BEZ235体外抑制宫颈癌增殖与迁移作用研究及Skp2过表达的临床意义
发布时间：2018-08-20 12:37
【摘要】：研究背景：宫颈癌作为女性第二大恶性肿瘤,发病率仅次于乳腺癌。BEZ235是磷脂酰肌醇-3(PI3K)及其下游因子哺乳动物雷帕毒素蛋白(mammalian target of rapamycin, mTOR)的双重靶向抑制剂,已表现出抑制多种肿瘤的作用,但在宫颈癌组织中的作用报道较少。S期激酶相关蛋白2(Skp2)是在调控细胞周期中发挥重要作用的蛋白,并参与细胞的增殖及凋亡。目前已发现Skp2蛋白在多种恶性肿瘤中呈过表达且导致肿瘤患者的不良预后。文献报道,Skp2对肿瘤发生发展的影响受PI3K信号通路的调控。在多种恶性肿瘤中,PI3K信号通路均可在转录和翻译前、后水平上对Skp2蛋白进行调控。 研究目的：探讨宫颈癌细胞中PI3K抑制剂BEZ235通过下调Skp2影响宫颈癌细胞的增殖及迁移能力；探讨Skp2蛋白在宫颈鳞状细胞癌组织中表达的临床病理学意义。 材料和方法：第一部分：宫颈癌Hela、C33A、SiHa细胞的传代培养；噻唑蓝(MTT)实验检测BEZ235对细胞生长及增殖情况的影响,并绘制生长曲线,筛选药物浓度；Hela和C33A细胞中加入0.1μM、0.4μM的BEZ235药物,对照组加入等体积的DMSO,作用48h后,提取RNA及蛋白,通过RT-PCR法检测宫颈癌细胞中Skp2mRNA的变化,并用Western-blot法检测宫颈癌细胞中Skp2、Ezrin、Six1蛋白的变化；划痕实验法检测BEZ235对Hela细胞迁移能力的影响。第二部分：免疫组化方法检测在25例正常宫颈上皮组织、84例宫颈上皮内瘤变(CIN)和163例宫颈鳞状细胞癌(SCC)中Skp2蛋白的表达情况；RT-PCR法检测人乳头瘤病毒(HPV)在宫颈癌不同组织中的表达情况；结合宫颈癌HPV 感染情况、临床分期等生物学特点检验Skp2蛋白在宫颈癌组织表达的临床病理学意义；统计学分析Skp2蛋白表达对患者生存时间的影响。 结果：第一部分：MTT实验结果表明,用BEZ235阻断PI3K信号通路可抑制宫颈癌细胞Hela、C33A和SiHa的增殖；RT-PCR和Western blot结果表明,宫颈癌Hela和C33A细胞中Skp2mRNA和Slp2、Ezrin、Sixl蛋白的表达水平均随BEZ235的浓度(0.1μM,0.4μM)增高而降低；划痕试验结果显示,对宫颈癌Hela细胞进行BEZ235药物处理24h、48h后,细胞移动距离较对照组明显缩短。第二部分：RT-PCR检测HPV mRNA在14例宫颈癌组织中有11例呈阳性表达；Skp2在CIN-1、CIN-2和CIN-3中的阳性率依次增高,且均高于正常组织(P均0.01)；在宫颈鳞状细胞癌中Skp2蛋白呈明显的弥漫性强阳性染色,其阳性率显著高于正常宫颈上皮组织(P0.01)；Skp2蛋白表达与FIGO分期及高危型HPV感染关系密切(P0.05)；Skp2蛋白阳性表达的宫颈鳞状细胞癌患者无瘤生存率及总生存率明显低于阴性表达的患者(P均0.01)。 结论：1.PI3K抑制剂BEZ235通过下调Skp2蛋白抑制宫颈癌细胞增殖与迁移能力；2.Skp2蛋白过表达可能是预示宫颈鳞状细胞癌患者的不良预后的检测指标,Skp2蛋白检测可作为宫颈鳞状细胞癌增殖指数测定及预后评估的有效分子标志物。
[Abstract]:Background: as the second largest malignant tumor in women, cervical cancer is a double targeting inhibitor of phosphatidylinositol -3 (PI3K) and its downstream mammal rapa toxin (mammalian target of rapamycin, mTOR). Although it has been shown to inhibit many kinds of tumors, the role of S phase kinase associated protein 2 (Skp2) in cervical carcinoma is less reported, which plays an important role in the regulation of cell cycle, and participates in cell proliferation and apoptosis. It has been found that Skp2 protein is overexpressed in many malignant tumors and leads to poor prognosis of tumor patients. The effect of Skp2 on tumor development is regulated by PI3K signaling pathway. PI3K signaling pathway can regulate Skp2 protein before and after translation in many kinds of malignant tumors. Objective: to investigate the effect of PI3K inhibitor BEZ235 on the proliferation and migration of cervical cancer cells by down-regulating Skp2, and to explore the clinicopathological significance of the expression of Skp2 protein in cervical squamous cell carcinoma. Materials and methods: the first part: the passage culture of HelaA33AnSiHa cell line of cervical cancer, the effect of BEZ235 on cell growth and proliferation was detected by thiazolyl (MTT) assay, and the growth curve was drawn, and the drug concentration was screened by adding 