Bmi-1调控人胆囊癌细胞增殖的效应及其WWOX信号通路的分子机制
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摘要
[研究目的]
1.探讨Bmi-1、WWOX及PUMA基因在胆囊癌组织和慢性胆囊炎伴轻-中度非典型增生组织及正常胆囊组织中的表达差异,分析Bmi-1/WWOX/PUMA表达与胆囊癌临床病理因素的关系,有望揭示其表达差异在胆囊癌发生、发展中的作用及其临床意义,为后续研究胆囊癌的发生机制奠定基础。
2.通过体外RNAi干扰技术成功构建靶向shRNA-Bmi-1重组载体抑制原癌基因Bmi-1表达,观察其对胆囊癌细胞增殖、凋亡及细胞周期的效应,探讨Bmi-1调控WWOX/P73/PUMA/Bax/Bcl-2/Caspase-3信号通路调节胆囊癌细胞生长的分子机制。
3.通过体外成功构建pcDNA3.0-WWOX真核表达载体,探讨过表达WWOX基因对胆囊癌细胞增殖,凋亡及细胞周期的影响,进一步研究WWOX/P73/PUMA/Bax/Bcl-2通路调控胆囊癌细胞生长的分子机制,为研究Bmi-1/WWOX通路间是否存有反馈环路奠定基础。
4.成功建立了人胆囊癌BALB/C裸鼠皮下移植瘤模型,通过体内试验评估靶向抑制Bmi-1对移植瘤的治疗效果,进一步探讨其对胆囊癌细胞生长的影响及调控Bmi-1/WWOX/P73/PUMA/Bax/Bcl-2通路的分子机制,验证体外实验结果。
5.本课题通过临床相关研究,体外及裸鼠体内试验研究,全面系统阐述Bmi-1调控人胆囊癌细胞增殖、凋亡及细胞周期的效应及其WWOX信号通路的分子机制,将有助于胆囊癌标志物的筛选及病情的预后和评估,为胆曩癌的早期诊断、分子靶向治疗提供理论依据和实验数据。
[研究方法]
1.临床相关研究：收集临床病理资料完整的原发性胆囊腺癌病人手术标本存档石蜡块55例,收集同期慢性胆囊炎伴轻-中度非典型增生组织标本30例及正常胆囊组织标本22例(均来自于肝内胆管结石或肝脏肿瘤行右半肝切除患者,胆囊内无结石及肿瘤侵袭)作对照,其中每组病例包括新鲜组织各5例。应用IHC-Max Vision法、RT-PCR及Real-time PCR等技术检测上述标本中Bmi-1/WWOX/PUMA mRNA及蛋白的表达差异,分析三者表达与胆囊癌临床病理因素的关系及意义。
2.体外研究,以原癌基因Bmi-1mRNA为靶点：通过体外RNAi干扰技术成功构建shRNA-Bmi-1重组载体转染胆囊癌GBC-SD细胞,应用倒置荧光显微镜及流式细胞技术评价其转染效率,以RT-PCR及Westernblot检测转染后胆囊癌细胞转录和翻译水平的变化,筛选出最佳干扰作用的Bmi-1靶序列重组质粒为后续实验组。通过倒置显微镜观察、Brdu实验、Annexin V/7-AAD单染和双染实验、透射电镜及细胞周期检测技术分析靶向shRNA抑制Bmi-1对胆囊癌细胞增殖活性、凋亡进程及细胞周期的影响机制及其生物学效应与时间梯度的关系,进一步通过Real-time PCR, Western Blot,免疫荧光染色及流式细胞检测等技术探讨靶向shRNA抑制Bmi-1表达对WWOX/P73/PUMA/Bax/Bcl-2/Caspase-3通路影响的分子机制。
3.体外研究,以抑癌基因WWOXmRNA为靶点：提取GBC-SD细胞总RNA,采用RT-PCR扩增WWOX全序列,将其克隆到真核表达载体pCDNA3.0中,通过限制性内切酶酶切及DNA测序鉴定,成功构建pcDNA3.0-WWOX重组真核表达载体。按脂质体介导转染GBC-SD细胞,同时转染空载体、脂质体为阴性对照及GBC-SD细胞为空白对照。以RT-PCR、Westemblot及免疫荧光技术检测转染后胆囊癌细胞转录和翻译水平的变化,以MTT法、AnnexinV/7-AAD双染、TUNNEL法、细胞周期检测技术、透射电镜技术、线粒体跨膜电位检测技术分析过表达WWOX对胆囊癌细胞增殖活性、凋亡及细胞周期的影响机制及其生物学效应与时间梯度的关系,进一步通过Real-time PCR, Western Blot,免疫荧光染色及流式细胞检测等技术探讨pcDNA3.0-WWOX真核表达载体对WWOX/P73/PUMA/Bax/Bcl-2通路调控的分子机制。
4.体内试验研究：以体外实验结果为基础,通过建立人胆囊癌BALB/C裸鼠皮下移植瘤模型,应用shRNA-Bmi-1重组质粒在移植瘤周围及瘤内多点注射,实验终止绘制移植瘤生长曲线,计算瘤体抑制率,通过标本解剖和组织病理检测手段探讨靶向shRNA抑制Bmi-1表达对裸鼠皮F移植瘤的抗瘤效果,通过Max Vision去、TUNEL法、透射电镜、RT-PCR及Western Blot等检测技术进一步探讨Bmi-1对胆囊癌细胞生长的影响及调控WWOX通路关键基因表达的分子机制。
[研究结果1
1.临床相关研究结果
(1)IHC-MaxVision检测结果显示：Bmi-1、WWOX及PUMA蛋白在胆囊癌组织中的阳性表达分别为83.6%(46/55)、38.2%(21/55)和34.5%(19/55),其中Bmi-1蛋白在胆囊癌组织中的阳性表达明显高于慢性胆囊炎伴非典型增生组织及正常胆囊组织,而WWOX及PUMA蛋白在胆囊癌组织中的阳性表达明显低于慢性胆囊炎伴非典型增生组织及正常胆囊组织(P<0.05)。(2)Bmi-1、WWOX及PUMA表达差异与临床病理因素的关系结果：Bmi-1、WWOX蛋白表达水平与胆囊癌的病理分期、分化程度及淋巴结转移有关(P<0.05),与胆囊癌患者性别、年龄、肿瘤大小、肿瘤部位、是否伴有肝硬化及胆囊结石无相关性(p>0.05)。PUMA蛋白表达水平与胆囊癌分化程度及淋巴结转移有关(P<0.05),而与患者性别、年龄、肿瘤大小、临床病理分期、肿瘤部位、是否伴有肝硬化及胆囊结石均无相关性(p>0.05)。(3) Realtime PCR和RT-PCR检测结果：Bmi-1mRNA表达为胆囊癌组织>慢性胆囊炎伴非典型增生组织>正常胆囊组织；而WWOX及PUMAmRNA表达为胆囊癌组织<慢性胆囊炎伴非典型增生组织<正常胆囊组织(P<0.05)。实验表明Bmi-1、WWOX及PUMA基因可能参与胆囊癌发生、发展过程,慢性胆囊炎伴非典型增生组织中Bmi-1持续高表达或WWOX/PUMA持续低表达警示胆囊癌发生的可能。
2.以原癌基因Bmi-1mRNA为靶点的体外研究结果
(1) shRNA-Bmi-1重组载体的构建及筛选结果：shRNA-Bmi-1重组载体构建后经基因测序证明所获得的4组Bmi-1基因扩增序列正确。应用倒置荧光显微镜及流式细胞技术检测细胞转染效率70%左右；将4组质粒分别转染胆囊癌GBC-SD细胞48h后,通过RT-PCR及Westernblot检测筛选出第四组序列在转录和翻译水平抑制显著,为后续实验组(即为shRNA-Bmi-1组)。
(2)靶向shRNA干扰Bmi-1表达对胆囊癌细胞增殖、凋亡及细胞周期的影响结果：通过RT-PCR及Westernblot检测显示靶向抑制Bmi-1,其mRNA及蛋白表达水平均明显低于对照组(p<0.05)；而对照组间表达量无显著差异(p>0.05)。倒置显微镜观察发现shRNA-Bmi-1对胆囊癌细胞的影响随时间的推移,增殖抑制率较对照组明显,其中以转染后72h抑制效果最显著,细胞死亡最多。Brdu检测显示shRNA-Bmi-1组细胞增殖活性较对照组降低,细胞增殖力呈时间依存性,随时间推移增殖力明显减弱,于转染72h细胞增殖抑制最强(p<0.05)。AnnexinV/7-AAD单染和双染检测显示转染shRNA-Bmi-1重组质粒24h-48h-72h后,shRNA-Bmi-1组细胞凋亡率均随时间推移明显增加(24h<48h<72h),且与晚期凋亡增加为主(p<0.05)。透射电镜检测发现shRNA-Bmi-1转染组胆囊癌细胞浓缩、核固缩形成凋亡小体。细胞周期检测显示shRNA-Bmi-1转染组G0/G1期细胞增多,G2/M和S期细胞减少,细胞阻滞于G0/G1,细胞凋亡率明显增高,增殖指数降低(p<0.05)。
(3)靶向shRNA干扰Bmi-1表达调控WWOX通路的分子机制研究结果：通过Real-time PCR、Western Blot及免疫荧光染色技术检测显示shRNA-Bmi-1转染组胆囊癌细胞Bmi-1mRNA及蛋白表达量及蛋白荧光强度较对照组减少,而WWOX、P73、PUMAmRNA及蛋白表达量较对照组均明显增加,蛋白荧光表达强度明显增强(p<0.05)；流式细胞仪技术检测显示shRNA-Bmi-1转染组胆囊癌细胞Bax及Caspase-3蛋白表达较对照组增加,而Bcl-2蛋白表达较对照组减少(p<0.05)。
3.以抑癌基因WWOX mRNA为靶点的体外实验研究结果
