ICAT对宫颈癌增殖和转移的作用及其机制研究

发布时间：2018-04-14 03:36

本文选题：ICAT + 宫颈癌　；参考：《重庆医科大学》2017年硕士论文

【摘要】：目的:研究ICAT在人宫颈癌组织中的表达情况,并探讨ICAT对宫颈癌增殖和转移的作用及其分子机制。方法:1.IHC检测宫颈癌组织(n=41)及正常宫颈组织(n=30)中ICAT蛋白的表达,Western blot法检测三株宫颈癌细胞系(CaSki、SiHa和He La)中ICAT的内源性表达。2.用ICAT过表达腺病毒(AdICAT)感染低表达ICAT的SiHa细胞,ICAT小干扰RNA(siICAT)转染高表达ICAT的CaSki细胞;分别用RT-PCR和Western blot法验证细胞系中ICAT在转录水平和蛋白水平表达;MTT、流式细胞术检测细胞的增殖能力;Transwell迁移和侵袭实验检测细胞迁移和侵袭能力;Western blot法检测各组细胞增殖相关CyclinD1和转移相关蛋白MMP9表达;Western blot法和IF检测过表达ICAT和降低内源性ICAT后宫颈癌细胞EMT标志物(E-cadherin和Vimentin)的表达;提取宫颈癌细胞的胞浆和胞核蛋白,Western blot法和IF法验证β-catenin在宫颈癌细胞的表达;IP实验检测ICAT对E-cadherin/β-catenin复合物的影响。3.体内实验分SiHa、SiHa/RFP、SiHa/ICAT三组,每组2×107个细胞,注射到裸鼠皮下,观察瘤体生长;剥离瘤体组织,石蜡包埋,切片,he染色;ihc检测各组瘤体ki-67、mmp9、e-cadherin和vimentin的表达。结果:1.标本评分显示,宫颈癌组织的icat的ihc平均分数为7.366±0.3916,而宫颈正常组织中为5.000±0.6215(p0.01);ihc结果显示,icat主要表达于癌细胞的胞浆;rt-pcr和westernblot结果一致,三株宫颈癌细胞系中,icat在转录水平和蛋白水平的表达siha最低,caski最高(p0.05)。2.adicat的感染可以显著促进siha细胞在转录和蛋白水平icat的表达(p0.01),siicat可以有效的干扰caski细胞在转录水平和蛋白水平icat的表达(p0.01);adicat感染后,与siha组和siha/rfp组相比,siha/icat组细胞的增殖明显加快(p0.01),s期所占比例明显增加(p0.05),穿膜细胞数增加两倍(p0.01);siicat转染后,与caski/sinc组相比,caski/siicat组细胞的吸光度值明显降低(p0.01),细胞s期所占比例减低(p0.05),穿膜细胞数减少一半(p0.01);过表达icat后,siha/icat组细胞内的e-cadherin表达降低,vimentin表达增加(p0.05);干扰掉内源性icat表达后,caski/siicat组细胞得到了相反的结果(p0.05);β-catenin在宫颈癌细胞中主要异常表达于胞浆;过表达icat后,复合物e-cadherin/β-catenin的表达降低。3.体内实验结果显示:siha/icat组瘤体明显大于siha组和SiHa/RFP组(P0.01);HE染色显示,SiHa/ICAT组细胞排列疏松、紊乱,核大深染;免疫组化结果显示,SiHa/ICAT组Ki-67、MMP9和Vimentin表达均增强而E-cadherin表达降低。结论:1.ICAT在宫颈癌组织中的表达显著高于正常组织且主要表达于癌细胞的胞浆。2.ICAT促进宫颈癌细胞的增殖、迁移、和侵袭。3.ICAT促进宫颈癌细胞的EMT,这一作用与ICAT竞争性结合β-catenin相关。
[Abstract]:Aim: to investigate the expression of ICAT in human cervical carcinoma, and to explore the role of ICAT in the proliferation and metastasis of cervical carcinoma and its molecular mechanism.Methods 1. IHC was used to detect the expression of ICAT protein in cervical carcinoma tissue (41) and normal cervix cervix (30). Western blot assay was used to detect the endogenous expression of ICAT in three cervical cancer cell lines (CaSkisiha and he La).ICAT overexpression adenovirus was used to infect SiHa cells with low expression of ICAT. Small interfering RNAs were transfected into CaSki cells with high expression of ICAT.RT-PCR and Western blot were used to verify the expression of ICAT at transcription level and protein level, respectively. Flow cytometry was used to detect cell proliferation ability. Transwell migration and invasion assay was used to detect cell migration and invasion ability. Western blot assay was used to detect cell migration and invasion.The expression of proliferation-associated CyclinD1 and metastasis associated protein MMP9 was detected by Western blot and if, and the expression of EMT markers E-cadherin and Vimentin in cervical cancer cells after endogenous ICAT was decreased.The expression of 尾 -catenin in cervical cancer cells was detected by IP assay. The effect of ICAT on E-cadherin/ 尾 -catenin complex was detected by IP assay.The mice were divided into three groups: SiHaHN / Si-RFP- SiHa- / ICAT. Each group of 2 脳 107 cells was injected into nude mice subcutaneously to observe the growth of the tumor, the tumor tissue was stripped, embedded in paraffin, and the expression of ki-67 mmp9e-cadherin and vimentin in each group was detected by HE staining.The result is 1: 1.The mean ihc score of icat in cervical cancer tissues was 7.366 卤0.3916, while that in normal cervical tissues was 5.000 卤0.6215p0.01p0.01ihc. The results of rt-PCR and westernblot were consistent.Expression of icat at transcriptional and protein levels in three cervical cancer cell lines the expression of icat in siha cells at the transcriptional and protein-level levels can be significantly enhanced by the infection of the lowest siha caski p0.05. 2.The expression of icat at the transcriptional and protein-level levels of caski cells can be effectively interfered with by p0.01siicat.And the expression of icat at the protein level after infection,Compared with the siha group and siha/rfp group, the proliferation of the cells increased significantly in the p0.01 / s phase, and the number of perforating cells increased twice after transfection.Compared with the caski/sinc group, the absorbance value of the cells in the caskey / siicat group was significantly lower than that in the caski/sinc group, the proportion of cells in the s phase was decreased by p0.05, the number of perforated cells was reduced by half, and the expression of e-cadherin in the cells of the icat overexpression group was decreased, and the expression of vimentin was increased by p0.05, which interfered with the endogenous icat surface.The results showed that 尾 -catenin was mainly expressed in the cytoplasm of cervical cancer cells.After overexpression of icat, the expression of e-cadherin/ 尾 -catenin decreased.The results of in vivo experiments showed that the tumor in the siha group was significantly larger than that in the siha group and the SiHa/RFP group. The results of HE staining showed that the cells in the SiHaR / ICAT group were loose, disordered and heavily stained, and the immunohistochemical results showed that the expression of Ki-67 MMP9 and Vimentin in the SiHaR / ICAT group was increased and the E-cadherin expression was decreased.Conclusion: 1. The expression of ICAT in cervical carcinoma is significantly higher than that in normal tissues and mainly expressed in the cytoplasm of cancer cells. 2. ICAT promotes the proliferation and migration of cervical cancer cells, and invasiveness. 3. ICAT promotes the EMTs of cervical cancer cells, which is related to the competitive binding of ICAT to 尾 -catenin.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.33

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