【作者】 张瑞涛；

【导师】 史惠蓉；

【作者基本信息】 郑州大学 ， 妇产科学， 2012， 博士

【摘要】 背景宫颈癌、卵巢癌和子宫内膜癌是女性生殖系统最常见的三大恶性肿瘤,其中卵巢癌的发病率虽然仅占女性恶性肿瘤的4%,但却是死亡率最高的妇科恶性肿瘤。卵巢癌具有起病隐匿、原发灶隐蔽、首次诊断多为晚期、易播散转移、术后易复发和易产生化疗耐药等特点,随着医学水平的进步、临床肿瘤细胞减灭术和联合化疗水平的提高,卵巢癌的五年生存率虽然得到了明显改善,但卵巢癌患者的总体预后仍不满意。多年来全世界投入大量人力、物力进行卵巢癌发病机制的有关研究,试图阐明卵巢癌发生发展的分子机制,尽管在卵巢癌的早期诊断、基因治疗和化疗耐药机制研究等方面取得了令人可喜的成绩和令人巨大的进展,但目前卵巢癌发生发展的分子生物学机制仍不清楚。经过多年的研究后,目前科学界趋向于认为肿瘤是一种由基因组多发性改变而引起的疾病,肿瘤发生的基础是癌基因功能区突变而激活和/或抑癌基因功能区突变而失活。持续的生长信号刺激、逃避生长抑制、抵抗程序性细胞死亡无限的复制潜能、诱导血管发生、侵袭和转移的激活、细胞能量代谢的重组和逃避免疫损伤是导致不同类型的肿瘤细胞恶性生长所必需的8种获得性细胞生理学改变。在肿瘤发展过程中大多甚至全部肿瘤都存在类似的细胞生理学特点的改变,因此与这些获得性细胞行为关系密切的癌基因和抑癌基因都成为肿瘤分子机制研究和癌症治疗研究的热点。结肠癌转移相关基因1(metastasis-associated in colon cancer-1, MACC1)是Stein等在2009年应用全基因组表达分析技术在结肠癌原发灶和转移灶中发现的一个以前未被描述过的基因,MACC1可在体外实验中促进结肠癌细胞的增殖、侵袭和肝细胞生长因子(hepatocyte growth factor, HGF)诱导的癌细胞播散,且可促进动物实验中结肠癌细胞移植瘤的生长,是可用于结肠癌转移和预后判断的独立标志物。目前的研究发现,MACC1与胃癌、肺癌和肝癌等恶性肿瘤的转移和预后密切相关。MACC1是HGF/C-met信号通路中MET基因的关键调节因子,HGF又称离散因子(scatter factor, SF),能促进多种肿瘤细胞的生长、粘附、移动及侵袭,且可刺激血管内皮细胞生长,在卵巢癌等一系列恶性肿瘤细胞的生长、侵袭及转移过程中发挥重要作用。目前,国内外尚无MACC1与卵巢癌相关关系的研究报道,MACC1是否参与卵巢癌的发生发展、其对卵巢癌的临床意义何在、其与卵巢癌细胞的生物学行为关系如何、其异常表达导致卵巢癌细胞侵袭和转移的分子机制如何等问题尚不清楚。目的通过检测不同性质卵巢组织中MACC1的表达,初步分析MACC1异常表达与卵巢癌临床病理参数的关系,评价卵巢癌组织中MACC1异常表达的临床意义。进而采用RNA干扰(RNA interference, RNAi)技术研究卵巢癌细胞中MACC1基因沉默后对卵巢癌细胞生长、凋亡、迁移和侵袭的影响,探讨其异常表达与卵巢癌细胞生物学行为的关系。最后,分析MACC1可能参与的信号传导系统,初步探讨MACC1参与卵巢癌侵袭和转移的可能分子机制。本研究分为以下四个部分：第一部分MACC1、HGF和C-met在卵巢上皮性癌组织中的表达及其意义方法1.采用RT-PCR技术检测20例正常卵巢组织、19例良性卵巢上皮性肿瘤和52例上皮性卵巢癌组织中MACC1 mRNA的表达情况。2.采用免疫组化和western blot技术检测20例正常卵巢组织、19例良性卵巢上皮性肿瘤和52例上皮性卵巢癌组织中MACC1、HGF和C-met蛋白的表达情况。3.分析MACC1、HGF和C-met的异常表达与卵巢癌临床病理参数的关系。4.探讨卵巢癌组织中MACC1、HGF和C-met的异常表达的相关性。结果1. MACC1 mRNA在卵巢癌组织中的表达显著高于良性肿瘤和正常卵巢组织中的表达(F=142.45,P=0.000)；卵巢癌临床分期越晚,MACC1 mRNA表达越高(F=51.305,P=0.000)。2.卵巢癌组织中MACC1、HGF和C-met蛋白的阳性率显著高于良性肿瘤和正常卵巢组织(χ2=35.845,P=0.000；χ2=24.532,P=0.000；χ2=28.893,P=0.000)。卵巢癌临床分期越晚,MACC1蛋白阳性率越高(χ2=9.707,P=0.024)3.卵巢癌组织中MACC1、HGF和C-met蛋白的相对表达量均显著高于良性肿瘤和正常卵巢组织(F=465.790,P=0.000；F=326.289,P=0.000；F=280.871,P=0.000)。4.在卵巢上皮性癌中,MACC1、HGF和C-met异常高表达与临床分期、组织分化和淋巴结转移相关,临床分期越晚、组织分化越差、伴随腹腔淋巴结转移的癌组织中MACC1、HGF和C-met表达越高(P<0.05)。5.在卵巢上皮性癌中,MACC1的异常表达与HGF和C-met的异常表达呈正相关(r=0.350,P=0.011：r=0.429,P=0.002),HGF表达与C-met表达表达呈正相关(r=0.487,P=0.000)。小结1. MACC1异常表达与卵巢癌发生发展关系密切,可作为卵巢上皮性癌诊断、治疗和预后分析的标志物。2. MACC1、HGF和C-met的异常表达可能协同参与了卵巢癌的恶性进展。第二部分MACC1基因特异性shRNA真核表达载体的构建及稳定转染重组载体OVCAR-3细胞的筛选方法1.设计合成MACC1基因特异性小发卡RNA (short hairpin RNA, shRNA)单链核苷酸序列。2.以真核荧光表达载体psuper-EGFP为基础构建MACC1基因特异性shRNA重组真核表达载体。3.重组shRNA真核表达载体转染卵巢癌细胞OVCAR-3, G418筛选稳定表达重组载体的卵巢癌细胞株。4.采用RT-PCR技术和western blot技术验证和筛选MACC1基因抑制效应最为显著的稳定转染细胞株。结果1.设计合成的MACC1基因特异性shRNA单链成功退火形成双链shRNA。2.经酶切和测序鉴定,双链shRNA插入真核表达载体psuper-EGFP位置正确。3.重组质粒成功转染OVCAR-3细胞,采用G418成功筛选获得稳定表达重组质粒的单克隆细胞株。4.经RT-PCR和western blot验证和筛选,获得MACC1基因抑制效果最为显著的单克隆稳定转染卵巢癌细胞株OVCAR-3-s3。小结1.成功构建针对MACC1基因的特异性重组shRNA真核表达载体。2.