Raf激酶抑制蛋白在胃癌发生发展中的作用及其机制研究
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摘要
研究背景：胃癌(gastric cancer, GC)是目前世界上发病率第四位的恶性肿瘤,其相关死亡率居所有恶性肿瘤的第二位,消化道恶性肿瘤的首位。由于其发病隐匿,临床症状在早期不明显,多数病人—经发现已处于临床晚期,总体5年生存率仅30%左右,严重威胁着人民的生命和健康。而迄今为止,胃癌的发病机制尚未完全明确,不能满足临床对胃癌进行有效预防及治疗的需要。因此,探索胃癌发病机制、寻找胃癌新的肿瘤标志物及基因治疗靶点用于胃癌的早期诊断、复发转移预测和预后评估对于胃癌防治具有重要作用。近年来,随着胃癌分子生物学研究的不断深入,针对肿瘤细胞生长、凋亡、细胞周期、侵袭浸润等分子生物靶点的寻找成为胃癌基础研究的重点和热点。
Raf激酶抑制蛋白(Raf kinase inhibitor protein,RKIP)属于磷脂酰乙醇胺结合蛋白(phosphatidylethanolamine-binding protein, PEBP)家族,是一种高度保守、广泛表达、具有多种生理与病理功能的小分子胞浆蛋白。参与多条信号转导通路的调控,如Raf-1-MAPK、NF-κB和G蛋白信号通路等,在细胞生长、增殖、分化、凋亡等生理过程中发挥重要作用。此外,近年来的研究表明RKIP是一个新的转移抑制基因,可以抑制前列腺癌、人乳腺癌和黑色素瘤等肿瘤细胞的转移,体外干预实验证实RKIP表达减少可以影响肿瘤转移、血管生成,抵抗凋亡,破坏染色体的完整性,在肿瘤的浸润与转移中起到重要的作用,可以作为判断某些肿瘤预后的分子标志物和肿瘤治疗的新靶标。本实验组张志强等的研究也显示RKIP在胃癌组织中表达下调或缺失,其表达改变可能与胃癌的发生、分化、浸润及转移有关[7]。但RKIP在胃癌发生、发展、浸润、转移中的具体作用及其机制如何,目前国内外研究报道较少。本研究是在前期研究的基础上,分析RKIP从癌前状态到最终癌变、转移这一系列胃粘膜癌变动态演变过程中的表达改变,寻找RKIP蛋白表达异常与胃粘膜癌变之间的关系。为明确RKIP基因在胃癌中的作用并考察其表达上调对胃癌恶性表型的影响,我们借助基因转染技术,构建RKIP(?)急定表达的胃癌细胞株SGC7901,研究RKIP基因在胃癌细胞增殖、生长、迁移、侵袭及凋亡中的作用,阐明RKIP在胃癌发生、分化及转移中扮演的角色。为进一步研究RKIP抑制胃癌的作用机制,我们分析了Cyclin D1、STAT3、ADAM-12这些与细胞增殖、凋亡、信号转导关系密切的基因及蛋白的表达与RKIP表达在胃粘膜癌变动态过程中的相互关系,探寻RKIP蛋白抑制胃癌发展、转移及肿瘤分化可能的作用机制,为胃癌的诊断、预后监测以及基因治疗提供依据和新的线索。
第一章胃粘膜癌变不同阶段组织中Raf激酶抑制蛋白的表达及其临床意义
目的：检测癌前状态、早期胃癌组织、进展期胃癌组织及转移淋巴结组织中RKIP蛋白的表达,并结合患者的临床病理资料进行统计学分析,探讨RKIP蛋白在癌前疾病、癌前病变到胃粘膜癌变不同阶段的表达变化,明确RKIP蛋白表达异常与胃粘膜癌变之间的关系,探讨RKIP在胃粘膜癌变中的可能作用。
方法：免疫组化检测癌前状态组织88例(包括胃腺瘤性息肉7例、胃溃疡27例、异型增生组织及肠化组织54例,其中轻中度异型增生17例,重度异型增生23例,伴肠化生26例)、早期胃癌组织48例、进展期胃癌组织121例及36例胃癌转移的淋巴组织中RKIP蛋白的表达,分析RKIP在胃粘膜癌变不同分化阶段的表达与临床病理学特征的关系。
结果：RKIP蛋白主要在胞浆表达,呈黄色到棕褐色颗粒,RKIP阳性表达如下：胃癌前状态88例,阳性表达62例(70.5%)；早期胃癌组织48例,阳性表达32例(66.7%)；进展期胃癌组织121例,阳性表达36例(29.8%)；36例胃癌转移的淋巴组织,阳性表达1例(2.8%)。RKIP阳性表达率在癌前状态组及早期胃癌组高表达,但两组比较无显著统计学差异(P>0.05),RKIP在不同程度的癌前病变(肠化及异型增生)中的阳性表达也无显著差异(P>0.05)。而在进展期胃癌组织中其表达水平较非癌良性癌前状态及早期胃癌组显著降低(P<0.05),在转移的淋巴结组织中其表达水平进一步降低,与癌前状态及早期、进展期胃癌组比较,均具有显著统计学差异(P<0.05)。免疫组化图片使用图像分析软件Image-Pro Plus6.0扫描,结果取累积光密度值(IOD)做统计分析,采用非参数秩和检验,统计分析显示RKIP表达水平在癌前状态组与早癌组织中没有显著差异,P=0.151；而在进展期胃癌及转移淋巴结组织中表达水平较癌前状态组显著下降,P<0.001；其在转移淋巴结组织阳性表达与进展期胃癌比较亦有显著下降,P<0.001。与免疫组化结果一致。
RKIP在不同类型的胃癌组织中的表达如下：RKIP蛋白在浸润至粘膜及粘膜下层、肌层、全层的胃癌组织中的表达率分别为68.8%、35.3%、20.8%；在高、中、低分化腺癌中表达率分别为：42.9%、41.9%、39.5%；在肠型和弥漫型胃癌中的表达率分别为：41.6%、38.2%；在Ⅰ期、Ⅱ期、Ⅲ/Ⅳ期胃癌中的表达率分别为：68.3%、47.8%、24.5%；在有淋巴结转移和无转移的胃癌组织中的表达分别为：22.7%、70.8%。RKIP蛋白表达与胃癌的浸润深度、TNM分期及淋巴结转移呈负相关(P<0.001),但RKIP蛋白表达与患者年龄、性别无关,RKIP在高、中、低分化腺癌中的表达以及在肠型及弥漫型胃癌中的表达水平无显著差异(P>0.05)。此外169例胃癌病理报告中,其中52例有Hp检测报告,32例Hp阳性,20例Hp阴性,合并Hp感染的胃癌中RKIP阳性表达率为43.8%,Hp阴性胃癌的RKIP阳性表达率为40.0%,统计分析显示RKIP在胃癌中的表达与Hp感染无明显相关(P>0.05)。结论：1.RKIP在癌前状态与早期胃癌组织高表达,在进展期胃癌及转移淋
巴组织中低表达。2.RKIP在肠型胃癌和弥漫型胃癌中表达无显著差异,与胃癌分化程
度及幽门螺杆菌感染亦无明显相关。3.RKIP表达水平与胃癌的浸润深度、TNM分期及淋巴结转移呈负相
关。可作为判断胃癌预后的重要指标。
第二章上调RKIP基因表达对胃癌细胞生物学行为的影响
目的：RKIP表达质粒稳定转染RKIP低表达胃癌细胞株SGC7901,体内外实验明确RKIP对胃癌细胞增殖、侵袭、凋亡、细胞周期及裸鼠成瘤的影响。
方法：采用脂质体介导转染技术,将RKIP真核表达重组质粒pcDNA3.1(+)/RKIP和空载体质粒分别导入RKIP低表达的胃癌细胞系SGC7901。q-RT-PCR和Western blot分别检测RKIP mRNA及蛋白质表达,并借助细胞生长曲线、软琼脂集落形成实验观察RKIP基因对胃癌细胞增殖的作用；划痕试验及Transwell实验检测胃癌细胞迁移能力和侵袭力的变化；流式细胞计数观察RKIP表达对胃癌细胞的细胞周期和凋亡率的影响；并建立人胃癌细胞裸鼠皮下移植瘤模型,观察RKIP对胃癌细胞增殖和转移能力的影响。
结果：转染RKIP基因的SGC7901细胞RKIP mRNA表达较未转染组增加4.41±0.56倍,Western blot分析显示RKIP蛋白质表达量显著增加,表明RKIP基因稳定表达的胃癌细胞系SGC7901构建成功。RKIP基因转染的SGC7901细胞生长速度明显减慢,从第4天起与空载体转染组及未转染组SGC7901细胞相比存在明显差异(P<0.001)。在软琼脂中集落形成RKIP基因转染组、空载体组和未经转染组集落形成数(×102)分别20±4,57±7和59±9,转染RKIP基因组克隆形成能力明显下降(P<0.05)。细胞划痕后18h.RKIP基因转染组细胞划痕宽度2.91±0.21mm,而空白及空载体对照组分别为1.26±0.41mm,1.31±0.34mm,RKIP基因转染组划痕恢复慢,与其他两组对比均有显著差异(P<0.05),转染RKIP基因使肿瘤细胞迁移能力下降。Transwell实验显示空白对照组及空载体组穿过基质膜细胞数分别为(256.5±37)个和(276.5±40)个,转染RKIP基因组则为(107.0±20)个,转染组穿过基质膜细胞数与其他两组对比显著减少(P<0.05),转染组侵袭能力明显受抑制。