(1)WWOX基因真核表达载体的构建：pcDNA3.0-WWOX重组质粒经双酶切鉴定及基因测序证明所获得的WWOX基因扩增序列正确。(2)pcDNA3.0-WWOX重组质粒对胆囊癌细胞增殖、凋亡及细胞周期影响结果：RT-PCR及Westernblot检测显示pcDNA3.0-WWOX转染48小时后WWOXmRNA及蛋白表达水平均明显高于对照组(p<0.05)；对照组间mRNA及蛋白表达量无显著差异(p>0.05)。免疫荧光检测结果显示pcDNA3.0-WWOX组细胞WWOX蛋白荧光强度在细胞核周围明显强于各对照组,而对照组间细胞WWOX蛋白荧光强度变化不明显。倒置显微镜观察发现pcDNA3.0-WWOX组贴壁的GBC-SD细胞数量明显减少,悬浮细胞及细胞碎片增加,而对照组细胞呈正常增殖状态,贴壁生长良好。MTT法检测显示pcDNA3.0-WWOX组细胞增殖活性明显降低,且随时间推移细胞增殖抑制效果更为显著,对照组细胞增殖抑制不明显(P<0.05)。BrdU检测显示pcDNA3.0-WWOX组细胞增殖率较对照组降低(P<0.05)。Annexin V/7-AAD双染检测发现pcDNA3.0-WWOX组胆囊癌细胞早、晚期凋亡率较对照组增高(p<0.05)；而对照组间早、晚期凋亡率未见显著差异(P>0.05)。TUNNEL法检测显示转染pcDNA3.0-WWOX重组质粒24h/48h/72h后,胆囊癌细胞凋亡比率较对照组增加,以转染48h后更为明显(p<0.05)。细胞周期检测发现pcDNA3.0-WWOX组G0/G1期细胞增多,G2/M和S期细胞减少,细胞凋亡率增高,增殖指数降低(p<0.05)；而对照组间未见显著性差异(p>0.05)。JC-1染色检测细胞线粒体跨膜电位(△Ψm)显示过表达WWOX基因胆囊癌细胞线粒体内绿色荧光信号显著高于空载体、脂质体及空白对照组,提示WWOX诱导胆囊癌细胞早期凋亡为主(p<0.05)。透射电镜观察pcDNA3.0-WWOX组细胞典型的凋亡形态学改变,出现核固缩、碎裂形成凋亡小体。(3)pcDNA-WWOX重组质粒对WWOX通路关键基因的影响结果：通过Real-time PCR,Western Blot及免疫荧光染色检测显示特异性上调WWOX表达,胆囊癌细胞中P73、PUMA在转录及翻译水平的表达量均较对照组增加(p<0.05)；流式细胞仪检测发现特异性上调WWOX表达,胆囊癌细胞中Bax蛋白表达水平较对照组明显增加,而Bcl-2蛋白表达水平较对照组减少(p<0.05)。
4.移植瘤体内试验研究结果
裸鼠皮下接种GBC-SD细胞株5-7天后成瘤率为100%。shRNA-Bmi-1组移植瘤生长速度较对照组减慢,治疗6周实验终止时各实验组体积较治疗前增大,但shRNA-Bmi-1组移植瘤体积明显小于各对照组(shRNA-Scramble组、Lipofectamine组、GBC-SD组),其瘤体抑制率为60.6%(p<0.05),对照组间体积无明显差异(P>0.05)。HE染色显示shRNA-Bmi-1组细胞坏死明显,细胞形态模糊,见炎性细胞浸润,组织结构不清晰,而各对照组细胞异型性明显,炎性细胞较少,组织结构清晰。MaxVision染色发现shRNA-Bmi-1组移植瘤组织中Ki67及VEGF蛋白表达较对照组减弱,提示shRNA-Bmi-1组肿瘤新生血管生成减少、细胞增殖抑制明显。TUNEL及透射电镜检测显示靶向shRNA抑制Bmi-1体内可诱导胆囊癌细胞凋亡,与体外实验相符合。通过Real-time PCR、Western Blot及免疫组织化学检测发现靶向shRNA干扰Bmi-1表达可特异性促进Bmi-1mRNA的降解及抑制其蛋白的表达,同时促进下游通路中关键基因WWOX/P73/PUMA/Bax表达,抑制Bcl-2的表达水平。
[研究结论]
1. Bmi-1、WWOX及PUMA的表达参与胆囊癌的发生发展过程,Bmi-1阳性表达率越高,或WWOX、PUMA阳性表达越低或表达缺失,提示肿瘤恶性程度增加、转移增快,Bmi-1、WWOX及PUMA的表达差异可能参与肿瘤的侵袭及演进。应用免疫组化联合检测胆囊癌组织中的Bmi-1、WWOX及PUMA的表达将有利于协助胆囊癌的早期诊断,有助于对其预后进行合理评估。
2.体外成功构建了shRNA-Bmi-1重组载体。靶向沉默Bmi-1表达能有效促进胆囊癌细胞Bmi-1mRNA降解及抑制其蛋白表达,能特异性抑制胆囊癌细胞增殖,诱导其凋亡,使细胞周期阻滞于G0/G1期,从而引起细胞DNA合成受阻,研究提示Bmi-1参与调控胆囊癌细胞的增殖和凋亡,维持细胞周期的运行,是抑制细胞凋亡和促进细胞异常增殖的重要因素。同时靶向沉默Bmi-1表达能有效促进WWOX通路中的关键基因WWOX/P73/PUMA/Bax/Caspase-3的表达,抑制Bcl-2蛋白表达,其作用可能与调控胆囊癌细胞生长效应的机制相关,Bmi-1及其Bmi-1/WWOX通路参与线粒体依赖的凋亡途径调控胆囊癌细胞的生长。
3.体外成功构建了pcDNA3.0-WWOX真核表达载体。过表达WWOX可有效抑制胆囊癌细胞WWOXmRNA降解、促进其蛋白表达,能有效抑制胆囊癌细胞的增殖活性、诱导其凋亡,使细胞周期阻滞于G0/G1期。同时上调并促进P73蛋白在胆囊癌细胞质中螯合积累,促进PUMA、Bax及抑制Bcl-2的转录和翻译过程,其作用可能是调控胆囊癌细胞生长的重要机制之一。WWOX/P73/PUMA通路通过调控线粒体依赖的凋亡途径来调节胆囊癌细胞的增殖、凋亡及细胞周期的变化,进一步证实了WWOX通路调控胆囊癌细胞的分子机制。
4.成功建立胆囊癌裸鼠皮下移植瘤动物模型。靶向沉默Bmi-1表达能诱导胆囊癌移植瘤细胞凋亡,显著抑制肿瘤的生长及新生血管的形成,影响裸鼠皮下移植瘤Bmi-1/WWOX/P73/PUMA/Bax/Bcl-2通路关键基因的表达,与体外实验相符合。体内实验进一步验证了Bmi-1/WWOX通路参与线粒体依赖的凋亡途径调控胆囊癌细胞的生长。
5.Bmi-1及WWOX可能为胆囊癌基因靶向治疗提供潜在的分子靶标,通过靶向沉默Bmi-1表达和特异性性上调WWOX的表达可能是胆囊癌联合基因治疗的潜在治疗手段之一,阻断Bmi-1/WWOX通路对胆囊癌靶向性治疗策略具有重要意义,同时也为探索胆囊癌早期诊断及多分子联合靶向治疗提供了新的实验依据。
[Objective]
1. To explore the expressions differences of the Bmi-1, WWOX and PUMA gene in gallbladder carcinoma, chronic cholecystitis tissue with mild-moderate atypical hyperplasia and normal gallbladder tissues, and to analyze the relationship between Bmi-1, WWOX and PUMA gene expressions and clinicopathological features of gallbladder cancer, and to reveal the role and Clinical significance of their expression differences in the occurrence and development process of gallbladder cancer, so as to lay the foundation for the follow-up study on the occurrence mechanism of gallbladder cancer.
2. To successfully construct targeting shRNA-Bmi-1recombinant vector to inhibit proto-oncogene Bmi-1expression by RNAi interference techniques in vitro experiment; to observe its effects on gallbladder cancer cell proliferation, apoptosis and cell cycle; To explore the molecular mechanisms of Bmi-1regulating gallbladder cancer cell growth through regulating WWOX/P73/PUMA/Bax/Bcl-2/Caspase-3signaling pathways.
