Lgr5与β-catenin在卵巢上皮性癌中的表达及对SKOV3细胞增殖迁移的影响

发布时间：2018-01-15 08:27

本文关键词：Lgr5与β-catenin在卵巢上皮性癌中的表达及对SKOV3细胞增殖迁移的影响　出处：《郑州大学》2017年硕士论文　论文类型：学位论文

【摘要】：背景与目的卵巢癌(ovarian cancer)是女性生殖器官三大恶性肿瘤之一,病理类型以上皮性肿瘤为主。卵巢癌的发病概率列居第三位,仅次于宫颈癌和子宫内膜癌,但其恶性程度最高,致死率位居第一,是妇女生命健康的最大杀手。虽然近年来有关卵巢癌的研究取得很大进展,但其发病机制尚不完全清楚。解决卵巢癌早期诊断,控制复发、转移和化疗耐药等问题成为卵巢癌研究的热点。G蛋白偶联受体(leucine-rich repeat-containing g-protein coupled receptor5,Lgr5)在胚胎发育和成体干细胞自我更新及维持中起重要作用,是多种组织和成体干细胞的标志物。研究表明,Lgr5可以加速细胞周期,促进细胞增殖,在许多恶性肿瘤(如胃肠道肿瘤、肝癌、基底细胞癌、宫颈癌等)中高表达,而正常组织中表达量较低。Wnt/β-catenin信号通路与肿瘤的发生发展密切相关,其核心分子β-catenin的异常高表达可能参与肿瘤干细胞的形成,影响卵巢癌患者的化疗耐受、肿瘤的复发、转移及预后。近年来研究发现,Lgr5在甲状腺癌、宫颈癌中可能通过上调β-catenin的表达以促进肿瘤细胞的增殖和迁移能力。因此,本研究对卵巢上皮性癌组织中Lgr5、β-catenin的表达以及靶向抑制Lgr5表达对对卵巢癌细胞SKOV3增殖迁移能力的影响进行了观察。方法实验收集了郑州大学第三附属医院2014年至2015年确诊卵巢癌患者30例,利用免疫组织化学技术检测Lgr5和β-catenin蛋白在卵巢癌组织中的表达情况。对卵巢癌SKOV3细胞转染靶向LGR5基因的干扰RNA(sh RNA),实验分为三组:空白对照组(未转染对照组)、阴性对照组(NC sh RNA转染组)和实验组(LGR5 sh RNA转染组)。Western Blot方法检测各组细胞处理前后lgr5和β-catenin蛋白表达水平,RT-PCR检测Lgr5和β-catenin的m RNA相对表达水平,分别采用CCK-8和划痕试验检测SKOV3细胞的增殖、迁移能力。采用SPSS22.0统计软件对数据进行统计学处理,各组定性资料间分别采用卡方检验,定量资料以均数±标准差表示,符合正态分布的两组数据间比较采用独立样本t检验,两组定性资料间的关联性分析采用Spearman分析。以α=0.05作为检验水准。结果卵巢癌组织的Lgr5阳性表达率为63.3%(19/30),β-catenin阳性表达率83.3%(25/30)二者表达水平均较良性肿瘤组、正常组显著升高(P0.05),且两者呈正相关(rs=0.40,P0.05);实验组SKOV3细胞转染sh RNA靶向沉默LGR5后,与空白对照组、阴性转染对照组相比,LGR5和β-catenin的m RNA水平均显著降低(P0.05);Lgr5和β-catenin的蛋白水平均显著降低(P0.05);靶向抑制LGR5表达后SKOV3细胞的增殖能力和迁移能力显著降低(P0.05)。结论(1)Lgr5和β-catenin在卵巢上皮性癌中均特异性高表达,且呈正相关性,提示两者可能起协同作用,参与上皮性卵巢癌的发生和发展。(2)在卵巢癌SKOV3细胞中Lgr5与β-catenin的表达密切相关,提示Lgr5可能通过调节β-catenin对SKOV3细胞发生作用。(3)在卵巢癌SKOV3细胞中Lgr5可能协同β-catenin调控细胞增殖及迁移能力。
[Abstract]:Background and objective: ovarian cancer (ovarian cancer) is one of the three malignant tumors of the female reproductive organs, pathological types of tumor. The skin above the incidence of ovarian cancer ranks the third, after cervical cancer and endometrial cancer, but the highest degree of malignancy and mortality ranked first, is the biggest killer of women's health. Although in recent years, great progress on research related to ovarian cancer, but its pathogenesis is not clear. To solve the early diagnosis of ovarian cancer, recurrence, metastasis and chemotherapy resistance problem has become a hot spot of.G protein coupled receptor in ovarian cancer research (leucine-rich repeat-containing G-protein coupled Receptor5, Lgr5) in embryonic and adult stem cells self renewal and maintenance plays an important role, is a variety of tissues and adult stem cell markers. The results show that Lgr5 can accelerate cell cycle, promote cell proliferation, in Many malignant tumors (such as gastrointestinal tumor, hepatocellular carcinoma, basal cell carcinoma, cervical cancer) is highly expressed in normal tissues, and the expression of the occurrence and development of low amount of.Wnt/ beta -catenin signal pathway and tumor is closely related to abnormal expression of the core molecule beta -catenin may be involved in the formation of cancer stem cells, effect of chemotherapy tolerance ovarian cancer, tumor recurrence, metastasis