流式细胞计数发现,RKIP转染组G0-G1的细胞百分比(69.34±1.37%)较空载体转染组(63.42±0.49%)和未转染组(62.45±0.91%)明显增多,而RKIP转染组G2-M,S的细胞百分比(G2-M：8.21±1.64%,S：19.78±1.14%)较空载体转染组(G2-M：11.91±0.23%,S：24.67±0.27%)和未转染组(G2-M：13.71±2.36%,S：23.85±2.41%)明显减少(P<0.05),转染RKIP组细胞凋亡率较未转染对照组及转染空载体组明显增加(3.59±0.51%vs1.07±0.29%,2.10±0.87%),差异有统计学意义(P=0.006)。裸鼠接种实验显示,空载体转染组和未经转染的SGC7901裸鼠组在接种后平均7天就可以长出明显的包块,而RKIP基因转染组长出肿瘤的时间显著延长,平均为21天,同时肿瘤瘤体生长缓慢。将瘤体积变化对时间绘制瘤体生长曲线,pcDNA3.1(+)-RKIP/SGC7901接种的移植瘤体积较空白组及空载体组明显减小,在第7,14,21,28,35,42天对三组移植瘤体积的均数进行统计学分析,结果表明具有显著性差异(P<0.05)。6周后处死裸鼠,取出瘤块,称取瘤质量。pcDNA3.1(+)-RKIP转染组肿瘤体积明显小于空载体转染组和未经转染组,差异有统计意义(7.36±2.10g vs13.63v1.61g,14.03±3.51g,F=6.503,P=0.03),说明RKIP重表达可以明显抑制SGC7901细胞在裸鼠中的移植瘤生长能力。
结论：
1)SGC7901胃癌细胞系RKIP mRNA和蛋白低表达,提示RKIP的低表达可能与胃癌有关。
2)RKIP能抑制SGC7901胃癌细胞的体外增殖能力和侵袭力。
3)成功建立转染组和对照组SGC7901胃癌细胞裸鼠皮下移植瘤模型,RKIP基因转染可以抑制裸鼠移植瘤的生长,降低SGC7901胃癌细胞的致瘤性。
4)RKIP对SGC7901胃癌细胞生长的抑制作用与其干预胃癌细胞周期、促进细胞凋亡有关。
5)RKIP基因重表达有助于SGC7901的恶性表型逆转,有望成为肿瘤治疗的有效靶点。
第三章Raf激酶抑制蛋白抑制胃癌侵袭转移的作用机制的初步研究
目的：探讨胃癌细胞及良性癌前状态、胃癌、转移淋巴结组织中的RKIP表达与Cyclin D1、STAT3、ADAM-12表达的关系,并探讨它们表达与胃癌临床病理的关系,为探索RKIP抑制胃癌侵袭转移的作用机制提供新的实验依据。
方法：q-RT-PCR检测稳定转染RKIP基因的胃癌细胞SGC7901中的Cyclin D1、STAT3、ADAM-12mRNA表达变化,在体外从细胞水平探讨RKIP基因抑制胃癌的作用机制是否与调控Cyclin D1、 STAT3及ADAM-12的表达有关。再采用免疫组化检测癌前状态组织88例；早期胃癌组织30例、进展期胃癌组织60例及25例胃癌转移的淋巴组织中Cyclin D1、STAT3、ADAM-12蛋白表达及其与胃癌的临床病理特征的关系,分析RKIP表达与Cyclin D1、STAT3、 ADAM-12表达在胃粘膜癌变过程中的相互关系。
结果：(1)RKIP基因转染组的CyclinD1、STAT3、ADAM12mRNA表达量分别为未转染组的0.405±0.073倍、0.578±0.274倍、0.673±0.447,转染载体组中三个基因mRNA表达量分别为未转染组的1.038±0.543倍、1.772±1.078倍、1.003±0.434倍。RKIP基因转染组胃癌细胞中的cyclinD1、STAT3基因表达显著下调(P<0.05),而RKIP基因转染后ADAM12基因表达与其他两组比较无明显差异(P>0.05)。(2)STAT3蛋白在胃癌前状态组、早期胃癌、进展期胃癌组织、转移淋巴组织的阳性表达率分别为例26.1%(23/88)、66.7%(20/30)、71.6%(43/60)、64.0%(16/25)。与胃癌前状态组比较,STAT3蛋白在早期胃癌、进展期胃癌及转移淋巴结组织其表达阳性率均显著升高,差异具有统计意义(P<0.001)。STAT3蛋白表达与胃癌的浸润深度、TNM分期、淋巴结转移及肿瘤分化程度呈正相关(P<0.05),但在肠型胃癌和弥漫型胃癌中表达无显著差异(P>0.05)。(3)CyclinDl蛋白在胃癌前状态组、早期胃癌、进展期胃癌组织、转移淋巴组织的阳性表达率分别为23.9%(21/88)、63.3%(19/30)、73.3%(44/60)、52.0%(13/25)。与胃癌前状态疾病比较,早期胃癌、进展期胃癌以及转移淋巴结组织中Cyclin D1蛋白阳性表达率均显著升高,差异具有统计意义(P<0.05)。CyclinD1蛋白表达与胃癌的分化程度及淋巴结转移呈正相关(P<0.05),并与胃癌分型有关,CyclinD1在弥漫型胃癌中阳性表达率较肠型胃癌高(P=0.001),与胃癌的浸润深度、TNM分期无明显相关(P>0.05)。(4)ADAM-12蛋白在癌前状态、早期胃癌、进展期胃癌、转移淋巴组织的阳性表达率分别为44.3%(39/88)、53.3%(16/30)、55.0%(33/60)、36%(9/25)。ADAM-12蛋白在癌前状态、早期、进展期胃癌以及转移淋巴结组织中的表达各组间均无显著差异(P>0.05)。(5)Cyclin D1、STAT3、ADAM-12累积光密度值(IOD)定量分析结果与免疫组化结果一致。(6)相关性分析表明RKIP蛋白与CyclinD1蛋白表达两者呈负相关(r=-0.411,P<0.001)；RKIP和STAT3两者表达呈负相关(r==-0.640,P<0.001)；CyclinD1、STAT3两者表达呈正相关(r=0.305P=0.004)。
结论：
1)RKIP基因转染可以抑制胃癌细胞株SGC7901中STAT3及cyclin D1、表达。
2)STAT3、CyclinD1蛋白在早期胃癌、进展期胃癌及转移淋巴结组织中高表达,与胃癌的分化程度及淋巴结转移呈正相关。
3)RKIP在胃癌组织中的表达与STAT3、CyclinD1蛋白的表达呈负相关,胃癌组织中STAT3、CyclinD1蛋白的表达正相关。
4)ADAM12在胃癌前状态、胃癌以及转移淋巴结组织各组间表达均无显著差异。
图52幅,表22个,参考文献148篇。
Backgroud:Gastric cancer is the fourth most common cancer and the second leading cause of cancer-related mortality worldwide, both its incidence rate and mortality occupy the first place among the gastrointestinal malignant tumors, which threatens human's health and lives severely. Stomach cancer is often asymptomatic or causes only nonspecific symptoms in its early stages. By the time symptoms occur, the cancer has often reached an advanced stage, of which the overall5-year survival rate is only about30%.And so far, the pathogenesis of gastric cancer has not completely clear, treatment of this cancer has not significantly break through, which can't meet the clinical requirement in the prevention and treatment of this disease. In this regard, it is important to identify new tumor markers and therapy targets for diagnosis, metastasis and recurrence prediction, prognosis assessment and gene therapy of this cancer. In recent years, looking for molecular biology targets of tumor cell growth, apoptosis, cell cycle, invasion and infiltration has become key points and hot spots in basic research of this cancer.