3. By successfully constructed pcDNA3.0-WWOX eukaryotic expression vector in vitro experiment, To explored the effects of WWOX gene overexpression on gallbladder cell proliferation, apoptosis and cell cycle, and to further study molecular mechanisms that WWOX/P73/PUMA/Bax/Bcl-2pathway regulates the cell growth of gallbladder cancers, and to lay a foundation for studying whether there is a feedback loop between Bmi-1/WWOX pathway.
4. To successfully establish subcutaneously transplantation tumor Model of human gallbladder carcinoma in BALB/c Nude Mice. To assess the therapeutic efficacy of targeting inhibition Bmi-1on transplanted tumor through in vivo experiment.to further investigate its impact on gallbladder cancer cell growth and molecular mechanism of regulating Bmi-1/WWOX/P73/PUMA/Bax/Bcl-2pathway, so that results in vitro can be verified.
5. Through clinical correlational research, experimental studies in vitro and in vivo on nude mice, to systematically describe effects and molecular mechanisms that Bmi-1regulates cell proliferation, apoptosis and cell cycle of human gallbladder cancer, and its WWOX signaling pathway, which will help screening markers of gallbladder cancer, assessment of prognosis, provide a theoretical basis and experimental data for the early diagnosis of gallbladder cancer and its molecule targeted therapy.
[Methods]
1. Clinical correlative research
55cases of patients with primary gallbladder adenocarcinoma of complete the clinicopathological data and its surgical specimens to keep in the archives with paraffin blocks were collected.30cases with chronic cholecystitis tissue with mild-moderate atypical hyperplasia from the same time period and22cases with normal gallbladder tissues from patients of the right hemihepatectomy with the calculus of intrahepatic duct or with liver tumor, which without stones and tumors invasion in the gallbladder were collected as controls.Five cases with fresh tissues were included In each group. Bmi-1/WWOX/PUMA mRNA and protein expression difference in the above specimens were determined by IHC-MaxVision, RT-PCR and Real-time PCR. The relationship between the protein and mRNA expression differences in three groups and the clinicopathological factors was analyzed.
2. In vitro studies, with proto-oncogene Bmi-1mRNA as a target
To successfully construct shRNA-Bmi-1recombinant vector and transfect it into gallbladder cancer GBC-SD cells by RNAi interference technology in vitro experiment. The transfection efficiencies were evaluated using an inverted fluorescence microscope and flow cytometry. The change levels of transcription and translation of gallbladder cancer cells after transfection were determined by RT-PCR and Westernblot assay. The best recombinant plasmid interfering Bmi-1target sequence was selected for subsequent experimental groups. Using an inverted microscope, Brdu essay, AnnexinV/7-AAD single and double-stained essay, transmission electron microscope and cell cycle analysis techniques, the influencing mechanisms of targeting shRNA inhibition of Bmi-1on gallbladder cancer cell proliferative activity, apoptosis progress and cell cycle and their relationship of biological effects and time gradient were investigated, to further explored the molecular mechanisms of regulation influence of targeting shRNA interfering with Bmi-1expression on WWOX/P73/PUMA/Bax/Bcl-2/Caspase-3pathways through Using Real-time PCR, Western Blot, immunofluorescence stain and flow cytometry techniques.
3. In vitro studies, with anti-oncogene WWOXmRNA as a target
The total RNA of GBC-SD cells was extracted. The complete WWOX sequence was amplified by RT-PCR and cloned into the eukaryotic expression vector pcDNA3.0. Through using restriction enzyme digesting and DNA sequencing to identify, the recombinant pcDNA3.0-WWOX eukaryotic expression vector was successfully constructed. GBC-SD cells were transfected by liposome-mediation, at same time, the empty vector and Liposomes was respectively transfected as a negative control and GBC-SD cells as a blank. The changes of transcription and translation levels of gallbladder cancer cells after transfection was determined by RT-PCR, Western blot and immunofluorescence techniques. Using MTT assay, Annexin V/7-AAD double staining, TUNNEL assay, cell cycle detection technology, transmission electron microscopy, mitochondrial transmembrane potential detection technique, the influencing mechanisms of over-expressed WWOX on proliferative activity, apoptosis and cell cycle of gallbladder cancer cell and their relationship with biological effects and time gradient were investigated. To further explored the molecular mechanisms of eukaryotic expression vector pCDNA3.0on regulating WWOX/P73/PUMA/Bax/Bcl-2pathways through Using Real-time PCR,Western Blot, immunofluorescence, and flow cytometry techniques.
4. In vivo studies
Based on results from in vitro studies, establishing subcutaneously transplantation tumor Model of human gallbladder carcinoma in BALB/c nude mice.By using shRNA-Bmi-1recombinant plasmid for multi-point injection within and around the transplantation tumor, the growth curve of the transplantation tumor was drawed, and the inhibition rate of the transplantation tumor volume was calculated at the end of the experiment.Through specimens anatomy and histopathology detection measure, anti-tumor effect of targeting shRNA interfering with Bmi-1expression on subcutaneously transplantation tumor in nude mice was explored.Through using Max Vision, TUNEL essay, transmission electron microscopy, RT-PCR and Western blot techniques, the molecular mechanisms were further investigated on the effect that Bmi-1regulated the gallbladder cancer cell growth and key gene expression of WWOX pathway.
[Results]
1. Results of clinical correlative studies
(1) IHC-MaxVision test results showed that the positive expressions of Bmi-1, WWOX and PUMA protein in gallbladder carcinoma tissue were respectively83.6%(46/55),38.2%(21/55) and34.5%(19/55). The positive expression of Bmi-1protein in gallbladder carcinoma tissue was significantly higher than chronic cholecystitis with atypical hyperplasia and normal gallbladder tissue, while the positive expression of WWOX and PUMA protein in gallbladder carcinoma tissue was significantly lower than chronic cholecystitis with atypical hyperplasia and normal gallbladder tissue (P <0.05).(2) Relationship between differential expression of Bmi-1, WWOX and PUMA and clinicopathological factors:Expression levels of Bmi-1and WWOX protein were correlated with the clinicopathological stage, tissue differentiation and lymph node metastasis of gallbladder cancer(P<0.05), but uncorrelated with gender, age, tumor size, tumor location, and existence of cirrhosis and gallstones(p>0.05). The expression level of PUMA protein was correlated with tissue differentiation and lymph node metastasis of gallbladder cancer (P<0.05), but uncorrelated with gender, age, tumor size, the clinicopathological stage, tumor location, and existence of cirrhosis and gallstones (p>0.05).(3) Real-time PCR and RT-PCR tests showed Bmi-lmRNA expression level is that gallbladder carcinoma>chronic cholecystitis with atypical hyperplasia> normal gallbladder tissue;while WWOX and PUMAmRNA expression levels are that gallbladder carcinomand PUMA genes might be involved in the occurrence and development of gallbladder cancer. Gallbladder carcinoma may occur if the persistent high expression of Bmi-1or the persistent lower expression of WWOX or PUMA in chronic cholecystitis with atypical hyperplasia tissue.
2. Results of in vitro study on the proto-oncogene Bmi-1mRNA as a target
(1) Constructing and screening results of shRNA-Bmi-lrecombinant vector:the amplified sequence of four groups of Bmi-1genes was proved to be correct by gene sequencing after recombinant vector shRNA-Bmi-1was constructed.The cell transfection efficiency was about70%using inverted fluorescence microscope and flow cytometry; After four groups plasmids were transfected into gallbladder GBC-SD cells for48h, the fourth group, in which the sequence had significant inhibition with the transcription and translation levels by RT-PCR and Westernblot essay, was selected as the subsequent experimental group (i.e. shRNA-Bmi-1group).
(2) The effect of targeting shRNA to interfere Bmi-1expression on gallbladder cancer cell proliferation, apoptosis and cell cycle:By Targeted inhibition of Bmi-1, the levels of Bmi-1mRNA and protein expression were significantly lower than the control groups determined by RT-PCR and Western blot (p<0.05). And there was no significant difference in the expression quantity between the control groups (p>0.05).The cell proliferation inhibition rate of shRNA-Bmi-1was very obvious over time than the control groups. And the strongest inhibitory effect and most death of cells were observed72h after transfection using inverted fluorescence microscope. Brdu tests show proliferation activity of gallbladder cancer cells in shRNA-Bmi-1group was lower than the control groups, and cell proliferation was time-dependent. The proliferation activity was significantly decreased with over time. The strongest inhibition of cell proliferation was observed72h after transfection (p<0.05). Annexin V/7-AAD single and double-stained show that cell apoptosis rate was significantly increased over time (24h<48h<72h) after transfected with shRNA-Bmi-1recombinant plasmid for24h,48h and72h. And late apoptotic rate increased mainly(p<0.05).The transmission electron microscope detections show concentration, pycnosis and became apoptotic body of gallbladder cells in shRNA-Bmi-1transfected group. Cell cycle detection show G0/G1phase of the cells were increased, while G2/M and S phase cells were decreased. The cells were blocked at the G0/G1phase and cell apoptosis rates were significantly increased and proliferation index were decreased(p<0.05).(3)Molecular mechanisms on targeting shRNA to interfere the expression of Bmi-1to regulate the WWOX pathway:Bmi-1mRNA and protein expression quantity and protein fluorescence intensity were decreased compared with the control groups, while expression quantities of WWOX, P73, PUMAmRNA and protein were significantly increased compared with the control groups and the expression intensity of the protein fluorescence was significantly enhanced in shRNA-Bmi-1transfection group determined by Real-time PCR, Western Blot essay, and immunofluorescence staining techniques (p<0.05); Flow cytometry technique analysis showed that protein expressions of Bax and Caspase-3of gallbladder cancer cells were increased compared with the control groups, while the protein expression of Bcl-2was decrease compared with the control groups (p<0.05).
