highlights the function of miR-182and miR-96in the leptin induced proliferation of ovarian cancer cells. These finding will help to know better about the mechanism of tumorigenesis and development of ovarian cancer and provide new targets for the early detection and therapy of ovarian cancer.
Part II THE IN VIVO EFFECT AND UNDERLYING MECHANISM OF ANTI-MIR-182COMPOUND ON GROWTH AND INVASION OF OVARIAN CANCER
Background
Ovarian cancer is the most lethal gynecologic malignancy and high grade serous ovarian carcinoma (HGSOC) is the most aggressive form of ovarian cancer, contributing to most ovarian cancer death due to early invasion and metastasis at the time of diagnosis. Current chemotherapies by reducing tumor burden may not benefit significantly in long term. Anti-microRNA treatment is emerging as a potential modality for cancer therapy. MicroRNAs (miRNAs) are a group of small, non-coding RNAs. Some tumor-related miRNAs may play a critical role in tumor growth, invasion and metastasis. MiR-182is an onco-miRs and its overexpression is associated with aggressive ovarian cancer, largely contributed by its negative regulation of multiple tumor suppressor genes involving in tumor growth, invasion, metastasis and DNA instability.
MiR-182is is overexpressed in HGSOC and is associated with aggressive tumor growth and invasion in HGSOC as well as the poor progression-free survival of patients. Our prelimitary data show that miR-182plays critical role in the proliferation and transformation of ovarian cancer cells mediated by leptin. Therefore, anti-miR-182may provide a promising therapy to reduce tumor burden and metastasis potential in those malignant neoplasms with miR-182overexpression. All in al, based on the domestic and overseas progress in this field and our preliminary research data, we tried for the first time to investigate and characterize the role of anti-miR-182treatment as a potential anti-invasion therapeutic strategy for ovarian cancer. The study selected the ovarian cancer cell line with miR-182overexpression and developed the xenografts by implanting cancer cells into intrabursa.
Gastric cancer (GC) ranks as the secondary leading cause of cancer-related death in the world. Adipocytes provide fatty acids for rapid tumor growth, and the dysfunction of lipid metabolism can lead to the pathogenesis of human GC. Leptin is an adipokine of the obesity (ob) gene, and its overexpression is closely correlated with GC metastasis, but its exact effect and the underlying mechanism in tumor progression remain unclear. Our previous findings reveal that leptin may promote GC cells invasion by upregulation of MT1-MMP. MT1-MMP may cleave intercellular adhesion molecule-1(ICAM-1), and then enhance tumor cells migration. ICAM-1is overexpressed and plays crucial roles in tumor metastasis. This study aimed to characterize the influence of leptin on ICAM-1expression in GC and elucidate its underlying molecular mechanism.
We previously determined that leptin could promote the migration of GC through upregraulation the expression of MT1-MMP,and other dates suggested that MT1-MMP could cut ICAM-1and promote the migration of tumor cells,but the exact mechanism of how leptin act on ICAM-1remains unclear.Overall,based on the worldwide study progress and the results we discovered previously,we collected GC tissue samples and detected the expression of leptin and ICAM-1,then analyzed the correlation between leptin and clinical parameters.And we also observed the influence of leptin on the effects of ICAM-1expression,discussed the function of ICAM-1in the process of GC migration.We finally observed the function of Rho/ROCK in ICAM-1expression regulated by leptin,simultaneously provided potential therapy target for the control of GC in early period. The tumor growth, invasion and metastasis were evaluated during anti-miR-182treatment by IVIS system and histopathology, followed by thorough analysis of miR182expression and its target gene expression. The treatment toxicity was also evaluated. Our study provides a potential therapeutic modality targeting at the aggressive tumor growth of HGSOC.
Objective
1. To produce a mouse model mimic to human ovarian cancer microenvironment.
2. To observe dynamicly the in vivo effect of anti-miR-182on the growth and invasion of ovarian cancer.
3. To evaluate the tumor growth, invasion and metastasis by histopathology.
4. To elucidate the molecular mechanism through which anti-miR-182regulate the growth and invasion of ovarian cancer.
Methods
1. Detection of the effect of anti-miR-182on the oncogenic properties of ovarian cancer.
The human ovarian cancer cells OVCAR3、HEY and SKOV3were used for the in vitro test. HEY and SKOV3with miR-182overexoression was established by the lentiviral system. The anti-miR-182was transfected into the above ovarian cancer cells and cutured for48hours at37℃. RT-PCR was employed to detect the interference efficacy of anti-miR-182on miR-182expression. Western blot was employed to measure the protein level of the miR-182target genes.
The anti-miR-182was transfected into the above ovarian cancer cells and cutured for24hours at37℃. Transwell assay was used to measure the invasion of cells. The proliferation of cells with or without anti-miR-182treatment was detected by WST-1for the next consecutive5days. The soft agar assay was employed to measure the anchorage-independent growth ability of cells.
2. Set up the mouse model mimic to the human primary ovarian cancer.
The luciferase gene was introduced into SKOV3cell with stable miR-182overexpression (SKOV3-miR-182-luc). The luciferase expression was used as tracer molecule for in vivo bioluminescence imaging system (analysis (IVIS) of tumor status.
The SKOV3-miR-182-luc cells (106cells) were embedded into collagen IV matrix, which finally shringe into tumor spherical nodules of about0.2centimeter in diameter. The tumor nodules were implanted into left ovarian intrabursal space in8weeks old mice.
3. Anti-miR-182administration in vivo.
After surgery, mice were randomly divided into two groups of control (20mice) test (20mice). On the third day after surgery, mice from control group were treated with scramble compound and mice from test group were treated with anti-miR-182(dose of25mg-kg-'body weight in0.2mL per injection) by intraperitoneum (I.P.) injection twice weekly. Among19mice from test group,4mice were treated with anti-miR-182for just one week (short term) and the other15mice were treated for eight weeks (long term).
4. Effect of anti-miR-182on the growth of ovarian carcinoma in vivo.
Tumor growth was monitored by IVIS each week for up to8weeks and the tumor size was reflected by the luciferin signal. All mice were euthanized at the end of8weeks. The bilateral ovaries and uterus were isolated and taken out from the pelvic cavity, and tumors in ovaries were measured and photographed. The serum and part of tumor tissue as well as liver was frozen at-80℃. The other organs were dissected and collected, including spleen, kidneys, liver, intestine, pancreas, omentum, stomach, omentum, pelvic lymph nodes, diaphragm lungs and heart. All cases of primary tumor tissue and organs were fixed and processed.
ELISA was used to detect the CA125level in mouse serum. RT-PCR and real-time PCR was employed to measure the mRNA expression of genes that is related with cell cycle or cell death (CDKN1A、CDKN1B、FOXO1、CHEK2).
5. Effect of anti-miR-182on the invasion and metastasis of ovarian carcinoma in vivo.
The mice were scanned weekly by the IVIS system to monitor tumor metastasis. All peritoneal organs as well as lungs and heart were sectioned and stained. The metastasis tumors were assessed and analyzed microscopically.
6. Molecular evaluation of the tumor tissue.
Total RNA was extracted from the serum and tumor tissue. RT and real-time PCR was employed to detect the expression of miR-182, as well as the polycistron cluster of miR-182family including miR-96and miR-183, in mice serum and tumor tissue. Western blot was used to detect the expression of miR-182target genes (BRCA1, MTSS1and FOXO3) and HMGA2in tumor tissue at the protein level.
7. The influence od anti-miR-182treatment on the tumor microenviorment.
The histology of all tumor xenografts and their relationship to ovary, oviducts and paraovarian fat pat. Immunohistochemitry was used to confirm the cell type in the tumor microenviroment. The human inflammatory gene array was used to measure the alteration of inflammatory gene profile. RT and real-time PCR was employed to detect the chemokines and cytokines in the control (n=8) and test (n=8) tumor at the mRNA level.
8. Toxicity analysis of the anti-miR-182treatment.
At the end of the treatment (8-week treatment), all organs (liver, pancreas, gut, kidneys, spleen and uterus) of mice were collected and processed. The histology and cytology of organs was assessed by histologic examination to evaluate the toxicity of the anti-miR-182treatment.
Results
1. Anti-miR-182inhibited the proliferation, invasion and anchorage-independent growth of ovarian cancer cells in vitro.
RT-PCR data reveals that when ovarian cancer cells (OVCAR3, HEY-miR-182and SKOV3-miR-182) were treated with anti-miR-182, the miR-182expression was significantly repressed in all cells (P<0.001). Weatern blot results shows that the target genes expression of miR-182were all upregulated by anti-miR-182(FOX03:1.47±0.18-fold, P<0.05; BRCA1:3.09±0.99-fold, P<0.05; MTSS1:1.39±0.18-fold, P<0.05), while the expression of miR-182induced gene HMGA2was down-regulated (62.6%±17.2%, P<0.01). The similar trend was found in OVCAR3and HEY-miR-182cells.
Transwell invasion assay reveals that anti-miR-182significantly inhibited tumor cell invasion/migration through Matrigel (57.1%±7.9%, P<0.001). Soft agar colony formation data shows that anti-miR-182significantly reduced the number and size of the single-cell colonies (54.8%±16.3%, P<0.01). WST-1assay show that anti-miR-182suppressed the proliferation of ovarian cancer cells in vitro (day3: P<0.05; day4:P<0.05; day5:P<0.01).
2. Intrabursal implantation of SKOV3tumor cells in nude mice.
SKOV3-miR-182-luc cells were embedded into collagen IV pellet and implanted intrabursaly into the nude mice. One day after surgery, IVIS scanning show that all mice dispayed the similar level of bioluminace signal, suggesting similar number cells implanted and no peritoneal leaking.
3. Anti-miR-182treatment reduces tumor growth of ovarian cancer in vivo.
The IVIS imaging shows the tumor size between control ((17.73±7.72) x109) and test groups ((5.20±6.63) x109, P<0.01) began different at week5and reached wider for the rest of weeks. The tumor size in anti-miR-182treatment group (5.42±1.00mm,90.59±37.31mm3) was significantly smaller than that in control (8.07±1.74mm,310.89±187.12mm3)(p<0.001). The treated tumors were32.8%(in diameter) or70.9%(in volume) smaller than that in vehicle control. ELISA data reveals that CA125level was lower in testing group than in control group (P=0.06).
Real-time PCR analysis show that anti-miR-182treatment siginificanly upregulated several cell cycle and cell death regulators that are predicted target genes of miR-182, including CDKN1A (P=0.0199), CDKN1B(P=0.0156), FOXO1(P<0.0001) and CHEK2(P<0.0001).