Raf kinase inhibitor protein (RKIP),belonging to the phosphatidylethanolamine-binding protein(PEBP) family, is a widely expressed and highly conserved small molecules cytoplasm protein with many physiological and pathological functions. In recent years, there has been an increased interest in RKIP due to the discovery of its ability to influence intracellular signaling cascades. RKIP binds to Raf-1, and blocks MAPK signaling pathway. Also it has been demonstrated to regulate G-protein-coupled receptor signaling pathway and NF-κB signaling. RKIP has been implicated in arange of normal and disease processes, such as cell growth, apoptosis, cell migration, angiogenesis, reproduction, and cancer metastasis. Evidence has emerged that RKIP can function as a suppressor of cancer metastasis, which suppresses metastasis in prostate cancer, breast cancer and melanoma cancer and so on. A variety of ablative interventions suggest that reduced RKIP function may influence metastasis, angiogenesis, resistance to apoptosis, and genome integrity. RKIP may constitute a useful prognostic marker for predicting the clinical outcome of certain cancers in human patients, and also may be a new therapeutic target in certain cancers.
Our previous study found that the expression of Raf kinase inhibitor protein (RKIP) in gastric cancer was reduced or absent, such changes may be connected with the occurrence, differentiation, invasion and metastasis of the gastric cancer, but those specific effects and mechanism of RKIP in the occurrence and metastasis of the gastric cancer have not been reported at home and abroad.
Based on the previous studies, we used immunohistochemistry to analyze expressions of RKIP protein in pre-cancerous conditions, precancerous lesions and gastric cancer tissues in this study, and to determine the the relationship between RKIP expression and carcinogenesis of gastric mucosa. Nextly, to explore the potential role of RKIP on the malignant biological characteristics of gastric carcinoma (GC) cell line SGC7901, cell proliferation, cell cycle, apoptosis rates and migration assays were analyzed after RKIP gene over expression GC cell lines in vitro or vivo. Finally, To investigate the molecular mechanisms by which RKIP inhibits GC cell proliferation or metastasis,we analysed Cyclin D1,STAT3and ADAM-12protein expression in normal gastric tissues, pre-cancerous conditions, precancerous lesions and gastric cancer tissues,and to detect the the relationship between RKIP and these proteins expression during the carcinogenesis of gastric mucosa. Our work would reveal the potential mechanisms that how RKIP inhibit the occurrence and development of GC, which provide the basis and the new clues for the diagnosis, prognosis monitoring and targeted gene therapy of GC.
Chapter one Expression of RKIP at different stages in the course of gastric carcinoma and its significance
Object:To investigate the expression of RKIP protein in pre-cancerous conditions, precancerous lesions and gastric cancer tissues, and to determine the relationship between RKIP expression and carcinogenesis of gastric mucosa.
Methods:There were88cases of pre-cancerous conditions of gastric cancer including7cases of gastric adenomatous polyp (GAP),27cases of gastric ulcer (GU) and54cases of dysplasia (DYS),169cases of gastric carcinoma (GC) and36cases of the corresponding intra-abdominal metastatic lymph node tissues of GC involved in this research. Among the dysplasia cases,17cases with mild to moderate dysplasia,23cases with severe dysplasia, and26cases with intestinal metaplasia(IM). In169gastric cancer's tissues,48were early gastric carcinoma (EGC),121were advanced gastric carcinoma(AGC). All of them were selected from Xiangya Hospital, Central South University and verified by pathology.All paraffin-embedded tissue samples were assessed by immunohistochemistry. We analyzed the diversification rule of RKIP expression from the precarcinomatous conditions to EGC and then to AGC in order to clarify the relationship between RKIP expression in the tumorigenesis of gastric mucosa and the clinical and pathological features of gastric cancer.
Results:By using immunohistochemistry,the positive rates of RKIP protein expression were significantly lower in AGC (29.8%,36/121) and in metastatic lymph node tissues (2.8%,1/36) than in pre-cancerous conditions (70.5%,62/88).There is little difference between pre-cancerous conditions and EGC (66.7%,32/48),and P>0.05.Meanwhile,the experimental results were analyzed by using Image-Pro Plus image analysis software. The two methods gave the same determination.The positive expression rate of of RKIP in the GC invasion within mucosa and submucosa,that beyond muscularis and that beyond serosa was68.8%,35.3%,20.8%,respectively. The positive expression rate of RKIP in cases of I grade,cases of II grade,cases of III/IVgrade was68.3%,47.8%,and24.5%,respectively. The positive expression rate of RKIP in65cases without lymph-node metastasis and97cases with lymph-node metastasis was70.8%and20.7%. Positive expression of RKIP protein was negatively correlated with deeper invasion,TNM stage and lymphoid node metastasis(P<0.001). However RKIP protein expression has nothing to do with age, sex, the histological differentiation and Lauren type (P>0.05).A total of52GC cases with Hp test results, of which32cases positive.In those32positive cases, RKIP positive expression rate was43.8%; and in other20negative cases,RKIP positive expression rate was40.0%.RKIP protein expression has nothing to do with Hp infection in GC(P>0.05).
Conclusion:RKIP protein was detected high expression in pre-cancerous conditions and EGC. RKIP protein expression has nothing to do with the histological differentiation,Lauren type and Hp infection in GC.The expression of RKIP protein in ACG and metastatic lymph node tissues was signifcantly lower than that in pre-cancerous conditions or EGC.Positive expression of RKIP protein is negatively correlated with deeper invasion,TNM stage and lymphoid node metastasis,which suggests that RKIP protein down-expression was associated with invasion and metastasis in GC. Abnormal alteration of RKIP is a relatively late event in stomach tumorigenesis and play an important role in the development of GC.Measurement of RKIP expression is of value in evaluating the prognosis of gastric carcinoma.
Chapter two Effect of RKIP Gene Transfection on The Malignant Biological characteristics of Gastric Carcinoma Cells
Object:To explore the potential role of RKIP on the malignant biological characteristics of gastric carcinoma (GC) cell line SGC7901.
Methods:A recombinant plasmid carrying RKIP (pcDNA3.1(+)-RKIP) was transfected into SGC7901cells by Lipofectin-mediated method. Cells untransfected SGC7901cells and those transfected with empty pcDNA3.1(+) plasmid were used as controls. The expression of RKIP mRNA and protein were detected by q-RT-PCR and Western blotting analysis respectively. The cell proliferation of the transfected SGC7901cells were demonstrated by drawing growth curve and soft agar assay. The invasion ability of the cells was detected by scratch assays and transwell assay. Cell cycle distribution and the percentage of apoptosis were determined by flow cytometric analysis. Tumor formation in nude mice was used to assess the tumorigenic characteristics in vivo.