3. The results of in vitro experiments with anti-oncogene WWOX mRNA as a target
(1) The construction of the WWOX gene eukaryotic expression vector:the amplified WWOX gene sequence was proved to be correct by double restriction enzyme digestion and gene sequencing of pcDNA3.0-WWOX recombinant plasmid.(2) The effect of pcDNA3.0-WWOX recombinant plasmid on gallbladder cancer cell proliferation, apoptosis and cell cycle:RT-PCR and Westernblot analysis showed WWOXmRNA and protein expression were significantly higher than the control groups48hours after transfection with pcDNA3.0-WWOX (p<0.05); There was no significant difference in WWOXmRNA and protein expression levels between the control groups (p>0.05). Immunofluorescence detection showed WWOX protein fluorescence intensity around the nucleus of gallbladder carcinoma cell in pcDNA3.0-WWOX group was stronger than the control groups, while the change in WWOX protein fluorescence intensity between the control groups was not significant. Inverted microscope observation found that the number of adherent GBC-SD cells in pcDNA3.0-WWOX group significantly reduced and suspended cells and cell debris increased, while it showed normal cell proliferation and good adherent growth in the control groups. MTT assay showed that cell proliferation of pcDNA3.0-WWOX group significantly decreased and the cell proliferation inhibition effect was more significant over time, while there was no significant inhibition of cell proliferation in the control groups (P<0.05). BrdU essay showed that the cell proliferation rate of pcDNA3.0-WWOX group was lower than the control groups (P<0.05). Annexin V/7-AAD double staining showed that the early and late apoptotic rates of gallbladder cancer cells in pcDNA3.0-WWOX group are higher than the control groups (p<0.05); while there was no significant difference in early and late apoptotic rates between the control groups (P>0.05). TUNNEL essay showed apoptosis ratio of gallbladder cancer cell increased compared with the control groups24h/48h/72h after transfection with the recombinant plasmid pcDNA3.0-WWOX, with more pronounced48h after transfection (p<0.05). Cell cycle analysis found that the G0/G1phase of gallbladder cancer cells in pcDNA3.0-WWOX group increased, the G2/M and S phase cells decreased, the apoptosis rate increased and proliferation index decreased (p<0.05); while there was no significant difference between the control groups (p>0.05). The transmembrane potential of chondriosome (ΔψFm) determined by JC-1staining showed the green fluorescence signal was significantly higher than the empty vector, liposomes and blank control group by overexpressed WWOX genes in the chondriosome of gallbladder cancer cells, suggesting that WWOX mainly induced early apoptosis of gallbladder cancer cells(p<0.05). The transmission electron microscope observed that typical apoptotic morphology of gallbladder cancer cells changed with pycnosis, nuclear fragmentation, formation of apoptotic bodies in the pcDNA3.0-WWOX group.(3) The effect of pcDNA-WWOX recombinant plasmid on the key gene of WWOX pathway:Real-time PCR, Western Blot and immunofluorescence assay showed that the expression levels of the transcription and translation of P73and PUMA in gallbladder cancer cells were higher than the control groups by WWOX expression upregulated pecifically (p<0.05); Flow cytometry essay showed that expression levels of Bax protein in gallbladder cancer cells were significantly higher than the control groups, while the expression levels of Bcl-2protein decreased compared with the control group by WWOX expression upregulated pecifically(p<0.05).
4. Results of in vivo experiments of transplantation tumors
The formation rate of the transplantation tumor was100%5-7days after nude mice inoculated subcutaneously with GBC-SD. The tumor growth in the shRNA-Bmi-1group was slower than the control groups. The tumor volume increased in each group after treatment for6weeks when the experiments were terminated, but the transplantation tumor volume in the shRNA-Bmi-1group was significantly smaller than the control groups (shRNA-Scramble group, Lipofectamine group, GBC-SD group). And its tumor inhibition rate was60.6%(p<0.05). There was no significant difference in the tumor volume between the control groups (P>0.05). HE staining essay showed obvious cell necrosis, fuzzy morphology, inflammatory cell infiltration, and unclear histological structure in the shRNA-Bmi-1group;while it showed obvious the cell heteromorphism, fewer inflammatory cells, a clear histological structure in the control groups. MaxVision stainings found that Ki67and VEGF protein expression of the transplantation tumor in the shRNA-Bmi-1group decreased compared with the control groups, suggesting reduced tumor angiogenesis and significantly inhibited cell proliferation in the shRNA-Bmi-1group. TUNEL and transmission electron microscope analysis showed that targeting shRNA to inhibit Bmi-1in vivo may induce gallbladder cancer cell apoptosis, which is consistent with the in vitro experiments. Real-time PCR, Western Blot and immunohistochemistry detection showed targeting shRNA interfering the Bmi-1expression can specifically promote Bmi-1mRNA degradation and inhibit the protein expression, and at the same time promote the expression of critical WWOX/P73/PUMA/Bax gene and inhibit the expression of Bcl-2gene in the downstream pathway on Bmi-1.
[Conclusions]
1. Bmi-1,WWOX and PUMA expression were involved in the occurrence and development process of gallbladder cancer. The increased positive expression rate of Bmi-1or decreased WWOX and PUMA positive expression rate or loss of expression indicate increased tumor malignancy and metastasis. The expression difference of Bmi-1,WWOX and PUMA may be involved in tumor invasion and progression.To combine detecting the expression of Bmi-1,WWOX and PUMA in gallbladder carcinoma tissues using immunohistochemical method was helpful to assist the early diagnosis and to help make a reasonable appraisal of their prognosis.
2. The shRNA-Bmi-1recombinant vector was successfully constructed in vitro, targeted silencing Bmi-1expression can effectively promote the degradation of Bmi-1mRNA and inhibit its protein expression in gallbladder cancer cells, specifically inhibit cell proliferation, induce apoptosis of gallbladder cancer cells, and arrest at G0/G1phase of cell cycle, resulting in DNA synthesis blocked in gallbladder cancer cells. Studies suggest that Bmi-1is involved in the regulation of proliferation and apoptosis of gallbladder cancer cells, and maintaining cell cycle operation.Bmi-1is an important factor inhibiting apoptosis and promoting abnormality cell proliferation. Meanwhile, targeting to silent the Bmi-1expression can effectively promote the expression of critical WWOX, P73, PUMA, Bax and Caspase-3gene and inhibit the expression of Bcl-2protein in the WWOX pathway, which may be related to the regulation mechanism of gallbladder cancer cell growth.Bmi-1and its Bmi-1/WWOX pathway was involved in the regulation of gallbladder cancer cell growth through the mitochondrial dependent apoptosis pathway.
3. The pcDNA3.0-WWOX eukaryotic expression vector was successfully constructed in vitro.The overexpression of WWOX gene can effectively inhibit degradation of WWOXmRNA, promote its protein expression in gallbladder cancer cells. It can effectively inhibit proliferation activity and induce the gallbladder cancer cell apoptosis. It can also block the cell cycle at the G0/G1phase. At the same time, it can up-regulate and promote the chelating accumulation of73protein in the cytoplasm of gallbladder cancer cells, promote the transcription and translation of PUMA and Bax, and inhibit the transcription and translation of Bcl-2, which may be one of important mechanisms regulating gallbladder cancer cell growth. WWOX/P73/PUMA pathway was involved in the regulation of gallbladder cancer cell proliferation, apoptosis and cell cycle changes through the mitochondrial dependent apoptosis pathway, which further verified the molecular mechanisms of WWOX pathway regulating gallbladder cancer cells.
4. The animal model of subcutaneous transplanted tumor of gallbladder cancer in nude mice was successfully constructed. Targeting to silent Bmi-1expression can induce the cell apoptosis of transplanted tumors of gallbladder cancers, significantly inhibit tumor growth and the formation of new vessels, and regulate the expression of key genes in Bmi-1/WWOX/P73/PUMA/Bax/Bcl-2pathway of subcutaneous transplanted tumor in nude mice. The findings are consistent with those by the in vitro experiments. It was further confirmed by in vivo experiments that Bmi-1/WWOX pathway is involved in the cell growth of gallbladder cancer through the mitochondrial dependent apoptosis pathway.
5. Bmi-1and WWOX may provide a potential molecular target for the gene targeted therapy of gallbladder cancer. It can be one of the potential therapeutic tools for gallbladder carcinoma combined with gene therapy through targeting to silent Bmi-1expression and specifically up-regulating the WWOX expression. Blocking the Bmi-1/WWOX pathway plays an important role in target therapeutic strategies for gallbladder cancer and also provides a new experimental basis for the diagnosis of early gallbladder cancer and multiple targeted molecular therapies for gallbladder cancer.