4. Anti-miR-182treatment inhibits metastasis of ovarian cancer in vivo.
The metastatic tumors detected by IVIS at8week were28%(5/18) control and7%(1/15) in testing groups. Histologic data shows that77.8%(14/18) of mice from control group and33.3%(5/15) of mice from testing group had at least one metastatic disease. A total of31metastases were found in controls, while only6metastases in treated mice. The rate of metastasis between test and control mice were statistically significant (P<0.05). The most common site of metastatic disease were spleen (44%(8in18) in control,20%(3in15) in test), omentum28%(5in18) in control and13%(2in15) in test, pancreas22%(4in18) in control and0%(0in15) in test and liver11%(2in18) in control and0%(0in15) in test. Metastases in pancreas were larger and more aggressive than other metastases. Only1metastasis was noted in control group. Although there was no metastasis into kidney parenchyma, as many as50%(9/18) ovarian tumors in control were adhered to ipsilateral kidney, but not seen in anti-miR-182treated mice (0/17). No lung metastasis was found in either control or testing groups.
5. Molecular analysis of target gene alterations in xenografts of tumors when treated by Anti-miR-182.
Real-time RT-PCR analysis revealed that miR-182expression was either in very low or non-detectable levels all tumors with anti-miR-182treatment in comparison to control (reduced94.9%±13.3%, P<0.001). Anti-miR-182could also significantly reduce miR-183expression (92.5%±9.7%, P<0.001), but not miR-96(P>0.05). Reduction of miR-182(53.2%±10.4%, P<0.01) and miR-183(84.3%±20.6%, P<0.01) was also found in mouse serum samples.
Western blot and qRT-PCR analyses reveals that the miR-182target genes were partially or completely restored by anti-miR-182treatment (BRCA1:2.63±0.35-fold, P<0.01; FOXO3:1.42±0.18-fold, P<0.01; MTSS1:2.63±1.29, P<0.01). While the expression of HMGA2was downregulated (71.5%±4.0%, P<0.01).
6. Anti-miR-182treatment induces inflammation which prevent tumor invasion.
About64%(11/17) of implanted site (paraovarian soft tissue) had extensive monocytic aggregate in mice with anti-miR-182treatment, while only17%(3/17) of control mice showed similar but much less monocytic aggregate. Immunostain for histolytic marker KP1confirmed the mononuclear histiocytes origin. Tumor implants with minimal monocytic infiltrates had clear infiltrating growth pattern and penetrating through intrabursa into surround fat, while those with heavy monocytic infiltrates could barely pass through intrabursal capsule.
The inflammatory gene array reveals that the inflammatory gene profile differed between control and testing tumors. Real-time PCR analysis shows that compared with the control tumors (n=8), the expression of CCL2(P<0.0001), IL-6(P=0.0019), IL-8(P=0.0002), TNF-a (P=0.0088) and IFN-y (P<0.0001) was siginificantly upregulated in the anti-miR-182treatment tumors (n=8). In SKOV3cell line with either low or high endogenous miR-182expression, administration of anti-miR-182could significantly increase CCL2expression (SKOV3:3.60±0.73-fold, P<0.001; SKOV3-miR-182:11.39±0.21-fold, P<0.001).
7. Anti-miR-182treatment has no genotoxic effects on mouse.
By histologic examination of organ system, we did not see significant histology and cytology changes after8weeks of treatment in liver, pancreas, gut, kidneys, spleen and uterus.
Conclusion
1. We produced successfully a mousemodel that can mimic well to the growth and invasion pattern of human primary ovarian cancer, which will provide good mouse model for future in vivo research on ovarian cancer.
2. Anti-miR-182treatment can reduce tumor growth, invasion and metastasis of ovarian cancer both in vitro and in vivo.
3. Anti-miR-182treatment can inhibit invasion of ovarian cancer by improving the tumor microenviroment.
All of those above provide the experimental support as well as new target and method for the early control of human ovarian cancer.
Significance
In this study produces a mouse model that can mimic to the development pattern of human ovarian cancer successfully. Our data illuminates the effect and underlying mechanism of anti-miR-182treatment on the tumor burden and invasion of ovarian cancer in vivo, and further confirms the molecular mechanism of the tumoregenesis of ovarian cancer. These findings provides new target for the diagnosis and therapy of primary and aggressive ovarian cancer, which will help to improve the current therapy model.
研究背景
卵巢癌是妇科常见的恶性肿瘤,严重威胁妇女健康,死亡率居妇科肿瘤之首。早期诊断困难及复发转移导致治疗手段缺乏,是卵巢癌高死亡率的主要原因。因此及早捕捉预警信号并阐明卵巢癌发生发展的相关分子机制,对卵巢癌的防治至关重要,但目前相关机制尚未阐明。流行病学数据表明肥胖增加激素依赖性肿瘤(如卵巢癌)的发生风险,瘦素(Leptin)作为重要的脂肪因子类激素之一,参与调控机体的脂肪代谢,并与多种肿瘤的发生有关。研究发现卵巢癌时瘦素水平升高,且与患者无病生存期密切相关,但目前瘦素与卵巢癌增殖、凋亡的关系及其在卵巢癌起源中的作用尚不明确。
MicroRNA (miRNA)是一类非编码小RNA,可在转录后水平通过抑制翻译或者诱导降解抑制靶mRNA的功能,调节细胞分化、增殖和凋亡过程。研究表明多种肿瘤组织存在miRNAs的异常表达或者突变,并影响肿瘤的生长、侵袭和转移。MiR-182是重要的miRNAs分子,其过表达于输卵管内上皮性浆液性卵巢癌和高级别浆液性卵巢癌,并促进良性和恶性肿瘤细胞的侵袭和转化,提示miR-182在卵巢癌的早期发生中具有重要意义,而对其进行有效调节将有助于卵巢癌的防治。研究表明瘦素可通过影响miRNAs的表达调节骨骼肌细胞的生长,但关于卵巢癌发生和发展过程中瘦素影响miRNAs表达及机制未见报道。
综上所述,基于相关领域国内外的研究进展,本课题体外研究了瘦素通路下游信号分子STAT5介导的miR-182上调对卵巢癌细胞生长和转化的影响及机制。本研究肯定了瘦素和miR-182在卵巢癌中的作用,进一步阐明了卵巢癌发生发展的机制,为卵巢癌的早期诊断和治疗提供新的线索。
研究目的
1.瘦素对卵巢癌细胞生长、增殖及转化的作用。
2.探索介导瘦素调节卵巢癌细胞生长、增殖及转化的miRNAs及miR-182和miR-96在瘦素作用中的影响。
3.观察瘦素下游信号通路对miR-182和miR-96的作用。
研究方法
1.卵巢癌细胞系瘦素及瘦素受体的表达
卵巢癌细胞(SKOV3和A2780)37℃C培养24小时,RT-PCR检测瘦素和瘦素长型受体OB-Rb和短型受体OB-Ra表达,Western blot检测瘦素及其受体的蛋白水平。培养72小时后收集上清,ELISA法检测内源性瘦素表达。
2.瘦素对卵巢癌细胞增殖、凋亡和转化的影响