成功筛选获得稳定表达重组shRNA质粒的细胞株,为下一步研究MACC1对HGF/C-met信号通路的调控机制及其与卵巢癌的关系奠定了实验基础。第三部分卵巢癌OVCAR-3细胞中MACC1基因抑制效应研究方法1.采用MTT法和单细胞平板克隆集落形成实验检测MACCl基因沉默前后OVCAR-3细胞增殖能力的变化。2.采用流式细胞术和TUNEL法检测MACC1基因沉默前后OVCAR-3细胞凋亡能力的变化。3.采用transwell小室和单层细胞划痕实验检测MACC1基因沉默前后OVCAR-3细胞迁移能力的变化。4.采用Matrigel侵袭实验和裸鼠移植瘤模型检测MACC1基因沉默前后OVCAR-3细胞侵袭能力的变化。结果1.与OVCAR-3、OVCAR-3-neo和OVCAR-3-NC细胞相比,OVCAR-3-s3细胞的增殖显著受到抑制；OVCAR-3-s3组的细胞克隆集落形成率显著下降(F=23.725,P=0.000)2.与OVCAR-3、OVCAR-3-neo和OVCAR-3-NC组相比,OVCAR-3-s3组细胞凋亡率明显增加(F=1679.406,P=0.000)；OVCAR-3-s3细胞中凋亡小体的数目显著增加(F=271.84,P=-0.000)3.与OVCAR-3、OVCAR-3-neo和OVCAR-3-NC组相比,无血清培养24h后OVCAR-3-s3细胞的划痕距离明显较宽；聚碳酸酯膜的下室面的OVCAR-3-s3细胞数明显减少(F=44.261,P=0.000)4.与OVCAR-3、OVCAR-3-neo和OVCAR-3-NC组相比,在包被Matrigel基质胶的transwell小室的聚碳酸酯膜下室面的OVCAR-3-s3细胞数明显减少(F=40.296,P=0.000)；裸鼠皮下注射35天后OVCAR-3-s3形成的移植瘤体积明显小于其他三组细胞(F=32.571,P=0.000)。小结1. MACC1基因沉默后可明显抑制卵巢癌OVCAR-3细胞的增殖、迁移、和侵袭能力,明显诱导卵巢癌OVCAR-3细胞的凋亡。2. MACC1具有作为卵巢癌预防和治疗基因靶点的潜在价值。第四部分MACC1异常参与卵巢癌发生发展分子机制研究方法1.HE染色观察OVCAR-3、OVCAR-3-neo、OVCAR-3-NC和OVCAR-3-s3细胞所致移植瘤肿瘤组织中瘤细胞的形态变化。2.免疫组化分析OVCAR-3、OVCAR-3-neo、OVCAR-3-NC和OVCAR-3-s3细胞所致移植瘤肿瘤组织中MACC1和C-met蛋白的表达。3. Western blot分析OVCAR-3、OVCAR-3-neo、OVCAR-3-NC和OVCAR-3-s3细胞中C-met、MEK1/2、p-MEK1/2、ERK1/2和p-ERK1/2蛋白的变化。4. RT-PCR检测OVCAR-3、OVCAR-3-neo、OVCAR-3-NC和OVCAR-3-s3细胞中cyclinD1、caspase3、caspase、survivin、MMP2、MMP9和VEGFA的表达。结果1.与OVCAR-3. OVCAR-3-neo和OVCAR-3-NC组相比,OVCAR-3-s3组瘤组织的瘤细胞形态学发生明显变化。2.与OVCAR-3、OVCAR-3-neo和OVCAR-3-NC组相比,OVCAR-3-s3细胞所致移植瘤组织中MACC1和C-met蛋白的阳性表达明显减弱。3.与OVCAR-3、OVCAR-3-neo和OVCAR-3-NC相比,OVCAR-3-s3细胞中C-met、p-MEK1/2和p-ERK1/2表达明显减低。4.与OVCAR-3、OVCAR-3-neo和OVCAR-3-NC组相比,OVCAR-3-s3细胞中cyclinD1、survivin、MMP2、MMP9和VEGFA表达明显减低,caspase3和caspase9表达明显增加。小结1. MACC1异常表达可能参与了卵巢癌细胞的恶性转化。2. MACC1异常可能通过HGF/C-met信号通路和MAPK信号通路介导其下游的肿瘤相关基因参与卵巢癌的侵袭和转移。结论1. MACC1基因表达异常与卵巢癌的发生发展关系密切,可能参与了卵巢癌细胞的恶性转化2. MACC1可能通过HGF/C-met信号通路和MAPK信号通路介导其下游的肿瘤相关基因参与卵巢癌的侵袭和转移3. MACC1基因有作为卵巢癌基因治疗靶点的潜在价值更多还原

【Abstract】 BackgroundCervical cancer, ovarian cancer and endometrial carcinoma are the three hackneyed malignancies in female genital system. The incidence rate of ovarain cancer only accounts to 4% of female malignancies, but the death rate of ovarian cancer is highest in female malignancies. Despite improvements in the application of aggressive cytoreductive surgery and combination chemotherapy, the five year survival rate of ovarian cancer is obvious improved, ovarian cancer has the most unfavorable total prognosis due to its insidious onset, concealed primary focus, diagnosis at late stage, dissemination, relapse, and tendency to develop chemotherapy resistance. Through considerable efforts aimed at elucidating the tumorigenesis of ovarian carcinoma, which gained thumping developments in early dianosisits, gene therapy and mechanism of chemotherapy resistance, the molecular mechanism of ovarian