1.探讨Bmi-1、WWOX及PUMA基因在胆囊癌组织和慢性胆囊炎伴轻-中度非典型增生组织及正常胆囊组织中的表达差异,分析Bmi-1/WWOX/PUMA表达与胆囊癌临床病理因素的关系,有望揭示其表达差异在胆囊癌发生、发展中的作用及其临床意义,为后续研究胆囊癌的发生机制奠定基础。
2.通过体外RNAi干扰技术成功构建靶向shRNA-Bmi-1重组载体抑制原癌基因Bmi-1表达,观察其对胆囊癌细胞增殖、凋亡及细胞周期的效应,探讨Bmi-1调控WWOX/P73/PUMA/Bax/Bcl-2/Caspase-3信号通路调节胆囊癌细胞生长的分子机制。
3.通过体外成功构建pcDNA3.0-WWOX真核表达载体,探讨过表达WWOX基因对胆囊癌细胞增殖,凋亡及细胞周期的影响,进一步研究WWOX/P73/PUMA/Bax/Bcl-2通路调控胆囊癌细胞生长的分子机制,为研究Bmi-1/WWOX通路间是否存有反馈环路奠定基础。
4.成功建立了人胆囊癌BALB/C裸鼠皮下移植瘤模型,通过体内试验评估靶向抑制Bmi-1对移植瘤的治疗效果,进一步探讨其对胆囊癌细胞生长的影响及调控Bmi-1/WWOX/P73/PUMA/Bax/Bcl-2通路的分子机制,验证体外实验结果。
5.本课题通过临床相关研究,体外及裸鼠体内试验研究,全面系统阐述Bmi-1调控人胆囊癌细胞增殖、凋亡及细胞周期的效应及其WWOX信号通路的分子机制,将有助于胆囊癌标志物的筛选及病情的预后和评估,为胆曩癌的早期诊断、分子靶向治疗提供理论依据和实验数据。
[研究方法]
1.临床相关研究：收集临床病理资料完整的原发性胆囊腺癌病人手术标本存档石蜡块55例,收集同期慢性胆囊炎伴轻-中度非典型增生组织标本30例及正常胆囊组织标本22例(均来自于肝内胆管结石或肝脏肿瘤行右半肝切除患者,胆囊内无结石及肿瘤侵袭)作对照,其中每组病例包括新鲜组织各5例。应用IHC-Max Vision法、RT-PCR及Real-time PCR等技术检测上述标本中Bmi-1/WWOX/PUMA mRNA及蛋白的表达差异,分析三者表达与胆囊癌临床病理因素的关系及意义。
2.体外研究,以原癌基因Bmi-1mRNA为靶点：通过体外RNAi干扰技术成功构建shRNA-Bmi-1重组载体转染胆囊癌GBC-SD细胞,应用倒置荧光显微镜及流式细胞技术评价其转染效率,以RT-PCR及Westernblot检测转染后胆囊癌细胞转录和翻译水平的变化,筛选出最佳干扰作用的Bmi-1靶序列重组质粒为后续实验组。通过倒置显微镜观察、Brdu实验、Annexin V/7-AAD单染和双染实验、透射电镜及细胞周期检测技术分析靶向shRNA抑制Bmi-1对胆囊癌细胞增殖活性、凋亡进程及细胞周期的影响机制及其生物学效应与时间梯度的关系,进一步通过Real-time PCR, Western Blot,免疫荧光染色及流式细胞检测等技术探讨靶向shRNA抑制Bmi-1表达对WWOX/P73/PUMA/Bax/Bcl-2/Caspase-3通路影响的分子机制。
3.体外研究,以抑癌基因WWOXmRNA为靶点：提取GBC-SD细胞总RNA,采用RT-PCR扩增WWOX全序列,将其克隆到真核表达载体pCDNA3.0中,通过限制性内切酶酶切及DNA测序鉴定,成功构建pcDNA3.0-WWOX重组真核表达载体。按脂质体介导转染GBC-SD细胞,同时转染空载体、脂质体为阴性对照及GBC-SD细胞为空白对照。以RT-PCR、Westemblot及免疫荧光技术检测转染后胆囊癌细胞转录和翻译水平的变化,以MTT法、AnnexinV/7-AAD双染、TUNNEL法、细胞周期检测技术、透射电镜技术、线粒体跨膜电位检测技术分析过表达WWOX对胆囊癌细胞增殖活性、凋亡及细胞周期的影响机制及其生物学效应与时间梯度的关系,进一步通过Real-time PCR, Western Blot,免疫荧光染色及流式细胞检测等技术探讨pcDNA3.0-WWOX真核表达载体对WWOX/P73/PUMA/Bax/Bcl-2通路调控的分子机制。
4.体内试验研究：以体外实验结果为基础,通过建立人胆囊癌BALB/C裸鼠皮下移植瘤模型,应用shRNA-Bmi-1重组质粒在移植瘤周围及瘤内多点注射,实验终止绘制移植瘤生长曲线,计算瘤体抑制率,通过标本解剖和组织病理检测手段探讨靶向shRNA抑制Bmi-1表达对裸鼠皮F移植瘤的抗瘤效果,通过Max Vision去、TUNEL法、透射电镜、RT-PCR及Western Blot等检测技术进一步探讨Bmi-1对胆囊癌细胞生长的影响及调控WWOX通路关键基因表达的分子机制。
[研究结果1
1.临床相关研究结果
(1)IHC-MaxVision检测结果显示：Bmi-1、WWOX及PUMA蛋白在胆囊癌组织中的阳性表达分别为83.6%(46/55)、38.2%(21/55)和34.5%(19/55),其中Bmi-1蛋白在胆囊癌组织中的阳性表达明显高于慢性胆囊炎伴非典型增生组织及正常胆囊组织,而WWOX及PUMA蛋白在胆囊癌组织中的阳性表达明显低于慢性胆囊炎伴非典型增生组织及正常胆囊组织(P<0.05)。(2)Bmi-1、WWOX及PUMA表达差异与临床病理因素的关系结果：Bmi-1、WWOX蛋白表达水平与胆囊癌的病理分期、分化程度及淋巴结转移有关(P<0.05),与胆囊癌患者性别、年龄、肿瘤大小、肿瘤部位、是否伴有肝硬化及胆囊结石无相关性(p>0.05)。PUMA蛋白表达水平与胆囊癌分化程度及淋巴结转移有关(P<0.05),而与患者性别、年龄、肿瘤大小、临床病理分期、肿瘤部位、是否伴有肝硬化及胆囊结石均无相关性(p>0.05)。(3) Realtime PCR和RT-PCR检测结果：Bmi-1mRNA表达为胆囊癌组织>慢性胆囊炎伴非典型增生组织>正常胆囊组织；而WWOX及PUMAmRNA表达为胆囊癌组织<慢性胆囊炎伴非典型增生组织<正常胆囊组织(P<0.05)。实验表明Bmi-1、WWOX及PUMA基因可能参与胆囊癌发生、发展过程,慢性胆囊炎伴非典型增生组织中Bmi-1持续高表达或WWOX/PUMA持续低表达警示胆囊癌发生的可能。
2.以原癌基因Bmi-1mRNA为靶点的体外研究结果
(1) shRNA-Bmi-1重组载体的构建及筛选结果：shRNA-Bmi-1重组载体构建后经基因测序证明所获得的4组Bmi-1基因扩增序列正确。应用倒置荧光显微镜及流式细胞技术检测细胞转染效率70%左右；将4组质粒分别转染胆囊癌GBC-SD细胞48h后,通过RT-PCR及Westernblot检测筛选出第四组序列在转录和翻译水平抑制显著,为后续实验组(即为shRNA-Bmi-1组)。
(2)靶向shRNA干扰Bmi-1表达对胆囊癌细胞增殖、凋亡及细胞周期的影响结果：通过RT-PCR及Westernblot检测显示靶向抑制Bmi-1,其mRNA及蛋白表达水平均明显低于对照组(p<0.05)；而对照组间表达量无显著差异(p>0.05)。倒置显微镜观察发现shRNA-Bmi-1对胆囊癌细胞的影响随时间的推移,增殖抑制率较对照组明显,其中以转染后72h抑制效果最显著,细胞死亡最多。Brdu检测显示shRNA-Bmi-1组细胞增殖活性较对照组降低,细胞增殖力呈时间依存性,随时间推移增殖力明显减弱,于转染72h细胞增殖抑制最强(p<0.05)。AnnexinV/7-AAD单染和双染检测显示转染shRNA-Bmi-1重组质粒24h-48h-72h后,shRNA-Bmi-1组细胞凋亡率均随时间推移明显增加(24h<48h<72h),且与晚期凋亡增加为主(p<0.05)。透射电镜检测发现shRNA-Bmi-1转染组胆囊癌细胞浓缩、核固缩形成凋亡小体。细胞周期检测显示shRNA-Bmi-1转染组G0/G1期细胞增多,G2/M和S期细胞减少,细胞阻滞于G0/G1,细胞凋亡率明显增高,增殖指数降低(p<0.05)。
(3)靶向shRNA干扰Bmi-1表达调控WWOX通路的分子机制研究结果：通过Real-time PCR、Western Blot及免疫荧光染色技术检测显示shRNA-Bmi-1转染组胆囊癌细胞Bmi-1mRNA及蛋白表达量及蛋白荧光强度较对照组减少,而WWOX、P73、PUMAmRNA及蛋白表达量较对照组均明显增加,蛋白荧光表达强度明显增强(p<0.05)；流式细胞仪技术检测显示shRNA-Bmi-1转染组胆囊癌细胞Bax及Caspase-3蛋白表达较对照组增加,而Bcl-2蛋白表达较对照组减少(p<0.05)。
3.以抑癌基因WWOX mRNA为靶点的体外实验研究结果