分别给予SKOV3和A2780细胞不同剂量瘦素(0、10、50、100和500ng/mL),每隔24小时收集细胞,WST-1法检测细胞增殖并绘制生长曲线。卵巢癌细胞SKOV3和A2780分为Control、Leptin、Anti-leptin、Leptin+Anti-leptin组,在0.3%的软琼脂中37℃C培养3周后,结晶紫染色观察不同组别软琼脂中形成的单细胞克隆数目。卵巢癌细胞分为Control、Leptin、Anti-leptin、Leptin+Anti-leptin组37℃培养48小时后,流式细胞术检测各组细胞的凋亡率,瘦素处理的结肠癌细胞-HCT-116用于阳性对照。
3.瘦素作用后卵巢癌细胞转录因子FOXO3表达的变化,并观察其在瘦素调节卵母巢癌细胞增殖中的作用
卵巢癌细胞(SKOV3和A2780)分为Control、Leptin、Anti-leptin、 Leptin+Anti-leptin组,37℃C培养48小时后,Western blot检测FOXO1和FOXO3蛋白水平变化,筛选表达变化的FOXO分子,然后Western Blot检测其下游靶分子蛋白水平表达。
siRNA干扰相应FOXO分子的表达,RT-PCR和Western Blot分别从基因水平和蛋白水平检测干扰效率,卵巢癌细胞分为Control、Leptin、Con-siRNA和FOXO-siRNA组,37℃培养48小时后,WST-1检测各组细胞的增殖变化。
4.瘦素对靶向作用于FOXO分子3'-UTR的miRNAs的影响,miRNAs在卵巢癌细胞增殖中的作用
PCR克隆扩增FOXO分子的3'-UTR并构建psiCHECK2荧光载体,用该载体及对照空载体分别转染卵巢癌细胞SKOV3和A2780。37℃培养24小时后,细胞分为Vector、Vector+Leptin、FOXO-3'-UTR, FOXO-3'-UTR+Leptin组并继续培养48小时,双荧光报告系统检测不同组别的荧光信号强度。
卵巢癌细胞SKOV3和A2780分为Control和Leptin组,培养48小时后,RT-PCR检测miRNAs分子的基因水平表达变化。用所测miRNAs的mimics转染卵巢癌细胞,37℃培养48小时,RT-PCR检测各组过表达效率,Western blot检测不同mimics转染细胞的FOXO分子蛋白水平的变化,WST-1检测细胞的增殖变化。用miRNAs抑制剂转染卵巢癌细胞,37℃培养24小时后RT-PCR检测各组抑制效率,细胞分为Control、Leptin、Leptin+miRNAs inhibitor并继续培养48小时,Western blot检测卵巢癌细胞的FOXO分子蛋白水平的变化,WST-1检测细胞的增殖变化。
5.瘦素受体下游信号传导分子STAT3、STAT5在瘦素调节的miRNAs表达上调中的作用
瘦素作用卵巢癌SKOV3和A2780细胞48小时后,Western blot检测瘦素长型受体OB-Rb表达水平的变化。
瘦素处理卵巢癌SKOV3和A2780细胞不同时间(0,15,30,60分钟)后,Western blot检测不同STAT信号通路蛋白(STAT3、STAT5)的表达变化。
卵巢癌SKOV3和A278048细胞分为Control、Leptin、STAT5-siRNA、 Cucurbitacin Ⅰ (p-STAT3抑制剂)、Leptin+STAT5-siRNA、Leptin+Cucurbitacin I组,RT-PCR检测目的miRNAs分子基因表达水平变化。
研究结果
1.卵巢癌细胞表达瘦素受体
实验表明,卵巢癌SKOV3和A2780细胞在蛋白水平和mRNA水平均可检测到瘦素、瘦素长型受体OB-Rb和短型受体OB-Ra的表达,两种细胞培养上清中也存在极低量的瘦素(分别为38.39±3.72pg/mL和35.40±2.44pg/mL),因远低于生理剂量,后续实验均给予外源性瘦素。
2.瘦素促进卵巢癌细胞的增殖和转化能力
瘦素以剂量依赖和时间依赖方式促进卵巢癌细胞SKOV3和A2780的增殖能力(P<0.05)。同时增加软琼脂中两种卵巢癌细胞克隆的数目和大小(SKOV3细胞：2.72--±0.34倍,P<0.05；A2780细胞：1.97±0.15倍,P<0.05),瘦素抗体可阻断瘦素的促细胞转化能力。流式细胞术显示瘦素不影响卵巢癌细胞SKOV3和A2780的凋亡率(P>0.05),但显著抑制结肠癌细胞HCT-116的凋亡(P=0.0169)。
3.瘦素抑制卵巢癌细胞FOXO3的表达,从而促进细胞增殖
Western blot结果表明瘦素(100ng/ml)显著抑制卵巢癌细胞SKOV3和A2780内FOXO3的表达(SKOV3细胞：55.1±1.8%,P<0.01；A2780细胞：48.4±10.1%,P<0.01), FOXO1的表达无显著变化(P>0.05)。进一步检测发现瘦素(100ng/ml)显著抑制细胞内FOXO3的两个下游靶基因Bim (SKOV3细胞：45.8±1.50%,P<0.01；A2780细胞：46.7±3.6%,P<0.01)和p27(SKOV3细胞：41.4±6.3%,P<0.01；A2780细胞：36.7±9.0%,P<0.01)的表达。利用FOXO3-siRNA干扰掉细胞内FOXO3基因和蛋白表达后,发现两种细胞的增殖能力均显著增强(SKOV3细胞：1.63±0.10倍,P<0.01；A2780细胞：1.59±0.16倍,P<0.05)。
4.瘦素通过上调靶向作用于FOXO33'端非翻译区(3'-UTR)的miRNAs抑制FOXO3表达,从而促进卵巢癌细胞增殖
利用包含FOXO33'-UTR的psiCHECK2荧光载体转染细胞后,双荧光报告系统显示瘦素(100ng/ml)显著降低卵巢癌细胞SKOV3和A2780内的荧光素酶强度(SKOV3细胞：53.4±2.5%,P=0.001；A2780细胞：41.6±8.5%,P=0.016)。RT-PCR结果显示瘦素(100ng/ml)显著上调SKOV3细胞中靶向作用于FOXO3的肿瘤相关miRNAs,包括miR-182(2.26±0.23倍,P<0.01)、miR-106(1.52±0.16倍,P=0.03)、miR-96(1.91±0.03倍,P<0.01)和miR-155(1.53±0.10倍,P=0.01),对A2780细胞中的上述miRNAs具有相似的作用。但两种细胞miR-93和miR-153的表达均不受瘦素影响(P>0.05)。
上述六种miRNAs的]mimics分别转染卵巢癌细胞SKOV3和A2780, Westernblot显示miR-182(SKOV3细胞：64.8%±11.4%,P<0.05；A2780细胞：51.8%±0.5%,P<0.05)和miR-96(SKOV3细胞：58.3%±2.5%,P<0.05；A2780细胞：42.2%±4.1%,P<0.05)两种mimics可以有效抑制两种细胞内FOX03的表达,miR-155和miR-106a的mimics则不具备此功能(P>0.05)。利用抑制剂干扰阻断两种细胞内miR-182和miR-96的表达后,Leptin对FOX03表达的抑制作用亦消失。WST-1检测两种细胞的增殖发现,miR-182(SKOV3细胞：2.65±0.05倍,P<0.01；A2780细胞：2.52±0.06倍,P<0.01)和miR-96(SKOV3细胞：2.03±0.08倍,P<0.01；A2780细胞：1.85±0.13倍,P<0.05)的mimics可促进两种细胞的增殖,而miR-182(SKOV3细胞：37.1%±3.3%,P<0.01；A2780细胞：37.4%±5.0%,P<0.01)和miR-96(SKOV3细胞：28.9%±6.2%,P<0.01；A2780细胞：29.9%±3.5%,P<0.01)抑制剂则可有效抑制瘦素对两种细胞的促增殖效应。
5.瘦素通过STAT5通路上调细胞中miR-182和miR-96的表达
瘦素(100ng/ml)能够促进卵巢癌细胞SKOV3和A2780瘦素长型受体OB-Rb的表达。同时瘦素可活化瘦素受体下游信号通路STAT3和STAT5,分别在60、30分钟活化达到高峰,特异性阻断两个通路发现cucurbitacin Ⅰ (STAT3抑制剂)不影响瘦素诱导的miR-182和miR-96表达上调(P>0.05),而STAT5-siRNA能够降低瘦素导致的miR-182(SKOV3细胞：43.5%±2.5%,P<0.01；A2780细胞：32.0%±9.4%,P<0.01)和miR-96(SKOV3细胞：34.1%±0.8%,P<0.01；A2780细胞：22.7%±4.2%,P<0.05)表达上调,表明STAT5通路参与miR-182和miR-96依赖的FOXO3抑制和细胞增殖。
结论
1.MiR-182和miR-96在瘦素抑制FOXO3表达、进而促进抑卵巢癌细胞增殖这一过程中发挥重要的介导作用。
2.瘦素通过下游STAT5信号途径上调卵巢癌细胞miR-182和miR-96的表达。
第二部分抗mi R-182化合物对卵巢癌体内生长和转移的影响及机制研究
研究背景
卵巢癌是女性死亡率最高的生殖系统肿瘤,高级别浆液性卵巢癌是卵巢癌中侵袭程度最高的类型,多数患者确诊时已属晚期,肿瘤已出现广泛转移。现有的手术治疗和化疗可在一定程度上减少肿瘤负荷,但对患者远期生存率并无显著影响。近年来抗microRNA治疗(anti-microRN A)成为癌症治疗的一种潜在有效的途径。miRNAs为一种小分子非编码RNA,某些miRNAs与肿瘤的生长、侵袭和远处转移密切相关。其中miR-182是一种原癌miRNAs,可负性调节多种与肿瘤生长、侵袭、转移调节有关的抑癌基因,从而促进多种肿瘤的发生发展。
研究表明miR-182过表达与高级别浆液性卵巢癌,与肿瘤的生长及侵袭密切相关,且与患者的不良预后有关。本课题组前期发现miR-182在瘦素介导的促卵巢癌细胞增殖和转化过程中发挥关键作用,因此在miR-182过表达的卵巢癌中,anti-miR-182治疗可能会减少肿瘤负荷、抑制侵袭转移。综上所述,基于本课题国内外的研究进展及课题组的前期工作基础,本课题组首次尝试应用anti-miR-182进行卵巢癌治疗。我们将人卵巢癌细胞固定植入小鼠卵巢包膜内,成功构建了小鼠原位卵巢癌模型,并给予小鼠anti-miR-182系统治疗,应用在体生物光学成像技术动态监测肿瘤的生长转移,治疗结束后用组织病理学手段确认了肿瘤在小鼠各脏器的侵袭转移情况及药物的作用,并进一步对肿瘤组织中miR-182靶基因的表达情况进行了分子生物学分析。本研究为侵袭性高级别浆液性卵巢癌提供了一种潜在的新的治疗手段。
研究目的
1.模拟人卵巢癌构建小鼠原位卵巢癌模型。
2.给与小鼠anti-miR-182治疗,动态观察药物对肿瘤生长和侵袭的影响。
3.组织学观察药物对肿瘤生长和侵袭转移的作用。
4.分析anti-miR-182影响肿瘤侵袭转移的分子机制。
研究方法
1.体外检测anti-miR-182对卵巢癌细胞的影响
体外实验利用人卵巢癌细胞系OVCAR3、HEY和SKOV3细胞,应用慢病毒包装转染系统构建miR-182稳定过表达的HEY和SKOV3细胞系。利用脂质体转染技术将anti-miR-182瞬时导入上述卵巢癌细胞系,细胞分为Control、 anti-miR-182组,37℃C培养48小时后RT-PCR检测细胞内miR-182的基因水平,Western blot检测miR-182靶基因的蛋白水平。
将anti-miR-182利用脂质体转入卵巢癌细胞OVCAR3、HEY和SKOV3,37℃培养24小时后,1)Transwell侵袭实验检测卵巢癌细胞侵袭能力的变化；2)将Control细胞和anti-miR-182分别种在5个96孔板上(2×103每孔,每种细胞每板5个复孔),利用WST-1连续五天每天检测细胞的增殖并绘制生长曲线；3)软琼脂克隆形成实验检测卵巢癌细胞非停泊性生长能力的变化。
2.构建裸鼠原位人卵巢癌模型
利用慢病毒包装转染系统将荧光素酶基因导入稳定过表达miR-182的SKOV3卵巢癌细胞(SKOV3-miR-182-luc),该荧光素酶与底物作用后可被在体生物光学成像系统检测(IVIS)。
SKOV3-miR-182-luc消化计数后,利用Ⅳ型胶原将肿瘤细胞(106个细胞)固定成直径约0.2厘米的球状小团,显微镜下手术将细胞团块植入8周龄裸鼠左侧卵巢包膜内。
3. Anti-miR-182治疗
术后小鼠随机分成对照组(20只)和anti-miR-182实验组(20只),并于术后第三天分别腹腔注射安慰剂或anti-miR-182(25mg/kg),每周两次,整个治疗周期为8周(其中实验组4只动物仅治疗1周)。
4. Anti-miR-182对卵巢原发肿瘤生长的影响
治疗期间每周一次应用IVIS技术动态监测对照组和实验组肿瘤的生长和侵袭,利用软件定量分析两组动物肿瘤信号强度。
8周后处死小鼠,测量对照组和实验组肿瘤实际长短径线,收集冰冻所有小鼠血清和部分肿瘤、肝脏组织,切除小鼠所有器官(肝、肾、脾、胰腺、肠道组织、子宫输卵管、卵巢肿瘤及对侧卵巢、肺、心脏)并固定备用。
ELISA检测对照组和实验组血清中CA125的水平。提取对照组和实验组肿瘤组织总RNA, RT-PCR检测两组肿瘤组织内与细胞周期调节或细胞死亡有关(CDKN1A、CDKN1B、FOXO1、CHEK2)的基因表达水平。