Results:QRT-PCR and Western blot analysis showed that the mRNA and protein levels of RKIP in the transfected cell lines were significantly higher than those in controls,which suggested that a stable cell line pcDNA3.1(+)-RKIP/SGC7901containing overexpressed RKIP gene was successfully established. Compare with vector transfected and untansfected SGC7901cells, the cell proliferation of pcDNA3.1(+)-RKIP/SGC7901cells was significantly inhibited from the4th day (P<0.001), colony formation numbers of pcDNA3.1(+)-RKIP/SGC7901cells in soft agar showed significant decreases (57±7,59±9vs20±4, both P<0.05). Scratch woundhealing assay show that the scratch width after18h was2.91±0.21mm in pcDNA3.1(+)-RKIP/SGC7901cells, significantly higher than that in pcDNA3.1(+)/SGC7901cells (1.31±0.34mm) and SGC7901cells (1.26±0.41mm), P<0.05. The results of Transwell invasion assay showed that the number of cells invaded the membrane was107.0±20in pcDNA3.1(+)-RKIP/SGC7901cells, significantly less than that of pcDNA3.1(+)/SGC7901cells (256.5±37) and SGC7901cells (276.5±40)(P<0.05). Flow cytometry analysis demonstrated that the percentage of cells in GO and G1phase was69.34±1.37%in pcDNA3.1(+)-RKIP/SGC7901cells, higher than that in pcDNA3.1(+)/SGC7901cells (63.42±0.49%) and SGC7901cells (62.55±0.91%). In contrast, the percentage of cells in G2and M phase was8.21±1.64%in pcDNA3.1(+)-RKIP/SGC7901cells, significantly lower than that in pcDNA3.1(+)/SGC7901cells (1.07±0.29%) and SGC7901cells (13.71±2.36%)(P<0.05). In addition, the apoptosis index was3.59±0.51%in pcDNA3.1(+)-RKIP/SGC7901cells, significantly higher than that in pcDNA3.1(+)/SGC7901cells (1.17±0.87%) and SGC7901cells (1.07±0.29%)(P<0.05). We employed xenograft nude mice model in which SGC7901and derived cells were injected subcutaneously. We observed that visual tumor developed7days in average after the injection in pcDNA3.1(+)/SGC7901and SGC7901groups, but developed21days in average after the injection in pcDNA3.1(+)-RKIP/SGC7901group. The tumors were dissected6weeks after the injection and their weight was significant less in pcDNA3.1(+)-RKIP/SGC7901group (7.36±2.10g) than in pcDNA3.1(+)/SGC7901group (13.63±1.61g) and SGC7901group (14.03±3.51g)(P<0.05,). In addition, tumor growth curve assay showed that transfection of RKIP expression plasmid inhibited the growth of xenografted SGC7901cells,the average volume of xenograft tumors on the7th,14th,21th,28th,35th,42th days was significantly lower than those in the controls at the same time points(P<0.05).
Conclusions:
1) SGC7901cell line display decreased expression of RKIP mRNA and protein,which indicated that down-regulation of RKIP expression is associated with gastric cancer.
2) Restoration of RKIP can reduce cell proliferation, migration and invasiveness in SGC7901cells in vitro.
3) Human gastric carcinoma implant tumor model in nude mice was successfully established. RKIP can suppress incidence of gastric cancer xenograft and decrease tumorigenicity in nude mice, inhibit growth of transplanted tumor in vivo.
4) RKIP inhibits SGC7901cell proliferation,migration and invasiveness, which is closely related with altering the cell cycle and promoting apoptosis.
5) RKIP gene may play an important role in gastric cancer cell proliferation, invasion and apoptosis; the expression of RKIP gene could favor the malignant phenotype revision of GC cells,which indicated that RKIP may serve as an effective target for cancer gene therapy.
Chapter three Mechanisms of RKIP inhibiting invasion and metastasis in GC
Object:Our investigation is aim to analyze the correlation among RKIP,Cyclin D1,STAT3and ADAM-12expression,especially to explore the correlation and significance with clinicopathological features of gastric carcinoma. These results provide a new experimental evidence for clarifying the mechanisms of RKIP inhibiting invasion and metastasis in GC.
Methods:Firstly,We analyzed Cyclin D1,STAT3and ADAM-12mRNA expression in the RKIP transfected SGC7901cell lines by q-RT-PCR in order to demonstrate whether RKIP inhibit invasion and metastasis in GC by regulating Cyclin D1,STAT3and ADAM-12expression. Secondly,we analyzed the rule of Cyclin D1,STAT3and ADAM-12protein expression from the precarcinomatous conditions to GC and then to metastatic lymph node tissues by using immunohistochemistry,in order to clarify the relationship between Cyclin D1,STAT3and ADAM-12protein expression in the tumorigenesis of gastric mucosa and the clinical and pathological features of gastric cancer.Furthermore the correlation among RKIP,Cyclin D1,STAT3and ADAM-12expression was analyzed.
Results:The mRNA expression of CyclinD1、STAT3、ADAM12in pcDNA3.1(+)-RKIP/SGC7901group was0.405±0.073times,0.578±0.274times,0.673±0.447times than those in SGC7901group, and the mRNA expression of CyclinD1、STAT3、ADAM12in pcDNA3.1(+)/SGC7901group was1.038±0.543times,1.772±1.078times,1.003±0.434times than those in SGC7901group. The mRNA expression of CyclinD1,STAT3in pcDNA3.1(+)-RKIP/SGC7901group was significantly lower than that of the control group or the vector group (P<0.05),however there were no significant differences in the expression levels of ADAM12mRNA between pcDNA3.1(+)-RKIP/SGC7901group and the control group or the vector group (P>0.05).By using immunohistochemistry,STAT3protein expression was significantly higher in EGC (66.7%,20/30),AGC (71.6%,43/60)and in metastatic lymph node tissue(64.0%,16/25)than that in precarcinomatous conditions (26.1%,23/88),P<0.001.Expression of STAT3protein was positive correlated with deeper invasion,TNM stage,the histological differentiation and lymphoid node metastasis (P<0.05),however,there was little difference between intestinal type and diffuse type gastric carcinoma (P>0.05). The positive rates of CyclinD1protein expression were significantly higher in EGC (63.3%,19/30) AGC (73.3%,44/66) and in metastatic lymph node tissue (52.0%,13/25) than those in precarcinomatous conditions (23.9%,21/88),P<0.05.Expression of CyclinD1protein was positive correlated with the histological differentiation, Lauren type and lymphoid node metastasis(P<0.05),however CyclinDl protein expression has nothing to do with deeper invasion,TNM stage(P>0.05). The positive rates of ADAM12protein expression in precarcinomatous conditions,EGC,AGC and metastatic lymph node tissues were44.3%(39/88),53.3%(16/30),55.0%(33/60),36(9/25),respectively.There was little difference among these groups (P>0.05).Meanwhile,the immunohistochemistry results were analyzed by using Image-Pro Plus image analysis software. The two methods gave the same determination.T h e expression of RKIP was negatively correlated with that of CyclinDl (r=-0.411,P<0.001) and STAT3(r=-0.640,P<0.001). Expression of CyclinD1and STAT3was positively correlated in GC (r=0.305, P=0.004).
Conclusion:
1.Overexpression of both STAT3and CyclinD1protein was observed in EGC,AGC and metastatic lymph node tissues. Expression of these protein was positive correlated with the histological differentiation and lymphoid node metastasis of GC.These observations suggest that the up-regulation of STAT3and CyclinDl may play important roles in the progression,invasion and matastasis of gastric cancer.