(1)WWOX基因真核表达载体的构建：pcDNA3.0-WWOX重组质粒经双酶切鉴定及基因测序证明所获得的WWOX基因扩增序列正确。(2)pcDNA3.0-WWOX重组质粒对胆囊癌细胞增殖、凋亡及细胞周期影响结果：RT-PCR及Westernblot检测显示pcDNA3.0-WWOX转染48小时后WWOXmRNA及蛋白表达水平均明显高于对照组(p<0.05)；对照组间mRNA及蛋白表达量无显著差异(p>0.05)。免疫荧光检测结果显示pcDNA3.0-WWOX组细胞WWOX蛋白荧光强度在细胞核周围明显强于各对照组,而对照组间细胞WWOX蛋白荧光强度变化不明显。倒置显微镜观察发现pcDNA3.0-WWOX组贴壁的GBC-SD细胞数量明显减少,悬浮细胞及细胞碎片增加,而对照组细胞呈正常增殖状态,贴壁生长良好。MTT法检测显示pcDNA3.0-WWOX组细胞增殖活性明显降低,且随时间推移细胞增殖抑制效果更为显著,对照组细胞增殖抑制不明显(P<0.05)。BrdU检测显示pcDNA3.0-WWOX组细胞增殖率较对照组降低(P<0.05)。Annexin V/7-AAD双染检测发现pcDNA3.0-WWOX组胆囊癌细胞早、晚期凋亡率较对照组增高(p<0.05)；而对照组间早、晚期凋亡率未见显著差异(P>0.05)。TUNNEL法检测显示转染pcDNA3.0-WWOX重组质粒24h/48h/72h后,胆囊癌细胞凋亡比率较对照组增加,以转染48h后更为明显(p<0.05)。细胞周期检测发现pcDNA3.0-WWOX组G0/G1期细胞增多,G2/M和S期细胞减少,细胞凋亡率增高,增殖指数降低(p<0.05)；而对照组间未见显著性差异(p>0.05)。JC-1染色检测细胞线粒体跨膜电位(△Ψm)显示过表达WWOX基因胆囊癌细胞线粒体内绿色荧光信号显著高于空载体、脂质体及空白对照组,提示WWOX诱导胆囊癌细胞早期凋亡为主(p<0.05)。透射电镜观察pcDNA3.0-WWOX组细胞典型的凋亡形态学改变,出现核固缩、碎裂形成凋亡小体。(3)pcDNA-WWOX重组质粒对WWOX通路关键基因的影响结果：通过Real-time PCR,Western Blot及免疫荧光染色检测显示特异性上调WWOX表达,胆囊癌细胞中P73、PUMA在转录及翻译水平的表达量均较对照组增加(p<0.05)；流式细胞仪检测发现特异性上调WWOX表达,胆囊癌细胞中Bax蛋白表达水平较对照组明显增加,而Bcl-2蛋白表达水平较对照组减少(p<0.05)。
4.移植瘤体内试验研究结果
裸鼠皮下接种GBC-SD细胞株5-7天后成瘤率为100%。shRNA-Bmi-1组移植瘤生长速度较对照组减慢,治疗6周实验终止时各实验组体积较治疗前增大,但shRNA-Bmi-1组移植瘤体积明显小于各对照组(shRNA-Scramble组、Lipofectamine组、GBC-SD组),其瘤体抑制率为60.6%(p<0.05),对照组间体积无明显差异(P>0.05)。HE染色显示shRNA-Bmi-1组细胞坏死明显,细胞形态模糊,见炎性细胞浸润,组织结构不清晰,而各对照组细胞异型性明显,炎性细胞较少,组织结构清晰。MaxVision染色发现shRNA-Bmi-1组移植瘤组织中Ki67及VEGF蛋白表达较对照组减弱,提示shRNA-Bmi-1组肿瘤新生血管生成减少、细胞增殖抑制明显。TUNEL及透射电镜检测显示靶向shRNA抑制Bmi-1体内可诱导胆囊癌细胞凋亡,与体外实验相符合。通过Real-time PCR、Western Blot及免疫组织化学检测发现靶向shRNA干扰Bmi-1表达可特异性促进Bmi-1mRNA的降解及抑制其蛋白的表达,同时促进下游通路中关键基因WWOX/P73/PUMA/Bax表达,抑制Bcl-2的表达水平。
[研究结论]
1. Bmi-1、WWOX及PUMA的表达参与胆囊癌的发生发展过程,Bmi-1阳性表达率越高,或WWOX、PUMA阳性表达越低或表达缺失,提示肿瘤恶性程度增加、转移增快,Bmi-1、WWOX及PUMA的表达差异可能参与肿瘤的侵袭及演进。应用免疫组化联合检测胆囊癌组织中的Bmi-1、WWOX及PUMA的表达将有利于协助胆囊癌的早期诊断,有助于对其预后进行合理评估。
2.体外成功构建了shRNA-Bmi-1重组载体。靶向沉默Bmi-1表达能有效促进胆囊癌细胞Bmi-1mRNA降解及抑制其蛋白表达,能特异性抑制胆囊癌细胞增殖,诱导其凋亡,使细胞周期阻滞于G0/G1期,从而引起细胞DNA合成受阻,研究提示Bmi-1参与调控胆囊癌细胞的增殖和凋亡,维持细胞周期的运行,是抑制细胞凋亡和促进细胞异常增殖的重要因素。同时靶向沉默Bmi-1表达能有效促进WWOX通路中的关键基因WWOX/P73/PUMA/Bax/Caspase-3的表达,抑制Bcl-2蛋白表达,其作用可能与调控胆囊癌细胞生长效应的机制相关,Bmi-1及其Bmi-1/WWOX通路参与线粒体依赖的凋亡途径调控胆囊癌细胞的生长。
3.体外成功构建了pcDNA3.0-WWOX真核表达载体。过表达WWOX可有效抑制胆囊癌细胞WWOXmRNA降解、促进其蛋白表达,能有效抑制胆囊癌细胞的增殖活性、诱导其凋亡,使细胞周期阻滞于G0/G1期。同时上调并促进P73蛋白在胆囊癌细胞质中螯合积累,促进PUMA、Bax及抑制Bcl-2的转录和翻译过程,其作用可能是调控胆囊癌细胞生长的重要机制之一。WWOX/P73/PUMA通路通过调控线粒体依赖的凋亡途径来调节胆囊癌细胞的增殖、凋亡及细胞周期的变化,进一步证实了WWOX通路调控胆囊癌细胞的分子机制。
4.成功建立胆囊癌裸鼠皮下移植瘤动物模型。靶向沉默Bmi-1表达能诱导胆囊癌移植瘤细胞凋亡,显著抑制肿瘤的生长及新生血管的形成,影响裸鼠皮下移植瘤Bmi-1/WWOX/P73/PUMA/Bax/Bcl-2通路关键基因的表达,与体外实验相符合。体内实验进一步验证了Bmi-1/WWOX通路参与线粒体依赖的凋亡途径调控胆囊癌细胞的生长。
5.Bmi-1及WWOX可能为胆囊癌基因靶向治疗提供潜在的分子靶标,通过靶向沉默Bmi-1表达和特异性性上调WWOX的表达可能是胆囊癌联合基因治疗的潜在治疗手段之一,阻断Bmi-1/WWOX通路对胆囊癌靶向性治疗策略具有重要意义,同时也为探索胆囊癌早期诊断及多分子联合靶向治疗提供了新的实验依据。
[Objective]
1. To explore the expressions differences of the Bmi-1, WWOX and PUMA gene in gallbladder carcinoma, chronic cholecystitis tissue with mild-moderate atypical hyperplasia and normal gallbladder tissues, and to analyze the relationship between Bmi-1, WWOX and PUMA gene expressions and clinicopathological features of gallbladder cancer, and to reveal the role and Clinical significance of their expression differences in the occurrence and development process of gallbladder cancer, so as to lay the foundation for the follow-up study on the occurrence mechanism of gallbladder cancer.
2. To successfully construct targeting shRNA-Bmi-1recombinant vector to inhibit proto-oncogene Bmi-1expression by RNAi interference techniques in vitro experiment; to observe its effects on gallbladder cancer cell proliferation, apoptosis and cell cycle; To explore the molecular mechanisms of Bmi-1regulating gallbladder cancer cell growth through regulating WWOX/P73/PUMA/Bax/Bcl-2/Caspase-3signaling pathways.