5.Anti-miR-182对卵巢肿瘤侵袭远处转移的影响
IVIS技术动态监测对照组和实验组原发瘤的转移信号。对两组动物所有器官进行组织切片和HE染色,显微镜下观察个脏器转移瘤的情况并进行统计分析。
6.肿瘤组织的分子生物学水平分析
提取对照组和实验组动物肿瘤及血清中的总RNA, RT和实时定量PCR检测肿瘤即血清中miR-182及其同一基因簇miR-183和miR-96的基因表达。提取两组动物肿瘤组织内的总蛋白,Western blot检测两组肿瘤组织内miR-182靶基因(BRCA1、MTSS1、FOX03)和诱导基因HMGA2的蛋白水平变化。
7. Anti-miR-182治疗对肿瘤微环境的影响
对对照组和实验组肿瘤组织切片和HE染色,镜下观察肿瘤与卵巢、输卵管和卵巢周围脂肪组织的关系,免疫组织化学染色确定肿瘤周围微环境中的细胞成分。炎症基因芯片技术观察两组肿瘤组织内炎症基因谱的变化。RT-PCR检测对照组和实验组(各8例)肿瘤内趋化因子和细胞因子(CCL2、IL-6、IL-8、TNF-α、 IFN-γ)的基因水平变化。
人卵巢癌细胞系SKOV3分为Control组、anti-miR-182组、miR-182过表达组、miR-182过表达+anti-miR-182组,37℃培养48小时后RT-PCR检测细胞内CCL2基因水平的变化。
8.药物毒性分析
8周治疗结束后,收集对照组和anti-miR-182实验动物的全部器官(肝脏、胰腺、胃肠道、肾、脾及子宫)并进行组织切片和HE染色,镜下观察两组组织内细胞的形态学变化,判断药物的毒性作用。
研究结果
1. Anti-miR-182抑制体外卵巢癌细胞的增殖、基质胶侵袭和转化能力
RT-PCR结果显示anti-miR-182显著抑制OVCAR3、HEY-miR-182和SKOV3-miR-182细胞内miR-182的基因水平(P<0.001)。Western blot结果显示,anti-miR-182显著上调SKOV3-miR-182细胞内miR-182靶基因FOXO3(1.47±0.18倍,P<0.05)、BRCAl (3.09±0.99倍,P<0.05)、和MTSS1(1.39±0.18倍,P<0.05)的蛋白水平,同时下调miR-182诱导的HMGA2(62.6%±17.2%,P<0.01)的蛋白表达,对OVCAR3和HEY-miR-182细胞具有相似效应。
基质胶侵袭实验结果显示anti-miR-182可显著抑制SKOV3-miR-182细胞的侵袭能力(抑制率57.1%±7.9%,P<0.001)。软琼脂克隆形成实验表明anti-miR-182可显著减少单细胞克隆形成数目和大小(抑制率54.8%±16.3%,P<0.01)。WST-1细胞增殖实验结果显示anti-miR-182抑制SKOV3-miR-182细胞的体外增殖(第3天,P<0.05；第四天P<0.05；第五天P<0.01)。
2.构建裸鼠原位人卵巢癌模型
将SKOV3-miR-182-luc细胞包埋入Ⅳ型胶原并植入裸鼠左侧卵巢包膜内,术后第二天,IVIS检测结果表明所有小鼠荧光信号相同(相对荧光强度3-3.5x109),表明卵巢癌细胞植入量在所有动物体内基本一致,无腹腔泄漏。
3. Anti-miR-182治疗抑制卵巢癌肿瘤的体内生长
术后第3天,小鼠被随机分为对照组和anti-miR-182实验组并分别给予安慰剂或anti-miR-182腹腔注射治疗,治疗剂量25mg/kg,一周两次,整个疗程为8周。IVIS图像显示从治疗第5周开始,实验组与对照组肿瘤大小之间开始出现差异(对照组信号强度为(17.73±7.72)×109,实验组信号强度为(5.20±6.63)×109,P<0.01),且该差异随着治疗时间延长而增大。
处死小鼠测量两组小鼠卵巢肿瘤实际大小发现表明anti-miR-182治疗组的肿瘤(5.42±1.00mm,90.59±37.31mm3)显著小于对照组(8.07±1.74mm,310.89±187.12mm3),其最长径线较对照组减少38%,体积较对照组减少70.9%(P<0.01). ELISA结果显示对照组小鼠血清CA125水平高于实验组小鼠(P=0.06)。
RT-实时定量PCR结果显示,mti-miR-182显著上调肿瘤组织内与细胞生长和死亡有关的miR-182假定靶基因CDKN1A(P=0.0199)、CDKN1B(P=0.0156)、 FOXO1(P<0.0001)、CHEK2(P<0.0001)。
4. Anti-miR-182治疗抑制小鼠体内卵巢癌细胞的侵袭和远处转移
治疗第8周IVIS图像显示,对照组肿瘤的远处转移率为28%(5/8),实验组的转移率为7%(1/15)。组织学分析结果显示对照组77.8%(14/18)的动物存在至少一处转移癌灶,实验组33.3%(5/15)的动物存在至少一处转移灶；对照组共发现31个转移瘤,实验组仅发现6个转移瘤。对照组和实验组肿瘤远处转移率具有显著的统计学意义(P<0.05)。转移瘤数目由多到少的部位是依次是脾脏(对照组阳性率为44%,8/18；实验组阳性率为20%,3/15)、大网膜(对照组阳性率为28%,5/18；实验组阳性率为13%,2/15)、胰腺(对照组阳性率为22%,4/18；实验组阳性率为0%)、和肝脏(对照组阳性率为11%,2/18；实验组阳性率为0%)。与其他部位的转移瘤相比,胰腺转移瘤更大、侵袭性更强。对照组有一只小鼠发生了对侧卵巢转移。虽然肾实质内未发现转移瘤,对照组50%(9/18)的动物的卵巢肿瘤与同侧发生肾粘连,而anti-miR-182治疗组动物未发现此现象(0/15)。对照组和实验组动物均未发现肺转移灶。
5. Anti-miR-182治疗后肿瘤组织的分子生物学分析
实时定量PCR结果显示与对照组肿瘤相比,anti-miR-182治疗组的肿瘤组织内miR-182表达水平极低(降低94.9%±13.3%)或低于测量范围(P<0.001),同时显著降低miR-183的表达(降低92.5%0±9.7%,P<0.001),但对miR-96的表达无影响(P>0.05)。小鼠血清内miR-182(降低53.2%±10.4%,P<0.01)和miR-183(降低84.3%±20.6%,P<0.01)的水平也显著降低。
Western blot结果显示经anti-miR-182治疗后肿瘤内miR-182的靶基因BRCA1(2.63±0.35倍,P<0.01)、FOXO3(1.4±0.18倍,P<0.01)和MTSS1(2.63±1.29倍,P<0.01)与对照组相比显著上调,而miR-182诱导的基因HMGA2表达下调(降低71.5%±4.0%,P<0.01)。
6. Anti-miR-182治疗诱导的炎症反应抑制肿瘤的侵袭
组织学观察发现anti-miR-182治疗组73.3%(11/15)的小鼠肿瘤细胞植入部位(卵巢旁软组织)有大量单核细胞浸润,对照组虽有16.7%(3/18)的动物存在单核细胞浸润,但单核细胞数量明显少于实验组。免疫组织化学染色证实了浸润细胞为KP1阳性小鼠单核细胞。单核细胞浸润少的肿瘤表现出浸润生长模式,并穿透卵巢包膜侵入周围脂肪组织,而周围有大量单核细胞浸润的肿瘤则局限于卵巢包膜内。
炎症基因谱分析显示对照组肿瘤与anti-miR-182治疗组肿瘤组织炎症基因谱发生改变。实时定量PCR结果表明与对照组相比,实验组肿瘤组织内CCL2(P<0.0001). IL-6(P=0.0019)、IL-8(P=0.0002)、TNF-α (P=0.0088)、IFN-γ(P<0.0001)的表达显著上调。体外实验表明,无论在初始SKOV3细胞还是SKOV3-miR-182细胞,anti-miR-182均可显著上调细胞内CCL2的基因表达(SKOV3细胞：3.6±0.73倍,P<0.001；SKOV3-miR-182细胞：11.39±0.21倍,P<0.001)。
7.Anti-miR-182治疗对小鼠无明显不良组织毒性作用
组织病理学观察发现经8周anti-miR-182治疗后,小鼠的肝脏、胰腺、胃肠道、肾、脾及子宫均无显著的组织学和细胞学变化。
结论
1.建立的裸鼠原位卵巢癌模型可成功模拟人原发性卵巢癌的生长和侵袭模式,适用于卵巢癌的体内研究。
2.Anti-miR-182体内、体外均可通过下调miR-182、恢复其靶分子的表达有效抑制人卵巢癌细胞的增殖、侵袭和远处转移；同时anti-miR-182可诱导肿瘤局部炎症反应抑制肿瘤侵袭转移。
3. Anti-miR-182体内应用无不良组织学毒性作用。
Part I THE EFFECT AND UNDERLYING MECHANISM OF MICRORNA-182ON LEPTIN INDUCED PROLIFERATION OF OVARIAN CANCER CELLS
Background
Ovarian cancer is the fifth leading cause of cancer-related death worldwide and is still the most lethal gynecologic malignancy among women. One of the main reasons for such a high mortality is that the majority of cases are diagnosed at an advanced stage. Therefore, it is critical to elucidate the mechanism of the tumorigenesis. The converging studies indicate that obesity is a risk factor for hormone-dependent cancers including ovarian cancer, and leptin, the product of obesity gene (Ob), is implicated in the tumorigenesis and development of many cancers. Leptin is elevated and is correlated with the poor progress-free survival of patient. But its exact effect and the underlying mechanism in ovarian cancer remains pooly understood.
microRNAs (miRNAs) are small non-coding RNAs that negatively regulate gene expression at post-transcriptional level by blocking of translation and/or degradation of target mRNAs.Tumor-related miRNAs may play a role in tumor cell differentiation, proliferation and apoptosis. Aberrant expression or mutation of miRNAs has been detected in several tumors. Of these miRNAs, miR-182is overexpressed in the high-grade type (HG-SOC) and can promote the invasion and tansformation of both benign and ovarian cancer cell and metastasis in vivo, suggesting the role of miR-182in early tumorigenesis of HG-SOC is of great important and it may be very helpful for the clinical therapy to find a way to manipulate the miR-182level. Some early carcinogenic factors, such as DNA damage and inflammation, may influence the expression of some miRNA. Leptin can regulate miRNAs expression profile in skeletal muscle cells. However, the exact effect and mechanism of leptin on the expression of miRNAs during ovarian cancer remain unknown.