cancer is still unclear.After a quarter century of rapid advances, cancer researches reveal cancer to be a disease involving dynamic changes in the genome. The foundation of cancer has been set in the activation of oncogenes with mutations of dominants and the inactivation of tumor suppressor genes with recessive loss of function. Sustaining proliferative signaling, evading growth suppressors, resisting cell death, enabling replicative immortality, inducing angiogenesis, activating invasion and metastasis, reprogramming of energy metabolism and evading immune destruction are eight acquired biological capabilities during the multistep development of different human tumors.Because these capabilities are shared in the development of most and perhaps all types of human tumors, the oncogenes and tumor suppressor genes which are closely related with these acquired capabilities become the hot spot of mechanism and therapy research of cancer.Metastasis-associated in colon cancer-1 (MACC1) is an undescribed gene ever before which was detected by genomewide expression analytical technique in primary focus and metastasis focus of colon cancer. MACC1 could promote proliferation, invasion and hepatocyte growth factor (HGF)-induced scattering of colon cancer cells in vitro, and promote the growth of colon cancer cell transplanted tumor, which could serve as a biomarker of metastasis and prognosis of colon cancer. MACC1 aslo were demonstrated to be closely related with the metastasis and prognosis of gastric cancer, lung cancer and liver cancer. MACC1 was proved as a regulator of MET in HGF/C-met signaling, HGF was also called scatter factor which could promote the growth, adhesion, migration and invasion of tumor cells, HGF also could stimulate the growth of vascular endothelial cells which played key roles in the growth, invasion and metastasis of many malignant tumor cells, including ovarian cancer. There was still none report on the relationships between MACC1 and ovarian cancer up to now, it is still unclear that whether MACC1 implicate in ovarian cancer, whether MACC1 has clinical significances to ovarian cancer, how about the relationships between MACC1 and the biological behaviors of ovarian cancer cells, how about the mechanism of MACC1 overexpression induced ovarian cancer cell malignant transformation.ObjectiveDetect the expression of MACC1 in different ovarian tissues, analyze the relations between overexpression of MACC1 and the clinical pathological features of ovarian cancer, and evaluate the clinical significances of overexpression of MACC 1 in ovarian cancer. Then inhibit the expression of MACC 1 in ovarian cancer cells by RNA interference, investigate the effects of MACC1 knockdown on the growth, apoptosis, migration and