MiR-30a-5p通过泛素水解酶22抑制人宫颈癌细胞上皮-间质转化功能

MiR-30a-5p Inhibits Epithelial Mesenchymal Transition Function in Human Cervical Cancer Cells by Targeting Ubiquitin-specific Protease 22

【目的】研究microRNA-30a-5p(miR-30a-5p)对人宫颈癌Hela细胞上皮-间质转化功能的影响及其相关机制。【方法】宫颈癌Hela细胞株分别转染目的mir的模拟物和阴性对照模拟物,分别以30a-5p组、NC组命名并标记细胞。同时,以未经过处理的Hela细胞作为对照(Control组)。分别用逆转录-聚合酶链反应法检测各组宫颈癌细胞的miR-30a-5p含量。Transwell实验检测3组细胞迁移能力和侵袭能力。Western-blot法检测3组细胞神经-钙粘素(N-cadherin)、α-连环蛋白(α-Catenin)和泛素水解酶22(USP22)表达水平。运用生物信息学方法预测miR-30a-5p的靶基因。采用Westernblot法检测USP22过表达对miR-30a-5p抑制EMT的拮抗作用。双荧光素酶实验检测miR-30a-5p与USP22的关系。建立皮下移植瘤模型观察miR-30a-5p的体内作用。【结果】30a-5p组宫颈癌细胞miR-30a-5p的表达水平明显上调,表达水平为Control组的853.82(862.26~843.11)倍(P<0.01)。30a-5p组侵袭细胞数量8.17(8.32~8.03)明显低于Control组(P<0.01)。30a-5p组细胞N-cadherin蛋白的细胞内含量明显下降,α-Catenin蛋白的细胞内含量明显上升,USP22蛋白表达量明显降低。合并USP22过表达处理的30a-5p组宫颈癌细胞中N-cadherin蛋白表达量明显升高,α-Catenin蛋白表达量明显降低。双荧光素酶检验结果显示USP22为miR-30a-5p的下游靶基因(P<0.01)。30a-5p组皮下移植瘤明显小于Control组(P<0.01)。与Control组肿瘤组织相比,30a-5p组肿瘤组织miR-30a-5p的相对含量升高,USP22蛋白含量降低,Ncadherin蛋白的含量降低,α-Catenin蛋白含量升高。【结论】miR-30a-5p在宫颈癌Hela细胞中,可能通过靶向识别下游靶基因USP22,进而抑制其翻译。最终实现对宫颈癌细胞EMT过程的抑制。

【Objective】To investigate the inhibitory effect and mechanism of microRNA-30a-5p(miR-30a-5p)on epithelial mesenchymal transition in cervical cancer Hela cells.【Methods】Hela cervical cancer cell lines were transfected with miR-30a-5p mimics or negative control mimic,respectively ,as 30a-5p or NC group. Control group was established with untreated Hela cervical cancer cells. miR-30a-5p content in each group was detected by RT-PCR assay. Transwell assay was used to detect the invasion ability of the 3 groups. Western-blot assay was used to detect the expressions of Ncadherin,α-Catenin and ubiquitin specific processing peptidase 22 (USP22)protein in the 3 groups. Prediction target genes of miR-30a-5p by bioinformatics methods. Antagonistic effect of USP22 over-expression on miR-30a-5p inhibition of EMT was detected by western blot assay. The relationship between miR-30a-5p and USP22 was detected by dual luciferase assay. Subcutaneous transplantation tumor model established,and the effect of miR-30a-5p in vivo was observed.【Results】The miR-30a-5p intracellular quantity in 30a-5p group Hela cells was up-regulated,and the expression level of miR-30a-5p was 853.82(862.26~843.11)times higher than that of Control group(P<0.01). The number of invasive cells in 30a-5p group was 8.17(8.32~8.03),which was significantly lower than that of Control group 62.33 (63.52~60.19)(P<0.01). USP22 may be the target gene of miR-30a-5p. In 30a-5p group,the intracellular quantity of N-cadherin protein was decreased,the intracellular quantity of α-Catenin protein was increased,and the intracellular quantity of USP22 protein was decreased. The intracellular quantity of N-cadherin protein in 30a-5p group was down-regulated,the intracellular quantity of α-Catenin protein was increased,and the intracellular quantity of USP22 protein was reduced. After USP22 over-expression,the intracellular quantity of N-cadherin protein in cervical cancer cells of 30a-5p group was up-regulated,and the intracellular quantity of α- Catenin protein were down-regulated. Dual luciferase assay showed that USP22 is a downstream target gene of miR-30a-5p(P <0.01). Subcutaneous transplantation tumors in 30a- 5p group were significantly smaller than those in Control group.【Conclusion】miR-30a-5p may inhibit the expression of EMT related protein through the downstream target gene USP22, and the epithelial mesenchymal transition function of cervical cancer Hela cells.