Based on the domestic and overseas progress in this field and our preliminary research data, in this study, we elucidated the effect and underlying mechanism of leptin on proliferation and transformation of ovarian cancer in vitro, explored the role of STAT5signaling pathway induced miR-182expression in this progress. Our results support that leptin and the upregulated-miR-182contribute to the ovarian cancer, which helps us to further know the mechanism of tumorigenesis of this specific cancer and provides molecular foundation for the early detection and therapy of ovarian cancer.
Objective
1. Observation of the effect of leptin on ovarian cancer cells proliferation and transformation.
2. Exploration of miRNAs regulated by leptin in ovarian cancer cells and their effect on ovarian cancer cells proliferation.
3. Elucidation the role of the downstream signaling pathway of leptin receptor in the expression of miR-182and miR-96.
Methods
1. The expression of leptin and leptin receptor in ovarian cancer cell. Ovarian cancer cells (SKOV3and A2780) were cultured for24hours at37℃. RT-PCR was employed to detect the mRNA expression of leptin, the long isoform leptin receptor OB-Rb and the short isoform leptin receptor OB-Ra. Western blot was employed to detect the protein level of leptin and leptin receptor. Ovarian cancer cells (SKOV3and A2780) were cultured for72hours and the supernatant was collected. ELISA was employed to detect the secreted leptin by ovarian cancer cells.
2. The effect of leptin on the proliferation, apoptosis and anchorage-independent growth of ovarian cancer cells.
Ovarian cancer cells (SKOV3and A2780) were treated with leptin (0,50,100,250,500ng/ml), and the proliferation of cells was measured using WST-1reagent for5consecutive days and the growth curve was traced. Ovarian cancer cells were divided into control、leptin、anti-leptin、leptin+anti-leptin groups and cultured in0.3%soft agar for3weeks at37℃. The single cell colonies were stained by crystal violet and counted. Ovarian cancer cells were divided into control、leptin、anti-leptin、 leptin+anti-leptin groups and cultured for48hours at37℃,flow cytometry was employed to detect the apoptosis and the leptin-treated colon carcinoma cell HCT-116was used as the positive control.
3. Detection the expression of the transcriptor FOXO family in ovarian cancer cells with leptin stimulation and observing their role in leptin-induced proliferation of ovarian cancer cells.
Ovarian cancer cells (SKOV3and A2780) were divided into control, leptin, anti-leptin and leptin+anti-leptin groups and cutured for48hours at37℃. Western blot was employed to measure the FOXO1and FOXO3expression at the protein level to screen the changed molecules. Then change of its or their downstream target molecules was measured using Western blot assay.
SiRNA was used to silence the function of the FOXO molecule(s) and RT-PCR and Western blot were used to detect the interference efficacy at mRNA and protein level, respectively. Ovarian cancer cells (SKOV3and A2780) were divided into control, leptin, Leptin+Con-siRNA and Leptin+FOXO-siRNA groups and cutured for48hours at37℃. The proliferation of cells was measured using the WST-1reagent.
4. The effect of leptin on miRNAs that target the3'-UTR of FOXO and the role of mIRNAs in the leptin induced ovarian cancer cell proliferation.
The3'-UTR reagion of human FOXO was amplified by PCR and cloned into the psiCHECK2luciferase vector. The constructed vector and the negative control was tranfected into ovarian cancer cells (SKOV3and A2780). After24hours at37℃, the cells were divided into Vector, Vector+Leptin, FOXO-3'-UTR and FOXO-3'-UTR+Leptin groups and further cultured for48hours at37℃. The relative luciferase activity was measured using the dual-luciferase reporter system.
Ovarian cancer cells were divided into Control and Leptin groups and cultured for48hours. RT-PCR was used to detect the mature miRNAs. The mimics of the deteced miRNAs was transfected into ovarian cancer cells and cultured for48hours. RT-PCR was employed to confirm the transfection efficacy. Western blot was used to detect the protein expression of FOXO. MiRNA inhibitors were used to knock out the expression of miRNAs and RT-PCR was used to confirm the interference efficacy. Ovarian cancer cells were divided into Control, Leptin, Leptin+miRNAs inhibitor groups and cultured for48hours. Western blot was employed to measure the expression of FOXO and WST-1was used to assess the cell proliferation.
5. The effect of Leptin receptor downstream signaling molecules STAT3and STAT5in leptin induced miRNAs expression.
Ovarian cancer cells (SKOV3and A2780) were treated with Leptin for48hours and Western blot was employed to measure the protein level of Leptin long isoform receptor OB-Rb.
Ovarian cancer cells were treated with leptin (100ng/ml) and cells were collected at different time point (0,15,30,60minutes). Western blot was employed to detect the level of the STAT3and STAT5signaling pathway.
Ovarian cancer cells were divide into Control, Leptin, Cucurbitacin I, STAT5-siRNA, Leptin+Cucurbitacin I and Leptin+STAT5-siRNA groups, RT-PCR was employed to measure the level of the target miRNAs.
Results
1. The Leptin receptors are constitutely expressed in ovarian cancer cells.
RT-PCR results indicated that both OB-Ra and OB-Rb were expressed on ovarian cancer cells SKOV3and A2780at mRNA level. Western blot results showed that these two were expressed at protein levels. In addition, Leptin mRNA and protein were detected at varied levels. Moreover, we detected secrete leptin in cultured supernatants of SKOV3(38.39±3.72pg/ml) and A2780(35.40±2.44pg/ml).
2. Leptin promoted the proliferation and anchorage-independent growth ability of ovarian cancer cells.
WST-1assay indicated that leptin enhanced the proliferation of SKOV3and A2780cells in a time-and dose-dependent manner (P<0.05). Leptin (100ng/mL) significantly promoted the anchorage-independent growth ability of A2780and SKOV3cells (SKOV3:2.72±0.34-fold, P<0.05; A2780:1.97±0.15-fold, P<0.05) as reflected by the significant increase in the number and size of colonies formed in soft agar,, and leptin neutralizing mAb attenuated this process. However, the apoptosis of the two cells were not affected by leptin (P>0.05), and the apoptosis of HCT-116(positive control) was inhibited by leptin (P=0.0169).
3. Leptin promoted ovarian cancer cell proliferation by inhibiting expression of FOXO3.
Western blot data indicated that leptin (100ng/mL) suppressed the expression of FOXO3significantly in both SKOV3cells (55.1±1.8%, P<0.01) and A2780cells (48.4±10.1%, P<0.01). However, the expression of FOXO1was not affected by leptin in both cell lines (P>0.05). Further data showed that leptin also inhibited the expression of Bim (45.8%±1.5%for SKOV3, P<0.01;46.7%±3.6%for A2780, P<0.01) and p27(41.4%±6.3%for SKOV3, P<0.01;36.7%±9.0%for A2780, P<0.01), respectively. When the mRNA and protein expression was knocked down by FOXO3-siRNA, the proliferation of both SKOV3(1.63±0.10-fold, P<0.01) and A2780cells (1.59±0.16-fold, P<0.05) was enhanced remarkedly.
4. Leptin promoted ovarian cell proliferation by upregulating miRNAs targeting the3'-UTR of FOX03.
Leptin (100ng/mL) can significantly reduced the normalized luciferase activity of SKOV3cells (53.4±2.5%, P=0.001) and A2780cells (41.6±8.5%, P=0.016) transfected with vector containing the3'-UTR of FOXO3, compared with the mock group. RT-PCR data indicated that leptin (100ng/ml) significantly increased the expression of miR-182(2.26±0.23-fold, P<0.01), miR-106a (1.52±0.16-fold, P=0.03), miR-96(1.91±0.03-fold, P<0.01) and miR-155(1.53±0.10-fold, P=0.01) in SKOV3 cells. Similar trend was observed in A2780cells. No difference was found on the expression of miR-93and miR-153(P>0.05).
Only miR-182(64.8%±11.4%for SKOV3, P<0.05;51.8%±0.5%for A2780, P<0.05) and miR-96(58.3%±2.5%for SKOV3, P<0.05;42.2%±4.1%for A2780, P<0.05) mimics inhibited FOXO3expression both cell lines. MiR-155and miR-106a mimics failed to downregulate FOXO3in two cells (P>0.05). MiR-182and miR-96inhibitors could rescue leptin-mediated downregulation of FOXO3. WST-1assay indicated that miR-182(2.65±0.05-fold SKOV3, P<0.01;2.52±0.06-fold for A2780, P<0.01) and miR-96mimics (2.03±0.08-fold SKOV3, P<0.01;1.85±0.13-fold for A2780, P<0.05) promoted the proliferation of both cell lines, and miR-182(37.1%±3.3%for SKOV3, P<0.01;37.4%±5.0%for A2780, P<0.01) and miR-96(28.9%±6.2%for SKOV3, P<0.01;29.9%±3.5%for A2780, P<0.01) inhibitors prevented the effect of leptin.
5. Leptin increased the expression of miR-182and miR-96through STAT5pathway.
The expression of the OB-Rb was enhanced by leptin (100ng/mL). Leptin induced the phosphorylation of STAT3and STAT5, whose activation reached a peak at60min and30min, respectively. The specific inhibition of STAT5by STAT5-siRNA prevented upregulation of precursor and mature miR-182(43.5%±2.5%for SKOV3, P<0.01;32.0%±9.4%for A2780, P<0.01) as well as miR-96(34.1.8%±0.8%for SKOV3, P<0.01;22.7%±4.2%for A2780, P<0.05) induced by leptin treatment. However, the specific STAT3inhibitor cucurbitacin I did not affect the miR-182and miR-96expression (P>0.05).
Conclusion
1. Leptin promotes the proliferation of ovarian cancer cells by suppressing the expression of FOXO3, but the apoptosis is not affeccted.
2. Leptin promotes the proliferation of ovarian cancer cells by upregulating miR-182and miR-96that targeting FOXO3. promotion of the gastric cancer cell MT1-MMP membrane expression is a KIF1B dependent process, involved in gastric cancer cell invasion process.
3. Elucidation that the role of the leptin receptor downstream signaling pathway molecules STAT5in the leptin induced miR-182and miR-96, which provides new target and experimental data for the early detection and therapy of ovarian cancer.
Significance
In his study, we elucidates the effect and underlying mechanism of leptin on FOXO3, explores the role of this process in the proliferation of ovarian cancer cells, and highlights the function of miR-182and miR-96in the leptin induced proliferation of ovarian cancer cells. These finding will help to know better about the mechanism of tumorigenesis and development of ovarian cancer and provide new targets for the early detection and therapy of ovarian cancer.