2.Inverse association between Raf Kinase Inhibitory Protein and signal transducers and activators of transcription3or cyclinD1expression in gastric cancer patients and inhibiting expression of cyclinD1and STAT3gene in gastric cell line SGC7901by overexpression RKIP gene suggest that RKIP inhibit the progression, invasion and matastasis of gastric cancer may through regulation of its downstream target genes STAT3and CyclinD1's expression and function. RKIP gene may play an important role in gastric cancer cell proliferation, invasion, apoptosis by inhibiting expression of STAT3and its downstream target gene CyclinD1's transcription.52Figures,22tables and148references.
Raf激酶抑制蛋白(Raf kinase inhibitor protein,RKIP)属于磷脂酰乙醇胺结合蛋白(phosphatidylethanolamine-binding protein, PEBP)家族,是一种高度保守、广泛表达、具有多种生理与病理功能的小分子胞浆蛋白。参与多条信号转导通路的调控,如Raf-1-MAPK、NF-κB和G蛋白信号通路等,在细胞生长、增殖、分化、凋亡等生理过程中发挥重要作用。此外,近年来的研究表明RKIP是一个新的转移抑制基因,可以抑制前列腺癌、人乳腺癌和黑色素瘤等肿瘤细胞的转移,体外干预实验证实RKIP表达减少可以影响肿瘤转移、血管生成,抵抗凋亡,破坏染色体的完整性,在肿瘤的浸润与转移中起到重要的作用,可以作为判断某些肿瘤预后的分子标志物和肿瘤治疗的新靶标。本实验组张志强等的研究也显示RKIP在胃癌组织中表达下调或缺失,其表达改变可能与胃癌的发生、分化、浸润及转移有关[7]。但RKIP在胃癌发生、发展、浸润、转移中的具体作用及其机制如何,目前国内外研究报道较少。本研究是在前期研究的基础上,分析RKIP从癌前状态到最终癌变、转移这一系列胃粘膜癌变动态演变过程中的表达改变,寻找RKIP蛋白表达异常与胃粘膜癌变之间的关系。为明确RKIP基因在胃癌中的作用并考察其表达上调对胃癌恶性表型的影响,我们借助基因转染技术,构建RKIP(?)急定表达的胃癌细胞株SGC7901,研究RKIP基因在胃癌细胞增殖、生长、迁移、侵袭及凋亡中的作用,阐明RKIP在胃癌发生、分化及转移中扮演的角色。为进一步研究RKIP抑制胃癌的作用机制,我们分析了Cyclin D1、STAT3、ADAM-12这些与细胞增殖、凋亡、信号转导关系密切的基因及蛋白的表达与RKIP表达在胃粘膜癌变动态过程中的相互关系,探寻RKIP蛋白抑制胃癌发展、转移及肿瘤分化可能的作用机制,为胃癌的诊断、预后监测以及基因治疗提供依据和新的线索。
第一章胃粘膜癌变不同阶段组织中Raf激酶抑制蛋白的表达及其临床意义
目的：检测癌前状态、早期胃癌组织、进展期胃癌组织及转移淋巴结组织中RKIP蛋白的表达,并结合患者的临床病理资料进行统计学分析,探讨RKIP蛋白在癌前疾病、癌前病变到胃粘膜癌变不同阶段的表达变化,明确RKIP蛋白表达异常与胃粘膜癌变之间的关系,探讨RKIP在胃粘膜癌变中的可能作用。
方法：免疫组化检测癌前状态组织88例(包括胃腺瘤性息肉7例、胃溃疡27例、异型增生组织及肠化组织54例,其中轻中度异型增生17例,重度异型增生23例,伴肠化生26例)、早期胃癌组织48例、进展期胃癌组织121例及36例胃癌转移的淋巴组织中RKIP蛋白的表达,分析RKIP在胃粘膜癌变不同分化阶段的表达与临床病理学特征的关系。
结果：RKIP蛋白主要在胞浆表达,呈黄色到棕褐色颗粒,RKIP阳性表达如下：胃癌前状态88例,阳性表达62例(70.5%)；早期胃癌组织48例,阳性表达32例(66.7%)；进展期胃癌组织121例,阳性表达36例(29.8%)；36例胃癌转移的淋巴组织,阳性表达1例(2.8%)。RKIP阳性表达率在癌前状态组及早期胃癌组高表达,但两组比较无显著统计学差异(P>0.05),RKIP在不同程度的癌前病变(肠化及异型增生)中的阳性表达也无显著差异(P>0.05)。而在进展期胃癌组织中其表达水平较非癌良性癌前状态及早期胃癌组显著降低(P<0.05),在转移的淋巴结组织中其表达水平进一步降低,与癌前状态及早期、进展期胃癌组比较,均具有显著统计学差异(P<0.05)。免疫组化图片使用图像分析软件Image-Pro Plus6.0扫描,结果取累积光密度值(IOD)做统计分析,采用非参数秩和检验,统计分析显示RKIP表达水平在癌前状态组与早癌组织中没有显著差异,P=0.151；而在进展期胃癌及转移淋巴结组织中表达水平较癌前状态组显著下降,P<0.001；其在转移淋巴结组织阳性表达与进展期胃癌比较亦有显著下降,P<0.001。与免疫组化结果一致。
RKIP在不同类型的胃癌组织中的表达如下：RKIP蛋白在浸润至粘膜及粘膜下层、肌层、全层的胃癌组织中的表达率分别为68.8%、35.3%、20.8%；在高、中、低分化腺癌中表达率分别为：42.9%、41.9%、39.5%；在肠型和弥漫型胃癌中的表达率分别为：41.6%、38.2%；在Ⅰ期、Ⅱ期、Ⅲ/Ⅳ期胃癌中的表达率分别为：68.3%、47.8%、24.5%；在有淋巴结转移和无转移的胃癌组织中的表达分别为：22.7%、70.8%。RKIP蛋白表达与胃癌的浸润深度、TNM分期及淋巴结转移呈负相关(P<0.001),但RKIP蛋白表达与患者年龄、性别无关,RKIP在高、中、低分化腺癌中的表达以及在肠型及弥漫型胃癌中的表达水平无显著差异(P>0.05)。此外169例胃癌病理报告中,其中52例有Hp检测报告,32例Hp阳性,20例Hp阴性,合并Hp感染的胃癌中RKIP阳性表达率为43.8%,Hp阴性胃癌的RKIP阳性表达率为40.0%,统计分析显示RKIP在胃癌中的表达与Hp感染无明显相关(P>0.05)。结论：1.RKIP在癌前状态与早期胃癌组织高表达,在进展期胃癌及转移淋
巴组织中低表达。2.RKIP在肠型胃癌和弥漫型胃癌中表达无显著差异,与胃癌分化程
度及幽门螺杆菌感染亦无明显相关。3.RKIP表达水平与胃癌的浸润深度、TNM分期及淋巴结转移呈负相
关。可作为判断胃癌预后的重要指标。
第二章上调RKIP基因表达对胃癌细胞生物学行为的影响
目的：RKIP表达质粒稳定转染RKIP低表达胃癌细胞株SGC7901,体内外实验明确RKIP对胃癌细胞增殖、侵袭、凋亡、细胞周期及裸鼠成瘤的影响。
方法：采用脂质体介导转染技术,将RKIP真核表达重组质粒pcDNA3.1(+)/RKIP和空载体质粒分别导入RKIP低表达的胃癌细胞系SGC7901。q-RT-PCR和Western blot分别检测RKIP mRNA及蛋白质表达,并借助细胞生长曲线、软琼脂集落形成实验观察RKIP基因对胃癌细胞增殖的作用；划痕试验及Transwell实验检测胃癌细胞迁移能力和侵袭力的变化；流式细胞计数观察RKIP表达对胃癌细胞的细胞周期和凋亡率的影响；并建立人胃癌细胞裸鼠皮下移植瘤模型,观察RKIP对胃癌细胞增殖和转移能力的影响。