and prognosis. In recent years, the study found that Lgr5 in thyroid cancer, may be to promote the proliferation and migration of tumor cells through upregulating the expression of beta -catenin in cervical cancer. Therefore, the study of Lgr5 in epithelial ovarian cancer, -catenin expression and targeting effect of Lgr5 expression on migration ability of ovarian cancer cell SKOV3 proliferation were observed. Methods the experiment collected 30 cases of the Third Affiliated Hospital of Zhengzhou University from 2014 to 2015 were diagnosed with ovarian cancer by immunohistochemistry The expression of textile chemical detection of Lgr5 and beta -catenin protein in ovarian cancer tissues. The ovarian cancer cells were transfected with SKOV3 targeting RNA LGR5 gene (SH RNA), were divided into three groups: blank control group (blank control group), negative control group (NC sh RNA transfection group) and experimental group (LGR5 SH RNA.Western Blot transfection group) method for the detection of cells in each group before and after treatment of Lgr5 and beta -catenin protein expression level, the relative expression level of M RNA RT-PCR detection of Lgr5 and beta -catenin, SKOV3 cells were used to detect CCK-8 and scratch test proliferation, migration ability. The data were analyzed with SPSS22.0 statistical software, group the qualitative data were chi square test, quantitative data to mean + standard deviation that is consistent with the data of the two groups of normal distribution were compared using independent samples t test, correlation analysis between two groups of qualitative data using Spearman A =0.05 analysis. As the standard test. Results the positive expression of Lgr5 in ovarian cancer tissue was 63.3% (19/30), beta -catenin positive expression rate was 83.3% (25/30) expression level were benign tumor group two, normal group increased significantly (P0.05), and there is a positive correlation (rs=0.40, P0.05); group SKOV3 the SH cell targeting RNA LGR5, compared with the blank control group, negative control group transfected with -catenin, LGR5 and beta m RNA levels were significantly decreased (P0.05); protein levels of Lgr5 and beta -catenin were significantly decreased (P0.05); targeting ability of proliferation and migration of SKOV3 cells after inhibition of LGR5 expression decreased significantly (P0.05). Conclusion (1) Lgr5 and beta -catenin in epithelial ovarian cancer were highly specific expression, and positive correlation, suggesting that they may play a synergistic effect, participate in the occurrence and development of epithelial ovarian cancer. (2) Lgr5 and ~ -catenin in SKOV3 cells of ovarian cancer Expression is closely related, suggesting that Lgr5 may play a role in regulating SKOV3 cells by regulating beta -catenin. (3) in ovarian cancer SKOV3 cells, Lgr5 may cooperate with beta -catenin to regulate cell proliferation and migration.

【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.31

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