Part II THE IN VIVO EFFECT AND UNDERLYING MECHANISM OF ANTI-MIR-182COMPOUND ON GROWTH AND INVASION OF OVARIAN CANCER
Background
Ovarian cancer is the most lethal gynecologic malignancy and high grade serous ovarian carcinoma (HGSOC) is the most aggressive form of ovarian cancer, contributing to most ovarian cancer death due to early invasion and metastasis at the time of diagnosis. Current chemotherapies by reducing tumor burden may not benefit significantly in long term. Anti-microRNA treatment is emerging as a potential modality for cancer therapy. MicroRNAs (miRNAs) are a group of small, non-coding RNAs. Some tumor-related miRNAs may play a critical role in tumor growth, invasion and metastasis. MiR-182is an onco-miRs and its overexpression is associated with aggressive ovarian cancer, largely contributed by its negative regulation of multiple tumor suppressor genes involving in tumor growth, invasion, metastasis and DNA instability.
MiR-182is is overexpressed in HGSOC and is associated with aggressive tumor growth and invasion in HGSOC as well as the poor progression-free survival of patients. Our prelimitary data show that miR-182plays critical role in the proliferation and transformation of ovarian cancer cells mediated by leptin. Therefore, anti-miR-182may provide a promising therapy to reduce tumor burden and metastasis potential in those malignant neoplasms with miR-182overexpression. All in al, based on the domestic and overseas progress in this field and our preliminary research data, we tried for the first time to investigate and characterize the role of anti-miR-182treatment as a potential anti-invasion therapeutic strategy for ovarian cancer. The study selected the ovarian cancer cell line with miR-182overexpression and developed the xenografts by implanting cancer cells into intrabursa.
Gastric cancer (GC) ranks as the secondary leading cause of cancer-related death in the world. Adipocytes provide fatty acids for rapid tumor growth, and the dysfunction of lipid metabolism can lead to the pathogenesis of human GC. Leptin is an adipokine of the obesity (ob) gene, and its overexpression is closely correlated with GC metastasis, but its exact effect and the underlying mechanism in tumor progression remain unclear. Our previous findings reveal that leptin may promote GC cells invasion by upregulation of MT1-MMP. MT1-MMP may cleave intercellular adhesion molecule-1(ICAM-1), and then enhance tumor cells migration. ICAM-1is overexpressed and plays crucial roles in tumor metastasis. This study aimed to characterize the influence of leptin on ICAM-1expression in GC and elucidate its underlying molecular mechanism.
We previously determined that leptin could promote the migration of GC through upregraulation the expression of MT1-MMP,and other dates suggested that MT1-MMP could cut ICAM-1and promote the migration of tumor cells,but the exact mechanism of how leptin act on ICAM-1remains unclear.Overall,based on the worldwide study progress and the results we discovered previously,we collected GC tissue samples and detected the expression of leptin and ICAM-1,then analyzed the correlation between leptin and clinical parameters.And we also observed the influence of leptin on the effects of ICAM-1expression,discussed the function of ICAM-1in the process of GC migration.We finally observed the function of Rho/ROCK in ICAM-1expression regulated by leptin,simultaneously provided potential therapy target for the control of GC in early period. The tumor growth, invasion and metastasis were evaluated during anti-miR-182treatment by IVIS system and histopathology, followed by thorough analysis of miR182expression and its target gene expression. The treatment toxicity was also evaluated. Our study provides a potential therapeutic modality targeting at the aggressive tumor growth of HGSOC.
Objective
1. To produce a mouse model mimic to human ovarian cancer microenvironment.
2. To observe dynamicly the in vivo effect of anti-miR-182on the growth and invasion of ovarian cancer.
3. To evaluate the tumor growth, invasion and metastasis by histopathology.
4. To elucidate the molecular mechanism through which anti-miR-182regulate the growth and invasion of ovarian cancer.
Methods
1. Detection of the effect of anti-miR-182on the oncogenic properties of ovarian cancer.
The human ovarian cancer cells OVCAR3、HEY and SKOV3were used for the in vitro test. HEY and SKOV3with miR-182overexoression was established by the lentiviral system. The anti-miR-182was transfected into the above ovarian cancer cells and cutured for48hours at37℃. RT-PCR was employed to detect the interference efficacy of anti-miR-182on miR-182expression. Western blot was employed to measure the protein level of the miR-182target genes.
The anti-miR-182was transfected into the above ovarian cancer cells and cutured for24hours at37℃. Transwell assay was used to measure the invasion of cells. The proliferation of cells with or without anti-miR-182treatment was detected by WST-1for the next consecutive5days. The soft agar assay was employed to measure the anchorage-independent growth ability of cells.
2. Set up the mouse model mimic to the human primary ovarian cancer.
The luciferase gene was introduced into SKOV3cell with stable miR-182overexpression (SKOV3-miR-182-luc). The luciferase expression was used as tracer molecule for in vivo bioluminescence imaging system (analysis (IVIS) of tumor status.
The SKOV3-miR-182-luc cells (106cells) were embedded into collagen IV matrix, which finally shringe into tumor spherical nodules of about0.2centimeter in diameter. The tumor nodules were implanted into left ovarian intrabursal space in8weeks old mice.
3. Anti-miR-182administration in vivo.
After surgery, mice were randomly divided into two groups of control (20mice) test (20mice). On the third day after surgery, mice from control group were treated with scramble compound and mice from test group were treated with anti-miR-182(dose of25mg-kg-'body weight in0.2mL per injection) by intraperitoneum (I.P.) injection twice weekly. Among19mice from test group,4mice were treated with anti-miR-182for just one week (short term) and the other15mice were treated for eight weeks (long term).
4. Effect of anti-miR-182on the growth of ovarian carcinoma in vivo.
Tumor growth was monitored by IVIS each week for up to8weeks and the tumor size was reflected by the luciferin signal. All mice were euthanized at the end of8weeks. The bilateral ovaries and uterus were isolated and taken out from the pelvic cavity, and tumors in ovaries were measured and photographed. The serum and part of tumor tissue as well as liver was frozen at-80℃. The other organs were dissected and collected, including spleen, kidneys, liver, intestine, pancreas, omentum, stomach, omentum, pelvic lymph nodes, diaphragm lungs and heart. All cases of primary tumor tissue and organs were fixed and processed.
ELISA was used to detect the CA125level in mouse serum. RT-PCR and real-time PCR was employed to measure the mRNA expression of genes that is related with cell cycle or cell death (CDKN1A、CDKN1B、FOXO1、CHEK2).
5. Effect of anti-miR-182on the invasion and metastasis of ovarian carcinoma in vivo.
The mice were scanned weekly by the IVIS system to monitor tumor metastasis. All peritoneal organs as well as lungs and heart were sectioned and stained. The metastasis tumors were assessed and analyzed microscopically.
6. Molecular evaluation of the tumor tissue.
Total RNA was extracted from the serum and tumor tissue. RT and real-time PCR was employed to detect the expression of miR-182, as well as the polycistron cluster of miR-182family including miR-96and miR-183, in mice serum and tumor tissue. Western blot was used to detect the expression of miR-182target genes (BRCA1, MTSS1and FOXO3) and HMGA2in tumor tissue at the protein level.
7. The influence od anti-miR-182treatment on the tumor microenviorment.
The histology of all tumor xenografts and their relationship to ovary, oviducts and paraovarian fat pat. Immunohistochemitry was used to confirm the cell type in the tumor microenviroment. The human inflammatory gene array was used to measure the alteration of inflammatory gene profile. RT and real-time PCR was employed to detect the chemokines and cytokines in the control (n=8) and test (n=8) tumor at the mRNA level.
8. Toxicity analysis of the anti-miR-182treatment.
At the end of the treatment (8-week treatment), all organs (liver, pancreas, gut, kidneys, spleen and uterus) of mice were collected and processed. The histology and cytology of organs was assessed by histologic examination to evaluate the toxicity of the anti-miR-182treatment.
Results
1. Anti-miR-182inhibited the proliferation, invasion and anchorage-independent growth of ovarian cancer cells in vitro.
RT-PCR data reveals that when ovarian cancer cells (OVCAR3, HEY-miR-182and SKOV3-miR-182) were treated with anti-miR-182, the miR-182expression was significantly repressed in all cells (P<0.001). Weatern blot results shows that the target genes expression of miR-182were all upregulated by anti-miR-182(FOX03:1.47±0.18-fold, P<0.05; BRCA1:3.09±0.99-fold, P<0.05; MTSS1:1.39±0.18-fold, P<0.05), while the expression of miR-182induced gene HMGA2was down-regulated (62.6%±17.2%, P<0.01). The similar trend was found in OVCAR3and HEY-miR-182cells.
Transwell invasion assay reveals that anti-miR-182significantly inhibited tumor cell invasion/migration through Matrigel (57.1%±7.9%, P<0.001). Soft agar colony formation data shows that anti-miR-182significantly reduced the number and size of the single-cell colonies (54.8%±16.3%, P<0.01). WST-1assay show that anti-miR-182suppressed the proliferation of ovarian cancer cells in vitro (day3: P<0.05; day4:P<0.05; day5:P<0.01).
2. Intrabursal implantation of SKOV3tumor cells in nude mice.
SKOV3-miR-182-luc cells were embedded into collagen IV pellet and implanted intrabursaly into the nude mice. One day after surgery, IVIS scanning show that all mice dispayed the similar level of bioluminace signal, suggesting similar number cells implanted and no peritoneal leaking.
3. Anti-miR-182treatment reduces tumor growth of ovarian cancer in vivo.
The IVIS imaging shows the tumor size between control ((17.73±7.72) x109) and test groups ((5.20±6.63) x109, P<0.01) began different at week5and reached wider for the rest of weeks. The tumor size in anti-miR-182treatment group (5.42±1.00mm,90.59±37.31mm3) was significantly smaller than that in control (8.07±1.74mm,310.89±187.12mm3)(p<0.001). The treated tumors were32.8%(in diameter) or70.9%(in volume) smaller than that in vehicle control. ELISA data reveals that CA125level was lower in testing group than in control group (P=0.06).
Real-time PCR analysis show that anti-miR-182treatment siginificanly upregulated several cell cycle and cell death regulators that are predicted target genes of miR-182, including CDKN1A (P=0.0199), CDKN1B(P=0.0156), FOXO1(P<0.0001) and CHEK2(P<0.0001).