invasion of ovarian cancer cells, approach the relationships between overexpression of MACC 1 and the biological behaviors of ovarian cancer cells. At last, analyze the possible signaling in which MACC1 implicated, discuss the possible mechanism of MACC 1 implicate in the development and progress of ovarian cancer. The present research includes four parts as follow:Part I Expressions and Significants of MACC1, HGF and C-met in Epithelial Ovarian CancerMethods1. The expressions of MACC1 mRNA in 20 specimens normal ovary tissues,19 specimens bengin ovarian tumors and 52 specimens epithelial ovarian cancer (EOC) tissues by RT-PCR.2. The expressions of MACC1, HGF and C-met protein in 20 specimens normal ovary tissues,19 specimens bengin ovarian tumors and 52 specimens ovarian cancer tissues by immunohistochemistry and western blot.3. The relationships between abnormal expressions of MACC1, HGF and C-met and the clinical pathological characters of ovarian cancer were analyzed.4. The correlation of abnormal expression in ovarian cancer among MACC 1, HGF and C-met were discussed.Results1. The expressions of MACC1 mRNA were significantly higher in EOC tissues than those in normal ovary and bengin ovarian tumor tissues (F=142.45, P=0.000); more advanced FIGO stage of EOC, higher expression of MACC1 mRNA (F=51.305,P=0.000).2. The expressions of MACC1, HGF and C-met protein were obviously higher in EOC tissues than those in normal ovary and bengin ovarian tumor tissues (χ2=35.845, P=0.000;χ2=24.532, P=0.000;χ2=28.893, P=0.000); more advanced FIGO stage of EOC, higher expression of MACC1 protein (χ2=9.707, P=0.024).3. The relative expression values of MACC1, HGF and C-met prtein were significantly higher in EOC tissues than those in normal ovary and bengin ovarian tumor tissues (F=465.790, P=0.000; F=326.289,P=0.000; F=280.871, P=0.000).4. The overexpressions of MACC1, HGF and C-met were related with FIGO stage, histological grade and lymph nodes metastasis, there were more advanced FIGO stage, worse histological grade and accompanied lymph nodes metastasis, there were more high overexpressions of MACC1, HGF and C-met in EOC (P< 0.05).5. There were positive correlations between overexpression of MACC1 and overexpressions of HGF and C-met (r=0.350, P=0.011; r=0.429, P=0.002); there was positive correlation between overexpression of HGF and overexpression of C-met (r=0.487, P=0.000).Summaries1. Overexpression of MACC1 was closely related with EOC, which could serve as a biomarker of diagnosis, therapy and prognosis of EOC.2. Overexpressions of MACC1, HGF and C-met might synergistically implicate in the malignant progression of EOC. Part II Construction of MACC1 Gene Specific shRNA Eukaryotic Expression Vector and Selection of OVCAR-3 Cells