结果：转染RKIP基因的SGC7901细胞RKIP mRNA表达较未转染组增加4.41±0.56倍,Western blot分析显示RKIP蛋白质表达量显著增加,表明RKIP基因稳定表达的胃癌细胞系SGC7901构建成功。RKIP基因转染的SGC7901细胞生长速度明显减慢,从第4天起与空载体转染组及未转染组SGC7901细胞相比存在明显差异(P<0.001)。在软琼脂中集落形成RKIP基因转染组、空载体组和未经转染组集落形成数(×102)分别20±4,57±7和59±9,转染RKIP基因组克隆形成能力明显下降(P<0.05)。细胞划痕后18h.RKIP基因转染组细胞划痕宽度2.91±0.21mm,而空白及空载体对照组分别为1.26±0.41mm,1.31±0.34mm,RKIP基因转染组划痕恢复慢,与其他两组对比均有显著差异(P<0.05),转染RKIP基因使肿瘤细胞迁移能力下降。Transwell实验显示空白对照组及空载体组穿过基质膜细胞数分别为(256.5±37)个和(276.5±40)个,转染RKIP基因组则为(107.0±20)个,转染组穿过基质膜细胞数与其他两组对比显著减少(P<0.05),转染组侵袭能力明显受抑制。流式细胞计数发现,RKIP转染组G0-G1的细胞百分比(69.34±1.37%)较空载体转染组(63.42±0.49%)和未转染组(62.45±0.91%)明显增多,而RKIP转染组G2-M,S的细胞百分比(G2-M：8.21±1.64%,S：19.78±1.14%)较空载体转染组(G2-M：11.91±0.23%,S：24.67±0.27%)和未转染组(G2-M：13.71±2.36%,S：23.85±2.41%)明显减少(P<0.05),转染RKIP组细胞凋亡率较未转染对照组及转染空载体组明显增加(3.59±0.51%vs1.07±0.29%,2.10±0.87%),差异有统计学意义(P=0.006)。裸鼠接种实验显示,空载体转染组和未经转染的SGC7901裸鼠组在接种后平均7天就可以长出明显的包块,而RKIP基因转染组长出肿瘤的时间显著延长,平均为21天,同时肿瘤瘤体生长缓慢。将瘤体积变化对时间绘制瘤体生长曲线,pcDNA3.1(+)-RKIP/SGC7901接种的移植瘤体积较空白组及空载体组明显减小,在第7,14,21,28,35,42天对三组移植瘤体积的均数进行统计学分析,结果表明具有显著性差异(P<0.05)。6周后处死裸鼠,取出瘤块,称取瘤质量。pcDNA3.1(+)-RKIP转染组肿瘤体积明显小于空载体转染组和未经转染组,差异有统计意义(7.36±2.10g vs13.63v1.61g,14.03±3.51g,F=6.503,P=0.03),说明RKIP重表达可以明显抑制SGC7901细胞在裸鼠中的移植瘤生长能力。
结论：
1)SGC7901胃癌细胞系RKIP mRNA和蛋白低表达,提示RKIP的低表达可能与胃癌有关。
2)RKIP能抑制SGC7901胃癌细胞的体外增殖能力和侵袭力。
3)成功建立转染组和对照组SGC7901胃癌细胞裸鼠皮下移植瘤模型,RKIP基因转染可以抑制裸鼠移植瘤的生长,降低SGC7901胃癌细胞的致瘤性。
4)RKIP对SGC7901胃癌细胞生长的抑制作用与其干预胃癌细胞周期、促进细胞凋亡有关。
5)RKIP基因重表达有助于SGC7901的恶性表型逆转,有望成为肿瘤治疗的有效靶点。
第三章Raf激酶抑制蛋白抑制胃癌侵袭转移的作用机制的初步研究
目的：探讨胃癌细胞及良性癌前状态、胃癌、转移淋巴结组织中的RKIP表达与Cyclin D1、STAT3、ADAM-12表达的关系,并探讨它们表达与胃癌临床病理的关系,为探索RKIP抑制胃癌侵袭转移的作用机制提供新的实验依据。
方法：q-RT-PCR检测稳定转染RKIP基因的胃癌细胞SGC7901中的Cyclin D1、STAT3、ADAM-12mRNA表达变化,在体外从细胞水平探讨RKIP基因抑制胃癌的作用机制是否与调控Cyclin D1、 STAT3及ADAM-12的表达有关。再采用免疫组化检测癌前状态组织88例；早期胃癌组织30例、进展期胃癌组织60例及25例胃癌转移的淋巴组织中Cyclin D1、STAT3、ADAM-12蛋白表达及其与胃癌的临床病理特征的关系,分析RKIP表达与Cyclin D1、STAT3、 ADAM-12表达在胃粘膜癌变过程中的相互关系。
结果：(1)RKIP基因转染组的CyclinD1、STAT3、ADAM12mRNA表达量分别为未转染组的0.405±0.073倍、0.578±0.274倍、0.673±0.447,转染载体组中三个基因mRNA表达量分别为未转染组的1.038±0.543倍、1.772±1.078倍、1.003±0.434倍。RKIP基因转染组胃癌细胞中的cyclinD1、STAT3基因表达显著下调(P<0.05),而RKIP基因转染后ADAM12基因表达与其他两组比较无明显差异(P>0.05)。(2)STAT3蛋白在胃癌前状态组、早期胃癌、进展期胃癌组织、转移淋巴组织的阳性表达率分别为例26.1%(23/88)、66.7%(20/30)、71.6%(43/60)、64.0%(16/25)。与胃癌前状态组比较,STAT3蛋白在早期胃癌、进展期胃癌及转移淋巴结组织其表达阳性率均显著升高,差异具有统计意义(P<0.001)。STAT3蛋白表达与胃癌的浸润深度、TNM分期、淋巴结转移及肿瘤分化程度呈正相关(P<0.05),但在肠型胃癌和弥漫型胃癌中表达无显著差异(P>0.05)。(3)CyclinDl蛋白在胃癌前状态组、早期胃癌、进展期胃癌组织、转移淋巴组织的阳性表达率分别为23.9%(21/88)、63.3%(19/30)、73.3%(44/60)、52.0%(13/25)。与胃癌前状态疾病比较,早期胃癌、进展期胃癌以及转移淋巴结组织中Cyclin D1蛋白阳性表达率均显著升高,差异具有统计意义(P<0.05)。CyclinD1蛋白表达与胃癌的分化程度及淋巴结转移呈正相关(P<0.05),并与胃癌分型有关,CyclinD1在弥漫型胃癌中阳性表达率较肠型胃癌高(P=0.001),与胃癌的浸润深度、TNM分期无明显相关(P>0.05)。(4)ADAM-12蛋白在癌前状态、早期胃癌、进展期胃癌、转移淋巴组织的阳性表达率分别为44.3%(39/88)、53.3%(16/30)、55.0%(33/60)、36%(9/25)。ADAM-12蛋白在癌前状态、早期、进展期胃癌以及转移淋巴结组织中的表达各组间均无显著差异(P>0.05)。(5)Cyclin D1、STAT3、ADAM-12累积光密度值(IOD)定量分析结果与免疫组化结果一致。(6)相关性分析表明RKIP蛋白与CyclinD1蛋白表达两者呈负相关(r=-0.411,P<0.001)；RKIP和STAT3两者表达呈负相关(r==-0.640,P<0.001)；CyclinD1、STAT3两者表达呈正相关(r=0.305P=0.004)。
结论：
1)RKIP基因转染可以抑制胃癌细胞株SGC7901中STAT3及cyclin D1、表达。
2)STAT3、CyclinD1蛋白在早期胃癌、进展期胃癌及转移淋巴结组织中高表达,与胃癌的分化程度及淋巴结转移呈正相关。
3)RKIP在胃癌组织中的表达与STAT3、CyclinD1蛋白的表达呈负相关,胃癌组织中STAT3、CyclinD1蛋白的表达正相关。
4)ADAM12在胃癌前状态、胃癌以及转移淋巴结组织各组间表达均无显著差异。
图52幅,表22个,参考文献148篇。
Backgroud:Gastric cancer is the fourth most common cancer and the second leading cause of cancer-related mortality worldwide, both its incidence rate and mortality occupy the first place among the gastrointestinal malignant tumors, which threatens human's health and lives severely. Stomach cancer is often asymptomatic or causes only nonspecific symptoms in its early stages. By the time symptoms occur, the cancer has often reached an advanced stage, of which the overall5-year survival rate is only about30%.And so far, the pathogenesis of gastric cancer has not completely clear, treatment of this cancer has not significantly break through, which can't meet the clinical requirement in the prevention and treatment of this disease. In this regard, it is important to identify new tumor markers and therapy targets for diagnosis, metastasis and recurrence prediction, prognosis assessment and gene therapy of this cancer. In recent years, looking for molecular biology targets of tumor cell growth, apoptosis, cell cycle, invasion and infiltration has become key points and hot spots in basic research of this cancer.