4. Anti-miR-182treatment inhibits metastasis of ovarian cancer in vivo.
The metastatic tumors detected by IVIS at8week were28%(5/18) control and7%(1/15) in testing groups. Histologic data shows that77.8%(14/18) of mice from control group and33.3%(5/15) of mice from testing group had at least one metastatic disease. A total of31metastases were found in controls, while only6metastases in treated mice. The rate of metastasis between test and control mice were statistically significant (P<0.05). The most common site of metastatic disease were spleen (44%(8in18) in control,20%(3in15) in test), omentum28%(5in18) in control and13%(2in15) in test, pancreas22%(4in18) in control and0%(0in15) in test and liver11%(2in18) in control and0%(0in15) in test. Metastases in pancreas were larger and more aggressive than other metastases. Only1metastasis was noted in control group. Although there was no metastasis into kidney parenchyma, as many as50%(9/18) ovarian tumors in control were adhered to ipsilateral kidney, but not seen in anti-miR-182treated mice (0/17). No lung metastasis was found in either control or testing groups.
5. Molecular analysis of target gene alterations in xenografts of tumors when treated by Anti-miR-182.
Real-time RT-PCR analysis revealed that miR-182expression was either in very low or non-detectable levels all tumors with anti-miR-182treatment in comparison to control (reduced94.9%±13.3%, P<0.001). Anti-miR-182could also significantly reduce miR-183expression (92.5%±9.7%, P<0.001), but not miR-96(P>0.05). Reduction of miR-182(53.2%±10.4%, P<0.01) and miR-183(84.3%±20.6%, P<0.01) was also found in mouse serum samples.
Western blot and qRT-PCR analyses reveals that the miR-182target genes were partially or completely restored by anti-miR-182treatment (BRCA1:2.63±0.35-fold, P<0.01; FOXO3:1.42±0.18-fold, P<0.01; MTSS1:2.63±1.29, P<0.01). While the expression of HMGA2was downregulated (71.5%±4.0%, P<0.01).
6. Anti-miR-182treatment induces inflammation which prevent tumor invasion.
About64%(11/17) of implanted site (paraovarian soft tissue) had extensive monocytic aggregate in mice with anti-miR-182treatment, while only17%(3/17) of control mice showed similar but much less monocytic aggregate. Immunostain for histolytic marker KP1confirmed the mononuclear histiocytes origin. Tumor implants with minimal monocytic infiltrates had clear infiltrating growth pattern and penetrating through intrabursa into surround fat, while those with heavy monocytic infiltrates could barely pass through intrabursal capsule.
The inflammatory gene array reveals that the inflammatory gene profile differed between control and testing tumors. Real-time PCR analysis shows that compared with the control tumors (n=8), the expression of CCL2(P<0.0001), IL-6(P=0.0019), IL-8(P=0.0002), TNF-a (P=0.0088) and IFN-y (P<0.0001) was siginificantly upregulated in the anti-miR-182treatment tumors (n=8). In SKOV3cell line with either low or high endogenous miR-182expression, administration of anti-miR-182could significantly increase CCL2expression (SKOV3:3.60±0.73-fold, P<0.001; SKOV3-miR-182:11.39±0.21-fold, P<0.001).
7. Anti-miR-182treatment has no genotoxic effects on mouse.
By histologic examination of organ system, we did not see significant histology and cytology changes after8weeks of treatment in liver, pancreas, gut, kidneys, spleen and uterus.
Conclusion
1. We produced successfully a mousemodel that can mimic well to the growth and invasion pattern of human primary ovarian cancer, which will provide good mouse model for future in vivo research on ovarian cancer.
2. Anti-miR-182treatment can reduce tumor growth, invasion and metastasis of ovarian cancer both in vitro and in vivo.
3. Anti-miR-182treatment can inhibit invasion of ovarian cancer by improving the tumor microenviroment.
All of those above provide the experimental support as well as new target and method for the early control of human ovarian cancer.
Significance
In this study produces a mouse model that can mimic to the development pattern of human ovarian cancer successfully. Our data illuminates the effect and underlying mechanism of anti-miR-182treatment on the tumor burden and invasion of ovarian cancer in vivo, and further confirms the molecular mechanism of the tumoregenesis of ovarian cancer. These findings provides new target for the diagnosis and therapy of primary and aggressive ovarian cancer, which will help to improve the current therapy model.
引文
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3. McMillen BD, Aponte MM, Liu Z, et al. Expression analysis of MIR182 and its associated target genes in advanced ovarian carcinoma. Mod Pathol,2012, 25(12):1644-1653.
4. Bendoraite A, Knouf EC, Garg KS, et al. Regulation of miR-200 family microRNAs and ZEB transcription factors in ovarian cancer:evidence supporting a mesothelial-to-epithelial transition. Gynecol Oncol,2010, 116(1):117-125.
5. Park SM, Gaur AB, Lengyel E, et al. The miR-200 family determines the epithelial phenotype of cancer cells by targeting the E-cadherin repressors ZEB1 and ZEB2. Genes Dev,2008,22(7):894-907.
6. Kalluri R Weinberg RA. The basics of epithelial-mesenchymal transition. J Clin Invest,2009,119(6):1420-1428.
7. Elloul S, Elstrand MB, Nesland JM, et al. Snail, Slug, and Smad-interacting protein 1 as novel parameters of disease aggressiveness in metastatic ovarian and breast carcinoma. Cancer,2005,103(8):1631-1643.
8. Symowicz J, Adley BP, Gleason KJ, et al. Engagement of collagen-binding integrins promotes matrix metalloproteinase-9-dependent E-cadherin ectodomain shedding in ovarian carcinoma cells. Cancer Res,2007, 67(5):2030-2039.
9. Moss NM, Barbolina MV, Liu Y, et al. Ovarian cancer cell detachment and multicellular aggregate formation are regulated by membrane type 1 matrix metalloproteinase:a potential role in I.p. metastatic dissemination. Cancer Res, 2009,69(17):7121-7129.
10. Davidson B, Goldberg I, Berner A, et al. Expression of membrane-type 1,2, and 3 matrix metalloproteinases messenger RNA in ovarian carcinoma cells in serous effusions. Am J Clin Pathol,2001,115(4):517-524.
11. Kenny HA Lengyel E. MMP-2 functions as an early response protein in ovarian cancer metastasis. Cell Cycle,2009,8(5):683-688.
12. Wu J, Liu Z, Shao C, et al. HMGA2 overexpression-induced ovarian surface epithelial transformation is mediated through regulation of EMT genes. Cancer Res,2011,71(2):349-359.
13. Rasheed SA, Teo CR, Beillard EJ, et al. MicroRNA-182 and MicroRNA-200a Control G-protein Subunit alpha-13 (GNA13) Expression and Cell Invasion Synergistically in Prostate Cancer Cells. J Biol Chem,2013, 288(11):7986-7995.
14. Teng Y, Mei Y, Hawthorn L, et al. WASF3 regulates miR-200 inactivation by ZEB1 through suppression of KISS1 leading to increased invasiveness in breast cancer cells. Oncogene,2013.
15. Zhang H, Pu J, Qi T, et al. MicroRNA-145 inhibits the growth, invasion, metastasis and angiogenesis of neuroblastoma cells through targeting hypoxia-inducible factor 2 alpha. Oncogene,2012.
16. Huynh C, Segura MF, Gaziel-Sovran A, et al. Efficient in vivo microRNA targeting of liver metastasis. Oncogene,2011,30(12):1481-1488.
17. Segura MF, Hanniford D, Menendez S, et al. Aberrant miR-182 expression promotes melanoma metastasis by repressing FOXO3 and microphthalmia-associated transcription factor. Proc Natl Acad Sci U S A, 2009,106(6):1814-1819.
18. Lei R, Tang J, Zhuang X, et al. Suppression of MIM by microRNA-182 activates RhoA and promotes breast cancer metastasis. Oncogene,2013.
19. Hirata H, Ueno K, Shahryari V, et al. MicroRNA-182-5p promotes cell invasion and proliferation by down regulating FOXF2, RECK and MTSS1 genes in human prostate cancer. PLoS One,2013,8(1):e55502.
20. Wu J Wei JJ. HMGA2 and high-grade serous ovarian carcinoma. J Mol Med (Berl),2013,91(10):1155-1165.
21. Xu X, Dong Z, Li Y, et al. The upregulation of signal transducer and activator of transcription 5-dependent microRNA-182 and microRNA-96 promotes ovarian cancer cell proliferation by targeting forkhead box 03 upon leptin stimulation. Int J Biochem Cell Biol,2013,45(3):536-545.
22. Lei H, Hemminki K, Altieri A, et al. Promoter polymorphisms in matrix metalloproteinases and their inhibitors:few associations with breast cancer susceptibility and progression. Breast Cancer Res Treat,2007,103(1):61-69.
23. Galimi F, Torti D, Sassi F, et al. Genetic and expression analysis of MET, MACC1, and HGF in metastatic colorectal cancer:response to met inhibition in patient xenografts and pathologic correlations. Clin Cancer Res,2011, 17(10):3146-3156.
24. Krishnan K, Steptoe AL, Martin HC, et al. MicroRNA-182-5p targets a network of genes involved in DNA repair. Rna,2013,19(2):230-242.
25. Chiang CH, Hou MF Hung WC. Up-regulation of miR-182 by beta-catenin in breast cancer increases tumorigenicity and invasiveness by targeting the matrix metalloproteinase inhibitor RECK. Biochim Biophys Acta,2013, 1830(4):3067-3076.
26. Zhang Y, Yang P, Sun T, et al. miR-126 and miR-126* repress recruitment of mesenchymal stem cells and inflammatory monocytes to inhibit breast cancer metastasis. Nat Cell Biol,2013,15(3):284-294.
27. Ma L, Reinhardt F, Pan E, et al. Therapeutic silencing of miR-lOb inhibits metastasis in a mouse mammary tumor model. Nat Biotechnol,2010, 28(4):341-347.
28. Fong MY Kakar SS. Ovarian cancer mouse models:a summary of current models and their limitations. J Ovarian Res,2009,2(1):12.
29. Heskamp S, Laverman P, Rosik D, et al. Imaging of human epidermal growth factor receptor type 2 expression with 18F-labeled affibody molecule ZHER2:2395 in a mouse model for ovarian cancer. J Nucl Med,2012, 53(1):146-153.
30. Zacchetti A, Martin F, Luison E, et al. Antitumor effects of a human dimeric antibody fragment 131I-AFRA-DFM5.3 in a mouse model for ovarian cancer. JNucl Med,2011,52(12):1938-1946.
31. Cordero AB, Kwon Y, Hua X, et al. In vivo imaging and therapeutic treatments in an orthotopic mouse model of ovarian cancer. J Vis Exp,2010(42).
32. Liu H, Du L, Wen Z, et al. Up-regulation of miR-182 expression in colorectal cancer tissues and its prognostic value. Int J Colorectal Dis,2013.
33. Gu C, Li X, Tan Q, et al. MiR-183 family regulates chloride intracellular channel 5 expression in inner ear hair cells. Toxicol In Vitro,2013, 27(1):486-491.