0.4 渭 M BEZ235 drug to Hela and C33A cells. The control group was treated with DMSO-containing the same volume for 48h, then RNA and protein were extracted, the changes of Skp2mRNA in cervical cancer cells were detected by RT-PCR method, and the changes of Skp2Ezrin1 protein in cervical cancer cells were detected by Western-blot assay. The effect of BEZ235 on the migration of Hela cells was detected by scratch test. The second part: immunohistochemical method was used to detect the expression of Skp2 protein in 84 cases of cervical intraepithelial neoplasia (CIN) and 163 cases of cervical squamous cell carcinoma (SCC) in 25 cases of normal cervical epithelial tissue and 163 cases of cervical squamous cell carcinoma. The expression of human papillomavirus (HPV) in different tissues of cervical cancer was detected by RT-PCR, and the clinicopathological significance of the expression of Skp2 protein in cervical carcinoma was examined by combining with the biological characteristics of cervical cancer HPV infection and clinical staging. The effect of Skp2 protein expression on survival time was analyzed statistically. Results: the results of the first part of the experiment showed that blocking the PI3K signaling pathway with BEZ235 could inhibit the proliferation of HelaC33A and SiHa cells by RT-PCR and Western blot. The expression level of Skp2mRNA and Slp2Ezrininosin Sixl protein in cervical cancer Hela and C33A cells decreased with the increase of BEZ235 concentration (0.1 渭 M, 0.4 渭 M), and the results of scratch test showed that the cell migration distance of cervical cancer Hela cells treated with BEZ235 for 24 h or 48 h was significantly shorter than that of the control group. In the second part, the positive rates of Skp2 in CIN-1 and CIN-2 and CIN-3 were increased in 11 of 14 cases of cervical carcinoma by RT-PCR, and were higher than those in normal tissues (P0.01). The positive rate of Skp2 protein in cervical squamous cell carcinoma was significantly higher than that in normal cervical epithelium (P0.01). The expression of Skp2 protein was closely related to FIGO stage and high risk HPV infection (P0.05). The tumor-free survival rate and overall survival rate of cervical squamous cell carcinoma patients with positive expression of Skp2 protein were significantly lower than those with negative expression (P0.01). Conclusion 1. PI3K inhibitor BEZ235 inhibits the proliferation and migration of cervical cancer cells by down-regulating Skp2 protein. Overexpression of 2.Skp2 protein may be an index to predict the poor prognosis of patients with cervical squamous cell carcinoma. The detection of Skp2 protein can be used as an effective molecular marker for the detection of proliferation index and prognosis evaluation of cervical squamous cell carcinoma.