Raf kinase inhibitor protein (RKIP),belonging to the phosphatidylethanolamine-binding protein(PEBP) family, is a widely expressed and highly conserved small molecules cytoplasm protein with many physiological and pathological functions. In recent years, there has been an increased interest in RKIP due to the discovery of its ability to influence intracellular signaling cascades. RKIP binds to Raf-1, and blocks MAPK signaling pathway. Also it has been demonstrated to regulate G-protein-coupled receptor signaling pathway and NF-κB signaling. RKIP has been implicated in arange of normal and disease processes, such as cell growth, apoptosis, cell migration, angiogenesis, reproduction, and cancer metastasis. Evidence has emerged that RKIP can function as a suppressor of cancer metastasis, which suppresses metastasis in prostate cancer, breast cancer and melanoma cancer and so on. A variety of ablative interventions suggest that reduced RKIP function may influence metastasis, angiogenesis, resistance to apoptosis, and genome integrity. RKIP may constitute a useful prognostic marker for predicting the clinical outcome of certain cancers in human patients, and also may be a new therapeutic target in certain cancers.
Our previous study found that the expression of Raf kinase inhibitor protein (RKIP) in gastric cancer was reduced or absent, such changes may be connected with the occurrence, differentiation, invasion and metastasis of the gastric cancer, but those specific effects and mechanism of RKIP in the occurrence and metastasis of the gastric cancer have not been reported at home and abroad.
Based on the previous studies, we used immunohistochemistry to analyze expressions of RKIP protein in pre-cancerous conditions, precancerous lesions and gastric cancer tissues in this study, and to determine the the relationship between RKIP expression and carcinogenesis of gastric mucosa. Nextly, to explore the potential role of RKIP on the malignant biological characteristics of gastric carcinoma (GC) cell line SGC7901, cell proliferation, cell cycle, apoptosis rates and migration assays were analyzed after RKIP gene over expression GC cell lines in vitro or vivo. Finally, To investigate the molecular mechanisms by which RKIP inhibits GC cell proliferation or metastasis,we analysed Cyclin D1,STAT3and ADAM-12protein expression in normal gastric tissues, pre-cancerous conditions, precancerous lesions and gastric cancer tissues,and to detect the the relationship between RKIP and these proteins expression during the carcinogenesis of gastric mucosa. Our work would reveal the potential mechanisms that how RKIP inhibit the occurrence and development of GC, which provide the basis and the new clues for the diagnosis, prognosis monitoring and targeted gene therapy of GC.
Chapter one Expression of RKIP at different stages in the course of gastric carcinoma and its significance
Object:To investigate the expression of RKIP protein in pre-cancerous conditions, precancerous lesions and gastric cancer tissues, and to determine the relationship between RKIP expression and carcinogenesis of gastric mucosa.
Methods:There were88cases of pre-cancerous conditions of gastric cancer including7cases of gastric adenomatous polyp (GAP),27cases of gastric ulcer (GU) and54cases of dysplasia (DYS),169cases of gastric carcinoma (GC) and36cases of the corresponding intra-abdominal metastatic lymph node tissues of GC involved in this research. Among the dysplasia cases,17cases with mild to moderate dysplasia,23cases with severe dysplasia, and26cases with intestinal metaplasia(IM). In169gastric cancer's tissues,48were early gastric carcinoma (EGC),121were advanced gastric carcinoma(AGC). All of them were selected from Xiangya Hospital, Central South University and verified by pathology.All paraffin-embedded tissue samples were assessed by immunohistochemistry. We analyzed the diversification rule of RKIP expression from the precarcinomatous conditions to EGC and then to AGC in order to clarify the relationship between RKIP expression in the tumorigenesis of gastric mucosa and the clinical and pathological features of gastric cancer.
Results:By using immunohistochemistry,the positive rates of RKIP protein expression were significantly lower in AGC (29.8%,36/121) and in metastatic lymph node tissues (2.8%,1/36) than in pre-cancerous conditions (70.5%,62/88).There is little difference between pre-cancerous conditions and EGC (66.7%,32/48),and P>0.05.Meanwhile,the experimental results were analyzed by using Image-Pro Plus image analysis software. The two methods gave the same determination.The positive expression rate of of RKIP in the GC invasion within mucosa and submucosa,that beyond muscularis and that beyond serosa was68.8%,35.3%,20.8%,respectively. The positive expression rate of RKIP in cases of I grade,cases of II grade,cases of III/IVgrade was68.3%,47.8%,and24.5%,respectively. The positive expression rate of RKIP in65cases without lymph-node metastasis and97cases with lymph-node metastasis was70.8%and20.7%. Positive expression of RKIP protein was negatively correlated with deeper invasion,TNM stage and lymphoid node metastasis(P<0.001). However RKIP protein expression has nothing to do with age, sex, the histological differentiation and Lauren type (P>0.05).A total of52GC cases with Hp test results, of which32cases positive.In those32positive cases, RKIP positive expression rate was43.8%; and in other20negative cases,RKIP positive expression rate was40.0%.RKIP protein expression has nothing to do with Hp infection in GC(P>0.05).
Conclusion:RKIP protein was detected high expression in pre-cancerous conditions and EGC. RKIP protein expression has nothing to do with the histological differentiation,Lauren type and Hp infection in GC.The expression of RKIP protein in ACG and metastatic lymph node tissues was signifcantly lower than that in pre-cancerous conditions or EGC.Positive expression of RKIP protein is negatively correlated with deeper invasion,TNM stage and lymphoid node metastasis,which suggests that RKIP protein down-expression was associated with invasion and metastasis in GC. Abnormal alteration of RKIP is a relatively late event in stomach tumorigenesis and play an important role in the development of GC.Measurement of RKIP expression is of value in evaluating the prognosis of gastric carcinoma.
Chapter two Effect of RKIP Gene Transfection on The Malignant Biological characteristics of Gastric Carcinoma Cells
Object:To explore the potential role of RKIP on the malignant biological characteristics of gastric carcinoma (GC) cell line SGC7901.
Methods:A recombinant plasmid carrying RKIP (pcDNA3.1(+)-RKIP) was transfected into SGC7901cells by Lipofectin-mediated method. Cells untransfected SGC7901cells and those transfected with empty pcDNA3.1(+) plasmid were used as controls. The expression of RKIP mRNA and protein were detected by q-RT-PCR and Western blotting analysis respectively. The cell proliferation of the transfected SGC7901cells were demonstrated by drawing growth curve and soft agar assay. The invasion ability of the cells was detected by scratch assays and transwell assay. Cell cycle distribution and the percentage of apoptosis were determined by flow cytometric analysis. Tumor formation in nude mice was used to assess the tumorigenic characteristics in vivo.