34. Hirata H, Ueno K, Shahryari V, et al. Oncogenic miRNA-182-5p targets Smad4 and RECK in human bladder cancer. PLoS One,2012,7(11):e51056.
35. Song L, Liu L, Wu Z, et al. TGF-beta induces miR-182 to sustain NF-kappaB activation in glioma subsets. J Clin Invest,2012,122(10):3563-3578.
36. Gong AY, Zhou R, Hu G, et al. MicroRNA-513 regulates B7-H1 translation and is involved in IFN-gamma-induced B7-H1 expression in cholangiocytes. J Immunol,2009,182(3):1325-1333.
37. Stittrich AB, Haftmann C, Sgouroudis E, et al. The microRNA miR-182 is induced by IL-2 and promotes clonal expansion of activated helper T lymphocytes. Nat Immunol,2010, 11(11):1057-1062.
38. Nieman KM, Kenny HA, Penicka CV, et al. Adipocytes promote ovarian cancer metastasis and provide energy for rapid tumor growth. Nat Med,2011, 17(11):1498-1503.
39. Dean RA, Cox JH, Bellac CL, et al. Macrophage-specific metalloelastase (MMP-12) truncates and inactivates ELR+CXC chemokines and generates CCL2,-7,-8, and-13 antagonists:potential role of the macrophage in terminating polymorphonuclear leukocyte influx. Blood,2008, 112(8):3455-3464.
40. Rollins BJ. Monocyte chemoattractant protein 1:a potential regulator of monocyte recruitment in inflammatory disease. Mol Med Today,1996, 2(5):198-204.
41. Kudo-Saito C, Shirako H, Ohike M, et al. CCL2 is critical for immunosuppression to promote cancer metastasis. Clin Exp Metastasis,2013, 30(4):393-405.
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1. Lengyel E. Ovarian cancer development and metastasis. Am J Pathol,2010, 177(3):1053-1064.
2. Liu Z, Liu J, Segura MF, et al. MiR-182 overexpression in tumourigenesis of high-grade serous ovarian carcinoma. J Pathol,2012,228(2):204-215.
3. McMillen BD, Aponte MM, Liu Z, et al. Expression analysis of MIR182 and its associated target genes in advanced ovarian carcinoma. Mod Pathol,2012, 25(12):1644-1653.
4. Bendoraite A, Knouf EC, Garg KS, et al. Regulation of miR-200 family microRNAs and ZEB transcription factors in ovarian cancer:evidence supporting a mesothelial-to-epithelial transition. Gynecol Oncol,2010, 116(1):117-125.
5. Park SM, Gaur AB, Lengyel E, et al. The miR-200 family determines the epithelial phenotype of cancer cells by targeting the E-cadherin repressors ZEB1 and ZEB2. Genes Dev,2008,22(7):894-907.
6. Kalluri R Weinberg RA. The basics of epithelial-mesenchymal transition. J Clin Invest,2009,119(6):1420-1428.
7. Elloul S, Elstrand MB, Nesland JM, et al. Snail, Slug, and Smad-interacting protein 1 as novel parameters of disease aggressiveness in metastatic ovarian and breast carcinoma. Cancer,2005,103(8):1631-1643.
8. Symowicz J, Adley BP, Gleason KJ, et al. Engagement of collagen-binding integrins promotes matrix metalloproteinase-9-dependent E-cadherin ectodomain shedding in ovarian carcinoma cells. Cancer Res,2007, 67(5):2030-2039.
9. Moss NM, Barbolina MV, Liu Y, et al. Ovarian cancer cell detachment and multicellular aggregate formation are regulated by membrane type 1 matrix metalloproteinase:a potential role in I.p. metastatic dissemination. Cancer Res, 2009,69(17):7121-7129.
10. Davidson B, Goldberg I, Berner A, et al. Expression of membrane-type 1,2, and 3 matrix metalloproteinases messenger RNA in ovarian carcinoma cells in serous effusions. Am J Clin Pathol,2001,115(4):517-524.
11. Kenny HA Lengyel E. MMP-2 functions as an early response protein in ovarian cancer metastasis. Cell Cycle,2009,8(5):683-688.
12. Wu J, Liu Z, Shao C, et al. HMGA2 overexpression-induced ovarian surface epithelial transformation is mediated through regulation of EMT genes. Cancer Res,2011,71(2):349-359.
13. Rasheed SA, Teo CR, Beillard EJ, et al. MicroRNA-182 and MicroRNA-200a Control G-protein Subunit alpha-13 (GNA13) Expression and Cell Invasion Synergistically in Prostate Cancer Cells. J Biol Chem,2013, 288(11):7986-7995.
14. Teng Y, Mei Y, Hawthorn L, et al. WASF3 regulates miR-200 inactivation by ZEB1 through suppression of KISS1 leading to increased invasiveness in breast cancer cells. Oncogene,2013.
15. Zhang H, Pu J, Qi T, et al. MicroRNA-145 inhibits the growth, invasion, metastasis and angiogenesis of neuroblastoma cells through targeting hypoxia-inducible factor 2 alpha. Oncogene,2012.
16. Huynh C, Segura MF, Gaziel-Sovran A, et al. Efficient in vivo microRNA targeting of liver metastasis. Oncogene,2011,30(12):1481-1488.
17. Segura MF, Hanniford D, Menendez S, et al. Aberrant miR-182 expression promotes melanoma metastasis by repressing FOXO3 and microphthalmia-associated transcription factor. Proc Natl Acad Sci U S A, 2009,106(6):1814-1819.
18. Lei R, Tang J, Zhuang X, et al. Suppression of MIM by microRNA-182 activates RhoA and promotes breast cancer metastasis. Oncogene,2013.
19. Hirata H, Ueno K, Shahryari V, et al. MicroRNA-182-5p promotes cell invasion and proliferation by down regulating FOXF2, RECK and MTSS1 genes in human prostate cancer. PLoS One,2013,8(1):e55502.
20. Wu J Wei JJ. HMGA2 and high-grade serous ovarian carcinoma. J Mol Med (Berl),2013,91(10):1155-1165.
21. Xu X, Dong Z, Li Y, et al. The upregulation of signal transducer and activator of transcription 5-dependent microRNA-182 and microRNA-96 promotes ovarian cancer cell proliferation by targeting forkhead box 03 upon leptin stimulation. Int J Biochem Cell Biol,2013,45(3):536-545.
22. Lei H, Hemminki K, Altieri A, et al. Promoter polymorphisms in matrix metalloproteinases and their inhibitors:few associations with breast cancer susceptibility and progression. Breast Cancer Res Treat,2007,103(1):61-69.
23. Galimi F, Torti D, Sassi F, et al. Genetic and expression analysis of MET, MACC1, and HGF in metastatic colorectal cancer:response to met inhibition in patient xenografts and pathologic correlations. Clin Cancer Res,2011, 17(10):3146-3156.
24. Krishnan K, Steptoe AL, Martin HC, et al. MicroRNA-182-5p targets a network of genes involved in DNA repair. Rna,2013,19(2):230-242.
25. Chiang CH, Hou MF Hung WC. Up-regulation of miR-182 by beta-catenin in breast cancer increases tumorigenicity and invasiveness by targeting the matrix metalloproteinase inhibitor RECK. Biochim Biophys Acta,2013, 1830(4):3067-3076.
26. Zhang Y, Yang P, Sun T, et al. miR-126 and miR-126* repress recruitment of mesenchymal stem cells and inflammatory monocytes to inhibit breast cancer metastasis. Nat Cell Biol,2013,15(3):284-294.
27. Ma L, Reinhardt F, Pan E, et al. Therapeutic silencing of miR-lOb inhibits metastasis in a mouse mammary tumor model. Nat Biotechnol,2010, 28(4):341-347.
28. Fong MY Kakar SS. Ovarian cancer mouse models:a summary of current models and their limitations. J Ovarian Res,2009,2(1):12.
29. Heskamp S, Laverman P, Rosik D, et al. Imaging of human epidermal growth factor receptor type 2 expression with 18F-labeled affibody molecule ZHER2:2395 in a mouse model for ovarian cancer. J Nucl Med,2012, 53(1):146-153.
30. Zacchetti A, Martin F, Luison E, et al. Antitumor effects of a human dimeric antibody fragment 131I-AFRA-DFM5.3 in a mouse model for ovarian cancer. JNucl Med,2011,52(12):1938-1946.
31. Cordero AB, Kwon Y, Hua X, et al. In vivo imaging and therapeutic treatments in an orthotopic mouse model of ovarian cancer. J Vis Exp,2010(42).
32. Liu H, Du L, Wen Z, et al. Up-regulation of miR-182 expression in colorectal cancer tissues and its prognostic value. Int J Colorectal Dis,2013.
33. Gu C, Li X, Tan Q, et al. MiR-183 family regulates chloride intracellular channel 5 expression in inner ear hair cells. Toxicol In Vitro,2013, 27(1):486-491.
34. Hirata H, Ueno K, Shahryari V, et al. Oncogenic miRNA-182-5p targets Smad4 and RECK in human bladder cancer. PLoS One,2012,7(11):e51056.
35. Song L, Liu L, Wu Z, et al. TGF-beta induces miR-182 to sustain NF-kappaB activation in glioma subsets. J Clin Invest,2012,122(10):3563-3578.
36. Gong AY, Zhou R, Hu G, et al. MicroRNA-513 regulates B7-H1 translation and is involved in IFN-gamma-induced B7-H1 expression in cholangiocytes. J Immunol,2009,182(3):1325-1333.
37. Stittrich AB, Haftmann C, Sgouroudis E, et al. The microRNA miR-182 is induced by IL-2 and promotes clonal expansion of activated helper T lymphocytes. Nat Immunol,2010, 11(11):1057-1062.
38. Nieman KM, Kenny HA, Penicka CV, et al. Adipocytes promote ovarian cancer metastasis and provide energy for rapid tumor growth. Nat Med,2011, 17(11):1498-1503.
39. Dean RA, Cox JH, Bellac CL, et al. Macrophage-specific metalloelastase (MMP-12) truncates and inactivates ELR+CXC chemokines and generates CCL2,-7,-8, and-13 antagonists:potential role of the macrophage in terminating polymorphonuclear leukocyte influx. Blood,2008, 112(8):3455-3464.
40. Rollins BJ. Monocyte chemoattractant protein 1:a potential regulator of monocyte recruitment in inflammatory disease. Mol Med Today,1996, 2(5):198-204.
41. Kudo-Saito C, Shirako H, Ohike M, et al. CCL2 is critical for immunosuppression to promote cancer metastasis. Clin Exp Metastasis,2013, 30(4):393-405.
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