【学位授予单位】：延边大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.33
本文编号：2193634
[Abstract]:Background: as the second largest malignant tumor in women, cervical cancer is a double targeting inhibitor of phosphatidylinositol -3 (PI3K) and its downstream mammal rapa toxin (mammalian target of rapamycin, mTOR). Although it has been shown to inhibit many kinds of tumors, the role of S phase kinase associated protein 2 (Skp2) in cervical carcinoma is less reported, which plays an important role in the regulation of cell cycle, and participates in cell proliferation and apoptosis. It has been found that Skp2 protein is overexpressed in many malignant tumors and leads to poor prognosis of tumor patients. The effect of Skp2 on tumor development is regulated by PI3K signaling pathway. PI3K signaling pathway can regulate Skp2 protein before and after translation in many kinds of malignant tumors. Objective: to investigate the effect of PI3K inhibitor BEZ235 on the proliferation and migration of cervical cancer cells by down-regulating Skp2, and to explore the clinicopathological significance of the expression of Skp2 protein in cervical squamous cell carcinoma. Materials and methods: the first part: the passage culture of HelaA33AnSiHa cell line of cervical cancer, the effect of BEZ235 on cell growth and proliferation was detected by thiazolyl (MTT) assay, and the growth curve was drawn, and the drug concentration was screened by adding 0.4 渭 M BEZ235 drug to Hela and C33A cells. The control group was treated with DMSO-containing the same volume for 48h, then RNA and protein were extracted, the changes of Skp2mRNA in cervical cancer cells were detected by RT-PCR method, and the changes of Skp2Ezrin1 protein in cervical cancer cells were detected by Western-blot assay. The effect of BEZ235 on the migration of Hela cells was detected by scratch test. The second part: immunohistochemical method was used to detect the expression of Skp2 protein in 84 cases of cervical intraepithelial neoplasia (CIN) and 163 cases of cervical squamous cell carcinoma (SCC) in 25 cases of normal cervical epithelial tissue and 163 cases of cervical squamous cell carcinoma. The expression of human papillomavirus (HPV) in different tissues of cervical cancer was detected by RT-PCR, and the clinicopathological significance of the expression of Skp2 protein in cervical carcinoma was examined by combining with the biological characteristics of cervical cancer HPV infection and clinical staging. The effect of Skp2 protein expression on survival time was analyzed statistically. Results: the results of the first part of the experiment showed that blocking the PI3K signaling pathway with BEZ235 could inhibit the proliferation of HelaC33A and SiHa cells by RT-PCR and Western blot. The expression level of Skp2mRNA and Slp2Ezrininosin Sixl protein in cervical cancer Hela and C33A cells decreased with the increase of BEZ235 concentration (0.1 渭 M, 0.4 渭 M), and the results of scratch test showed that the cell migration distance of cervical cancer Hela cells treated with BEZ235 for 24 h or 48 h was significantly shorter than that of the control group. In the second part, the positive rates of Skp2 in CIN-1 and CIN-2 and CIN-3 were increased in 11 of 14 cases of cervical carcinoma by RT-PCR, and were higher than those in normal tissues (P0.01). The positive rate of Skp2 protein in cervical squamous cell carcinoma was significantly higher than that in normal cervical epithelium (P0.01). The expression of Skp2 protein was closely related to FIGO stage and high risk HPV infection (P0.05). The tumor-free survival rate and overall survival rate of cervical squamous cell carcinoma patients with positive expression of Skp2 protein were significantly lower than those with negative expression (P0.01). Conclusion 1. PI3K inhibitor BEZ235 inhibits the proliferation and migration of cervical cancer cells by down-regulating Skp2 protein. Overexpression of 2.Skp2 protein may be an index to predict the poor prognosis of patients with cervical squamous cell carcinoma. The detection of Skp2 protein can be used as an effective molecular marker for the detection of proliferation index and prognosis evaluation of cervical squamous cell carcinoma.
【学位授予单位】：延边大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.33
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