3. By successfully constructed pcDNA3.0-WWOX eukaryotic expression vector in vitro experiment, To explored the effects of WWOX gene overexpression on gallbladder cell proliferation, apoptosis and cell cycle, and to further study molecular mechanisms that WWOX/P73/PUMA/Bax/Bcl-2pathway regulates the cell growth of gallbladder cancers, and to lay a foundation for studying whether there is a feedback loop between Bmi-1/WWOX pathway.
4. To successfully establish subcutaneously transplantation tumor Model of human gallbladder carcinoma in BALB/c Nude Mice. To assess the therapeutic efficacy of targeting inhibition Bmi-1on transplanted tumor through in vivo experiment.to further investigate its impact on gallbladder cancer cell growth and molecular mechanism of regulating Bmi-1/WWOX/P73/PUMA/Bax/Bcl-2pathway, so that results in vitro can be verified.
5. Through clinical correlational research, experimental studies in vitro and in vivo on nude mice, to systematically describe effects and molecular mechanisms that Bmi-1regulates cell proliferation, apoptosis and cell cycle of human gallbladder cancer, and its WWOX signaling pathway, which will help screening markers of gallbladder cancer, assessment of prognosis, provide a theoretical basis and experimental data for the early diagnosis of gallbladder cancer and its molecule targeted therapy.
[Methods]
1. Clinical correlative research
55cases of patients with primary gallbladder adenocarcinoma of complete the clinicopathological data and its surgical specimens to keep in the archives with paraffin blocks were collected.30cases with chronic cholecystitis tissue with mild-moderate atypical hyperplasia from the same time period and22cases with normal gallbladder tissues from patients of the right hemihepatectomy with the calculus of intrahepatic duct or with liver tumor, which without stones and tumors invasion in the gallbladder were collected as controls.Five cases with fresh tissues were included In each group. Bmi-1/WWOX/PUMA mRNA and protein expression difference in the above specimens were determined by IHC-MaxVision, RT-PCR and Real-time PCR. The relationship between the protein and mRNA expression differences in three groups and the clinicopathological factors was analyzed.
2. In vitro studies, with proto-oncogene Bmi-1mRNA as a target
To successfully construct shRNA-Bmi-1recombinant vector and transfect it into gallbladder cancer GBC-SD cells by RNAi interference technology in vitro experiment. The transfection efficiencies were evaluated using an inverted fluorescence microscope and flow cytometry. The change levels of transcription and translation of gallbladder cancer cells after transfection were determined by RT-PCR and Westernblot assay. The best recombinant plasmid interfering Bmi-1target sequence was selected for subsequent experimental groups. Using an inverted microscope, Brdu essay, AnnexinV/7-AAD single and double-stained essay, transmission electron microscope and cell cycle analysis techniques, the influencing mechanisms of targeting shRNA inhibition of Bmi-1on gallbladder cancer cell proliferative activity, apoptosis progress and cell cycle and their relationship of biological effects and time gradient were investigated, to further explored the molecular mechanisms of regulation influence of targeting shRNA interfering with Bmi-1expression on WWOX/P73/PUMA/Bax/Bcl-2/Caspase-3pathways through Using Real-time PCR, Western Blot, immunofluorescence stain and flow cytometry techniques.
3. In vitro studies, with anti-oncogene WWOXmRNA as a target
The total RNA of GBC-SD cells was extracted. The complete WWOX sequence was amplified by RT-PCR and cloned into the eukaryotic expression vector pcDNA3.0. Through using restriction enzyme digesting and DNA sequencing to identify, the recombinant pcDNA3.0-WWOX eukaryotic expression vector was successfully constructed. GBC-SD cells were transfected by liposome-mediation, at same time, the empty vector and Liposomes was respectively transfected as a negative control and GBC-SD cells as a blank. The changes of transcription and translation levels of gallbladder cancer cells after transfection was determined by RT-PCR, Western blot and immunofluorescence techniques. Using MTT assay, Annexin V/7-AAD double staining, TUNNEL assay, cell cycle detection technology, transmission electron microscopy, mitochondrial transmembrane potential detection technique, the influencing mechanisms of over-expressed WWOX on proliferative activity, apoptosis and cell cycle of gallbladder cancer cell and their relationship with biological effects and time gradient were investigated. To further explored the molecular mechanisms of eukaryotic expression vector pCDNA3.0on regulating WWOX/P73/PUMA/Bax/Bcl-2pathways through Using Real-time PCR,Western Blot, immunofluorescence, and flow cytometry techniques.
4. In vivo studies
Based on results from in vitro studies, establishing subcutaneously transplantation tumor Model of human gallbladder carcinoma in BALB/c nude mice.By using shRNA-Bmi-1recombinant plasmid for multi-point injection within and around the transplantation tumor, the growth curve of the transplantation tumor was drawed, and the inhibition rate of the transplantation tumor volume was calculated at the end of the experiment.Through specimens anatomy and histopathology detection measure, anti-tumor effect of targeting shRNA interfering with Bmi-1expression on subcutaneously transplantation tumor in nude mice was explored.Through using Max Vision, TUNEL essay, transmission electron microscopy, RT-PCR and Western blot techniques, the molecular mechanisms were further investigated on the effect that Bmi-1regulated the gallbladder cancer cell growth and key gene expression of WWOX pathway.
[Results]
1. Results of clinical correlative studies
(1) IHC-MaxVision test results showed that the positive expressions of Bmi-1, WWOX and PUMA protein in gallbladder carcinoma tissue were respectively83.6%(46/55),38.2%(21/55) and34.5%(19/55). The positive expression of Bmi-1protein in gallbladder carcinoma tissue was significantly higher than chronic cholecystitis with atypical hyperplasia and normal gallbladder tissue, while the positive expression of WWOX and PUMA protein in gallbladder carcinoma tissue was significantly lower than chronic cholecystitis with atypical hyperplasia and normal gallbladder tissue (P <0.05).(2) Relationship between differential expression of Bmi-1, WWOX and PUMA and clinicopathological factors:Expression levels of Bmi-1and WWOX protein were correlated with the clinicopathological stage, tissue differentiation and lymph node metastasis of gallbladder cancer(P<0.05), but uncorrelated with gender, age, tumor size, tumor location, and existence of cirrhosis and gallstones(p>0.05). The expression level of PUMA protein was correlated with tissue differentiation and lymph node metastasis of gallbladder cancer (P<0.05), but uncorrelated with gender, age, tumor size, the clinicopathological stage, tumor location, and existence of cirrhosis and gallstones (p>0.05).(3) Real-time PCR and RT-PCR tests showed Bmi-lmRNA expression level is that gallbladder carcinoma>chronic cholecystitis with atypical hyperplasia> normal gallbladder tissue;while WWOX and PUMAmRNA expression levels are that gallbladder carcinoma
2. Results of in vitro study on the proto-oncogene Bmi-1mRNA as a target
(1) Constructing and screening results of shRNA-Bmi-lrecombinant vector:the amplified sequence of four groups of Bmi-1genes was proved to be correct by gene sequencing after recombinant vector shRNA-Bmi-1was constructed.The cell transfection efficiency was about70%using inverted fluorescence microscope and flow cytometry; After four groups plasmids were transfected into gallbladder GBC-SD cells for48h, the fourth group, in which the sequence had significant inhibition with the transcription and translation levels by RT-PCR and Westernblot essay, was selected as the subsequent experimental group (i.e. shRNA-Bmi-1group).
(2) The effect of targeting shRNA to interfere Bmi-1expression on gallbladder cancer cell proliferation, apoptosis and cell cycle:By Targeted inhibition of Bmi-1, the levels of Bmi-1mRNA and protein expression were significantly lower than the control groups determined by RT-PCR and Western blot (p<0.05). And there was no significant difference in the expression quantity between the control groups (p>0.05).The cell proliferation inhibition rate of shRNA-Bmi-1was very obvious over time than the control groups. And the strongest inhibitory effect and most death of cells were observed72h after transfection using inverted fluorescence microscope. Brdu tests show proliferation activity of gallbladder cancer cells in shRNA-Bmi-1group was lower than the control groups, and cell proliferation was time-dependent. The proliferation activity was significantly decreased with over time. The strongest inhibition of cell proliferation was observed72h after transfection (p<0.05). Annexin V/7-AAD single and double-stained show that cell apoptosis rate was significantly increased over time (24h<48h<72h) after transfected with shRNA-Bmi-1recombinant plasmid for24h,48h and72h. And late apoptotic rate increased mainly(p<0.05).The transmission electron microscope detections show concentration, pycnosis and became apoptotic body of gallbladder cells in shRNA-Bmi-1transfected group. Cell cycle detection show G0/G1phase of the cells were increased, while G2/M and S phase cells were decreased. The cells were blocked at the G0/G1phase and cell apoptosis rates were significantly increased and proliferation index were decreased(p<0.05).(3)Molecular mechanisms on targeting shRNA to interfere the expression of Bmi-1to regulate the WWOX pathway:Bmi-1mRNA and protein expression quantity and protein fluorescence intensity were decreased compared with the control groups, while expression quantities of WWOX, P73, PUMAmRNA and protein were significantly increased compared with the control groups and the expression intensity of the protein fluorescence was significantly enhanced in shRNA-Bmi-1transfection group determined by Real-time PCR, Western Blot essay, and immunofluorescence staining techniques (p<0.05); Flow cytometry technique analysis showed that protein expressions of Bax and Caspase-3of gallbladder cancer cells were increased compared with the control groups, while the protein expression of Bcl-2was decrease compared with the control groups (p<0.05).