Stably Transfected with Recombinant VectorMethods1. Designed and synthesized MACC1 specific shRNA oligonucleotide strands.2. Constructed MACC1 gene specific shRNA recombinant vector based on eukaryotic fluorescence expression plasmid psuper-EGFP.3. Transfected OVCAR-3 cells with recombinant shRNA vectors, selected OVCAR-3 cells stably transfected with recombinant shRNA vectors by G418.4. Identified the best inhibited effect of MACC1 in transfected OVCAR-3 cells by RT-PCR and western blot.Results1. MACC1 specific shRNA oligonucleotides were annealed and ligated to be double-strand shRNA successfully.2. Identified by digestion and sequencing, double-strand shRNAs were inserted into psuper-EGFP at right sites.3. Transfected OVCAR-3 cells with recombinant vectors, and harvested monoclonal cell lines of OVCAR-3 cells with stably trasnfected recombinant vectors by G418 successfully.4. Identified by RT-PCR and western blot, OVCAR-3-s3 were the cell line with best inhibited effect of MACC1.Summaries1. Constructed specific shRNA recombinant eukaryotic fluorescence expression plasmid against MACC1 gene successfully.2. Harvested OVCAR-3 cell lines with stable expression recombinant shRNA vectors successfully, which lay a foundation for the further investigation about the relationships between MACC1 and the development and progression of ovarian cancer and the mechanism of MACC1 regulated HGF/C-met signaling.PartⅢEffects of MACC1 Gene Inhibition on the Ovarian Cancer Cell Line OVCAR-3 CellsMethods1. The proliferation of OVCAR-3 cells before and after inhibition of MACC1 was measured by MTT assay and monocell plate colony formation assay.2. The apoptosis of OVCAR-3 cells before and after inhibition of MACC1 was measured by FCM assay and TUNEL assay.3. The migration of OVCAR-3 cells before and after inhibition of MACC1 was measured by cell monolayer scratch assay and transwell assay.4. The invasion of OVCAR-3 cells before and after inhibition of MACC1 was measured by Matrigel assay and xenograft modle assay.Results1. Compared with OVCAR-3, OVCAR-3-neo and OVCAR-3-NC cells, the proliferation of OVCAR-3-s3 was suppressed obviously, and the rate of colony formation was decreased significantly (F=23.725, P=0.000).2. Compared with OVCAR-3, OVCAR-3-neo and OVCAR-3-NC cells, the apoptosis rate of OVCAR-3-s3 was increased obviously (F=1679.406, P=0.000), and the numbers of apoptosis body were increased significantly (F=271.84, P=0.000).3. Compared with OVCAR-3, OVCAR-3-neo and OVCAR-3-NC cells, the scrathes of OVCAR-3-s3 healed more slowly obviously after cultivation 24h without FBS, and the numbers of OVCAR-3-s3 cell on the lower polycarbonate membrane of transwell chamber were decreased significantly (F=44.261,P=0.000).4. Compared with