Results:QRT-PCR and Western blot analysis showed that the mRNA and protein levels of RKIP in the transfected cell lines were significantly higher than those in controls,which suggested that a stable cell line pcDNA3.1(+)-RKIP/SGC7901containing overexpressed RKIP gene was successfully established. Compare with vector transfected and untansfected SGC7901cells, the cell proliferation of pcDNA3.1(+)-RKIP/SGC7901cells was significantly inhibited from the4th day (P<0.001), colony formation numbers of pcDNA3.1(+)-RKIP/SGC7901cells in soft agar showed significant decreases (57±7,59±9vs20±4, both P<0.05). Scratch woundhealing assay show that the scratch width after18h was2.91±0.21mm in pcDNA3.1(+)-RKIP/SGC7901cells, significantly higher than that in pcDNA3.1(+)/SGC7901cells (1.31±0.34mm) and SGC7901cells (1.26±0.41mm), P<0.05. The results of Transwell invasion assay showed that the number of cells invaded the membrane was107.0±20in pcDNA3.1(+)-RKIP/SGC7901cells, significantly less than that of pcDNA3.1(+)/SGC7901cells (256.5±37) and SGC7901cells (276.5±40)(P<0.05). Flow cytometry analysis demonstrated that the percentage of cells in GO and G1phase was69.34±1.37%in pcDNA3.1(+)-RKIP/SGC7901cells, higher than that in pcDNA3.1(+)/SGC7901cells (63.42±0.49%) and SGC7901cells (62.55±0.91%). In contrast, the percentage of cells in G2and M phase was8.21±1.64%in pcDNA3.1(+)-RKIP/SGC7901cells, significantly lower than that in pcDNA3.1(+)/SGC7901cells (1.07±0.29%) and SGC7901cells (13.71±2.36%)(P<0.05). In addition, the apoptosis index was3.59±0.51%in pcDNA3.1(+)-RKIP/SGC7901cells, significantly higher than that in pcDNA3.1(+)/SGC7901cells (1.17±0.87%) and SGC7901cells (1.07±0.29%)(P<0.05). We employed xenograft nude mice model in which SGC7901and derived cells were injected subcutaneously. We observed that visual tumor developed7days in average after the injection in pcDNA3.1(+)/SGC7901and SGC7901groups, but developed21days in average after the injection in pcDNA3.1(+)-RKIP/SGC7901group. The tumors were dissected6weeks after the injection and their weight was significant less in pcDNA3.1(+)-RKIP/SGC7901group (7.36±2.10g) than in pcDNA3.1(+)/SGC7901group (13.63±1.61g) and SGC7901group (14.03±3.51g)(P<0.05,). In addition, tumor growth curve assay showed that transfection of RKIP expression plasmid inhibited the growth of xenografted SGC7901cells,the average volume of xenograft tumors on the7th,14th,21th,28th,35th,42th days was significantly lower than those in the controls at the same time points(P<0.05).
Conclusions:
1) SGC7901cell line display decreased expression of RKIP mRNA and protein,which indicated that down-regulation of RKIP expression is associated with gastric cancer.
2) Restoration of RKIP can reduce cell proliferation, migration and invasiveness in SGC7901cells in vitro.
3) Human gastric carcinoma implant tumor model in nude mice was successfully established. RKIP can suppress incidence of gastric cancer xenograft and decrease tumorigenicity in nude mice, inhibit growth of transplanted tumor in vivo.
4) RKIP inhibits SGC7901cell proliferation,migration and invasiveness, which is closely related with altering the cell cycle and promoting apoptosis.
5) RKIP gene may play an important role in gastric cancer cell proliferation, invasion and apoptosis; the expression of RKIP gene could favor the malignant phenotype revision of GC cells,which indicated that RKIP may serve as an effective target for cancer gene therapy.
Chapter three Mechanisms of RKIP inhibiting invasion and metastasis in GC
Object:Our investigation is aim to analyze the correlation among RKIP,Cyclin D1,STAT3and ADAM-12expression,especially to explore the correlation and significance with clinicopathological features of gastric carcinoma. These results provide a new experimental evidence for clarifying the mechanisms of RKIP inhibiting invasion and metastasis in GC.
Methods:Firstly,We analyzed Cyclin D1,STAT3and ADAM-12mRNA expression in the RKIP transfected SGC7901cell lines by q-RT-PCR in order to demonstrate whether RKIP inhibit invasion and metastasis in GC by regulating Cyclin D1,STAT3and ADAM-12expression. Secondly,we analyzed the rule of Cyclin D1,STAT3and ADAM-12protein expression from the precarcinomatous conditions to GC and then to metastatic lymph node tissues by using immunohistochemistry,in order to clarify the relationship between Cyclin D1,STAT3and ADAM-12protein expression in the tumorigenesis of gastric mucosa and the clinical and pathological features of gastric cancer.Furthermore the correlation among RKIP,Cyclin D1,STAT3and ADAM-12expression was analyzed.
Results:The mRNA expression of CyclinD1、STAT3、ADAM12in pcDNA3.1(+)-RKIP/SGC7901group was0.405±0.073times,0.578±0.274times,0.673±0.447times than those in SGC7901group, and the mRNA expression of CyclinD1、STAT3、ADAM12in pcDNA3.1(+)/SGC7901group was1.038±0.543times,1.772±1.078times,1.003±0.434times than those in SGC7901group. The mRNA expression of CyclinD1,STAT3in pcDNA3.1(+)-RKIP/SGC7901group was significantly lower than that of the control group or the vector group (P<0.05),however there were no significant differences in the expression levels of ADAM12mRNA between pcDNA3.1(+)-RKIP/SGC7901group and the control group or the vector group (P>0.05).By using immunohistochemistry,STAT3protein expression was significantly higher in EGC (66.7%,20/30),AGC (71.6%,43/60)and in metastatic lymph node tissue(64.0%,16/25)than that in precarcinomatous conditions (26.1%,23/88),P<0.001.Expression of STAT3protein was positive correlated with deeper invasion,TNM stage,the histological differentiation and lymphoid node metastasis (P<0.05),however,there was little difference between intestinal type and diffuse type gastric carcinoma (P>0.05). The positive rates of CyclinD1protein expression were significantly higher in EGC (63.3%,19/30) AGC (73.3%,44/66) and in metastatic lymph node tissue (52.0%,13/25) than those in precarcinomatous conditions (23.9%,21/88),P<0.05.Expression of CyclinD1protein was positive correlated with the histological differentiation, Lauren type and lymphoid node metastasis(P<0.05),however CyclinDl protein expression has nothing to do with deeper invasion,TNM stage(P>0.05). The positive rates of ADAM12protein expression in precarcinomatous conditions,EGC,AGC and metastatic lymph node tissues were44.3%(39/88),53.3%(16/30),55.0%(33/60),36(9/25),respectively.There was little difference among these groups (P>0.05).Meanwhile,the immunohistochemistry results were analyzed by using Image-Pro Plus image analysis software. The two methods gave the same determination.T h e expression of RKIP was negatively correlated with that of CyclinDl (r=-0.411,P<0.001) and STAT3(r=-0.640,P<0.001). Expression of CyclinD1and STAT3was positively correlated in GC (r=0.305, P=0.004).
Conclusion:
1.Overexpression of both STAT3and CyclinD1protein was observed in EGC,AGC and metastatic lymph node tissues. Expression of these protein was positive correlated with the histological differentiation and lymphoid node metastasis of GC.These observations suggest that the up-regulation of STAT3and CyclinDl may play important roles in the progression,invasion and matastasis of gastric cancer.
2.Inverse association between Raf Kinase Inhibitory Protein and signal transducers and activators of transcription3or cyclinD1expression in gastric cancer patients and inhibiting expression of cyclinD1and STAT3gene in gastric cell line SGC7901by overexpression RKIP gene suggest that RKIP inhibit the progression, invasion and matastasis of gastric cancer may through regulation of its downstream target genes STAT3and CyclinD1's expression and function. RKIP gene may play an important role in gastric cancer cell proliferation, invasion, apoptosis by inhibiting expression of STAT3and its downstream target gene CyclinD1's transcription.52Figures,22tables and148references.
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