BEZ235體外抑制宮頸癌增殖與遷移作用研究及Skp2過表達的臨床意義
發(fā)布時間：2018-08-20 12:37
【摘要】：研究背景：宮頸癌作為女性第二大惡性腫瘤,發(fā)病率僅次于乳腺癌。BEZ235是磷脂酰肌醇-3(PI3K)及其下游因子哺乳動物雷帕毒素蛋白(mammalian target of rapamycin, mTOR)的雙重靶向抑制劑,已表現(xiàn)出抑制多種腫瘤的作用,但在宮頸癌組織中的作用報道較少。S期激酶相關蛋白2(Skp2)是在調(diào)控細胞周期中發(fā)揮重要作用的蛋白,并參與細胞的增殖及凋亡。目前已發(fā)現(xiàn)Skp2蛋白在多種惡性腫瘤中呈過表達且導致腫瘤患者的不良預后。文獻報道,Skp2對腫瘤發(fā)生發(fā)展的影響受PI3K信號通路的調(diào)控。在多種惡性腫瘤中,PI3K信號通路均可在轉錄和翻譯前、后水平上對Skp2蛋白進行調(diào)控。 研究目的：探討宮頸癌細胞中PI3K抑制劑BEZ235通過下調(diào)Skp2影響宮頸癌細胞的增殖及遷移能力；探討Skp2蛋白在宮頸鱗狀細胞癌組織中表達的臨床病理學意義。 材料和方法：第一部分：宮頸癌Hela、C33A、SiHa細胞的傳代培養(yǎng)；噻唑藍(MTT)實驗檢測BEZ235對細胞生長及增殖情況的影響,并繪制生長曲線,篩選藥物濃度；Hela和C33A細胞中加入0.1μM、0.4μM的BEZ235藥物,對照組加入等體積的DMSO,作用48h后,提取RNA及蛋白,通過RT-PCR法檢測宮頸癌細胞中Skp2mRNA的變化,并用Western-blot法檢測宮頸癌細胞中Skp2、Ezrin、Six1蛋白的變化；劃痕實驗法檢測BEZ235對Hela細胞遷移能力的影響。第二部分：免疫組化方法檢測在25例正常宮頸上皮組織、84例宮頸上皮內(nèi)瘤變(CIN)和163例宮頸鱗狀細胞癌(SCC)中Skp2蛋白的表達情況；RT-PCR法檢測人乳頭瘤病毒(HPV)在宮頸癌不同組織中的表達情況；結合宮頸癌HPV 感染情況、臨床分期等生物學特點檢驗Skp2蛋白在宮頸癌組織表達的臨床病理學意義；統(tǒng)計學分析Skp2蛋白表達對患者生存時間的影響。 結果：第一部分：MTT實驗結果表明,用BEZ235阻斷PI3K信號通路可抑制宮頸癌細胞Hela、C33A和SiHa的增殖；RT-PCR和Western blot結果表明,宮頸癌Hela和C33A細胞中Skp2mRNA和Slp2、Ezrin、Sixl蛋白的表達水平均隨BEZ235的濃度(0.1μM,0.4μM)增高而降低；劃痕試驗結果顯示,對宮頸癌Hela細胞進行BEZ235藥物處理24h、48h后,細胞移動距離較對照組明顯縮短。第二部分：RT-PCR檢測HPV mRNA在14例宮頸癌組織中有11例呈陽性表達；Skp2在CIN-1、CIN-2和CIN-3中的陽性率依次增高,且均高于正常組織(P均0.01)；在宮頸鱗狀細胞癌中Skp2蛋白呈明顯的彌漫性強陽性染色,其陽性率顯著高于正常宮頸上皮組織(P0.01)；Skp2蛋白表達與FIGO分期及高危型HPV感染關系密切(P0.05)；Skp2蛋白陽性表達的宮頸鱗狀細胞癌患者無瘤生存率及總生存率明顯低于陰性表達的患者(P均0.01)。 結論：1.PI3K抑制劑BEZ235通過下調(diào)Skp2蛋白抑制宮頸癌細胞增殖與遷移能力；2.Skp2蛋白過表達可能是預示宮頸鱗狀細胞癌患者的不良預后的檢測指標,Skp2蛋白檢測可作為宮頸鱗狀細胞癌增殖指數(shù)測定及預后評估的有效分子標志物。
[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.
【學位授予單位】：延邊大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R737.33
本文編號：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.
【學位授予單位】：延邊大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R737.33
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