OVCAR-3, OVCAR-3-neo and OVCAR-3-NC cells, the numbers of OVCAR-3-s3 cell on the lower polycarbonate membrane of transwell chamber covered matirgel were decreased significantly (F=40.296, P=0.000) (F=23.725, P=0.000), and the volumes of xenograft tumors of OVCAR-3-s3 cells after injection 35 days were obviously smaller (F=32.571, P=0.000).Summaries1. Inhibition of MACC1 gene could obviously suppress the proliferation, migration and invasion of OVCAR-3 cells, but obviously induce the apoptosis of OVCAR-3 cells.2. MACC1 could serve as a potential gene target for prevention and therapy of ovarian cancer.Part IV Studies on the Overexpression of MACC1 and the Pathogenesis of Development and Progress of Ovarian CancerMethods1. The morphologic changes of xenograft tumors cells caused by OVCAR-3, OVCAR-3-neo,OVCAR-3-NC and OVCAR-3-s3 cells were observed by HE stain.2. The expressions of MACC1 and C-met protein in xenograft tumors caused by OVCAR-3, OVCAR-3-neo, OVCAR-3-NC and OVCAR-3-s3 cells were observed by immunohistochemistry.3. The expressions of C-met, MEK1/2, p-MEK1/2, ERK1/2 and p-ERK1/2 protein in OVCAR-3, OVCAR-3-neo,OVCAR-3-NC and OVCAR-3-s3 cells were measured by western blot.4. The expressions of cyclinDl, caspase3, caspase9, survivin, MMP2, MMP9 and VEGFA mRNA in OVCAR-3, OVCAR-3-neo, OVCAR-3-NC and OVCAR-3-s3 cells were measured by RT-PCR.Results1. Compared with OVCAR-3, OVCAR-3-neo and OVCAR-3-NC cells, the morphology of OVCAR-3-s3 cell changed obviously.2. Compared with OVCAR-3, OVCAR-3-neo and OVCAR-3-NC cells, the expressions of MACC1 and C-met protein in xenograft tumors caused by OVCAR-3-s3 cells were decreased obviously.3. Compared with OVCAR-3, OVCAR-3-neo and OVCAR-3-NC cells, the expressions of C-met, p-MEK1/2 and p-ERK1/2 protein in OVCAR-3-s3 cells were decreased significantly.4. Compared with OVCAR-3, OVCAR-3-neo and OVCAR-3-NC cells, the expressions of cyclinD1, survivin, MMP2, MMP9 and VEGFA mRNA in OVCAR-3-s3 cells were decreased significantly, but the expression of caspase3 and caspase9 mRNA were increased obviously.Summaries1. Overexpression of MACC1 might implicate in the malignant transformation of ovarian cancer cells.2. Overexpression of MACC1 might implicate in the invasion and metastasis of ovarian cancer via HGF/C-met signaling and MAPK signaling mediated downstream tumor associated genes. Conclusions1. Overexpression of MACC1 was closely related with EOC, which might implicate in the malignant transformation of ovarian cancer cells.2. Overexpression of MACC1 might implicate in the invasion and metastasis of ovarian cancer via HGF/C-met signaling and MAPK signaling mediated downstream tumor associated genes.3. MACC1 could serve as a potential gene target for prevention and therapy of ovarian cancer.更多还原