3. The results of in vitro experiments with anti-oncogene WWOX mRNA as a target
(1) The construction of the WWOX gene eukaryotic expression vector:the amplified WWOX gene sequence was proved to be correct by double restriction enzyme digestion and gene sequencing of pcDNA3.0-WWOX recombinant plasmid.(2) The effect of pcDNA3.0-WWOX recombinant plasmid on gallbladder cancer cell proliferation, apoptosis and cell cycle:RT-PCR and Westernblot analysis showed WWOXmRNA and protein expression were significantly higher than the control groups48hours after transfection with pcDNA3.0-WWOX (p<0.05); There was no significant difference in WWOXmRNA and protein expression levels between the control groups (p>0.05). Immunofluorescence detection showed WWOX protein fluorescence intensity around the nucleus of gallbladder carcinoma cell in pcDNA3.0-WWOX group was stronger than the control groups, while the change in WWOX protein fluorescence intensity between the control groups was not significant. Inverted microscope observation found that the number of adherent GBC-SD cells in pcDNA3.0-WWOX group significantly reduced and suspended cells and cell debris increased, while it showed normal cell proliferation and good adherent growth in the control groups. MTT assay showed that cell proliferation of pcDNA3.0-WWOX group significantly decreased and the cell proliferation inhibition effect was more significant over time, while there was no significant inhibition of cell proliferation in the control groups (P<0.05). BrdU essay showed that the cell proliferation rate of pcDNA3.0-WWOX group was lower than the control groups (P<0.05). Annexin V/7-AAD double staining showed that the early and late apoptotic rates of gallbladder cancer cells in pcDNA3.0-WWOX group are higher than the control groups (p<0.05); while there was no significant difference in early and late apoptotic rates between the control groups (P>0.05). TUNNEL essay showed apoptosis ratio of gallbladder cancer cell increased compared with the control groups24h/48h/72h after transfection with the recombinant plasmid pcDNA3.0-WWOX, with more pronounced48h after transfection (p<0.05). Cell cycle analysis found that the G0/G1phase of gallbladder cancer cells in pcDNA3.0-WWOX group increased, the G2/M and S phase cells decreased, the apoptosis rate increased and proliferation index decreased (p<0.05); while there was no significant difference between the control groups (p>0.05). The transmembrane potential of chondriosome (ΔψFm) determined by JC-1staining showed the green fluorescence signal was significantly higher than the empty vector, liposomes and blank control group by overexpressed WWOX genes in the chondriosome of gallbladder cancer cells, suggesting that WWOX mainly induced early apoptosis of gallbladder cancer cells(p<0.05). The transmission electron microscope observed that typical apoptotic morphology of gallbladder cancer cells changed with pycnosis, nuclear fragmentation, formation of apoptotic bodies in the pcDNA3.0-WWOX group.(3) The effect of pcDNA-WWOX recombinant plasmid on the key gene of WWOX pathway:Real-time PCR, Western Blot and immunofluorescence assay showed that the expression levels of the transcription and translation of P73and PUMA in gallbladder cancer cells were higher than the control groups by WWOX expression upregulated pecifically (p<0.05); Flow cytometry essay showed that expression levels of Bax protein in gallbladder cancer cells were significantly higher than the control groups, while the expression levels of Bcl-2protein decreased compared with the control group by WWOX expression upregulated pecifically(p<0.05).
4. Results of in vivo experiments of transplantation tumors
The formation rate of the transplantation tumor was100%5-7days after nude mice inoculated subcutaneously with GBC-SD. The tumor growth in the shRNA-Bmi-1group was slower than the control groups. The tumor volume increased in each group after treatment for6weeks when the experiments were terminated, but the transplantation tumor volume in the shRNA-Bmi-1group was significantly smaller than the control groups (shRNA-Scramble group, Lipofectamine group, GBC-SD group). And its tumor inhibition rate was60.6%(p<0.05). There was no significant difference in the tumor volume between the control groups (P>0.05). HE staining essay showed obvious cell necrosis, fuzzy morphology, inflammatory cell infiltration, and unclear histological structure in the shRNA-Bmi-1group;while it showed obvious the cell heteromorphism, fewer inflammatory cells, a clear histological structure in the control groups. MaxVision stainings found that Ki67and VEGF protein expression of the transplantation tumor in the shRNA-Bmi-1group decreased compared with the control groups, suggesting reduced tumor angiogenesis and significantly inhibited cell proliferation in the shRNA-Bmi-1group. TUNEL and transmission electron microscope analysis showed that targeting shRNA to inhibit Bmi-1in vivo may induce gallbladder cancer cell apoptosis, which is consistent with the in vitro experiments. Real-time PCR, Western Blot and immunohistochemistry detection showed targeting shRNA interfering the Bmi-1expression can specifically promote Bmi-1mRNA degradation and inhibit the protein expression, and at the same time promote the expression of critical WWOX/P73/PUMA/Bax gene and inhibit the expression of Bcl-2gene in the downstream pathway on Bmi-1.
[Conclusions]
1. Bmi-1,WWOX and PUMA expression were involved in the occurrence and development process of gallbladder cancer. The increased positive expression rate of Bmi-1or decreased WWOX and PUMA positive expression rate or loss of expression indicate increased tumor malignancy and metastasis. The expression difference of Bmi-1,WWOX and PUMA may be involved in tumor invasion and progression.To combine detecting the expression of Bmi-1,WWOX and PUMA in gallbladder carcinoma tissues using immunohistochemical method was helpful to assist the early diagnosis and to help make a reasonable appraisal of their prognosis.
2. The shRNA-Bmi-1recombinant vector was successfully constructed in vitro, targeted silencing Bmi-1expression can effectively promote the degradation of Bmi-1mRNA and inhibit its protein expression in gallbladder cancer cells, specifically inhibit cell proliferation, induce apoptosis of gallbladder cancer cells, and arrest at G0/G1phase of cell cycle, resulting in DNA synthesis blocked in gallbladder cancer cells. Studies suggest that Bmi-1is involved in the regulation of proliferation and apoptosis of gallbladder cancer cells, and maintaining cell cycle operation.Bmi-1is an important factor inhibiting apoptosis and promoting abnormality cell proliferation. Meanwhile, targeting to silent the Bmi-1expression can effectively promote the expression of critical WWOX, P73, PUMA, Bax and Caspase-3gene and inhibit the expression of Bcl-2protein in the WWOX pathway, which may be related to the regulation mechanism of gallbladder cancer cell growth.Bmi-1and its Bmi-1/WWOX pathway was involved in the regulation of gallbladder cancer cell growth through the mitochondrial dependent apoptosis pathway.
3. The pcDNA3.0-WWOX eukaryotic expression vector was successfully constructed in vitro.The overexpression of WWOX gene can effectively inhibit degradation of WWOXmRNA, promote its protein expression in gallbladder cancer cells. It can effectively inhibit proliferation activity and induce the gallbladder cancer cell apoptosis. It can also block the cell cycle at the G0/G1phase. At the same time, it can up-regulate and promote the chelating accumulation of73protein in the cytoplasm of gallbladder cancer cells, promote the transcription and translation of PUMA and Bax, and inhibit the transcription and translation of Bcl-2, which may be one of important mechanisms regulating gallbladder cancer cell growth. WWOX/P73/PUMA pathway was involved in the regulation of gallbladder cancer cell proliferation, apoptosis and cell cycle changes through the mitochondrial dependent apoptosis pathway, which further verified the molecular mechanisms of WWOX pathway regulating gallbladder cancer cells.
4. The animal model of subcutaneous transplanted tumor of gallbladder cancer in nude mice was successfully constructed. Targeting to silent Bmi-1expression can induce the cell apoptosis of transplanted tumors of gallbladder cancers, significantly inhibit tumor growth and the formation of new vessels, and regulate the expression of key genes in Bmi-1/WWOX/P73/PUMA/Bax/Bcl-2pathway of subcutaneous transplanted tumor in nude mice. The findings are consistent with those by the in vitro experiments. It was further confirmed by in vivo experiments that Bmi-1/WWOX pathway is involved in the cell growth of gallbladder cancer through the mitochondrial dependent apoptosis pathway.
5. Bmi-1and WWOX may provide a potential molecular target for the gene targeted therapy of gallbladder cancer. It can be one of the potential therapeutic tools for gallbladder carcinoma combined with gene therapy through targeting to silent Bmi-1expression and specifically up-regulating the WWOX expression. Blocking the Bmi-1/WWOX pathway plays an important role in target therapeutic strategies for gallbladder cancer and also provides a new experimental basis for the diagnosis of early gallbladder cancer and multiple targeted molecular therapies for gallbladder cancer.
引文
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