微小RNA-101通过USP22抑制人肝癌MHCC97H细胞的上皮-间质转化功能

MicroRNA-101 inhibits epithelial-mesenchymal transition in human liver carcinoma MHCC97H cells via USP22

  • 摘要:
      目的   研究微小核糖核酸(miR)-101对人肝细胞癌(肝癌)MHCC97H细胞的上皮-间质转化功能的影响及其相关机制。
      方法   MHCC97H肝癌细胞株分别转染miR-101 mimics和negative control mimic,分别作为M101组、NCM组,并设立未转染对照(control)组,采用逆转录聚合酶链反应(RT-PCR)法检测各组细胞miR-101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vimentin、α-catenin、E-cadherin和USP22表达水平。双荧光素酶实验检测miR-101与USP22的关系。
      结果   M101组miR-101的表达水平明显上调,表达水平约为control组的761倍(P < 0.05)。M101组迁移细胞数量明显低于control组的(15.7±1.6)个比(94.1±1.8)个,P < 0.05。M101组侵袭细胞数量明显低于control组的(9.1±0.4)个比(51.6±0.9)个,P < 0.05。M101组细胞vimentin蛋白表达量明显降低,α-catenin及E-cadherin蛋白表达量明显升高,USP22蛋白表达量明显降低。双荧光素酶检验结果显示USP22为miR-101的下游靶基因。
      结论   miR-101可能通过降低下游靶基因USP22水平影响EMT相关蛋白表达,抑制MHCC97H肝癌细胞的上皮-间质转化功能。

     

    Abstract:
      Objective   To investigate the effect and underlying mechanism of micro ribonucleic acid (miR)-101 on the epithelial-mesenchymal transition in human hepatocellular carcinoma MHCC97H cells.
      Methods   MHCC97H cell lines were transfected with miR-101 mimics (M101 group) and negative control mimic (NCM group), and the cell lines without transfection were used as the control group. The expression levels of miR-101 in cells of 3 groups were quantitatively measured using reverse transcription polymerase chain reaction (RT-PCR). Transwell assay was performed to evaluate the cell migration and invasion ability of 3 groups. The expression levels of vimentin, -catenin, E-cadherin and USP22 proteins in cells of 3 groups were measured by Western blot. The relationship between miR-101 and USP22 was analyzed by dual-luciferase assay.
      Results   In the M101 group, the expression level of miR-101 was significantly up-regulated, which was approximately 761 times of that in the control group (P < 0.05). In the M101 group, the quantity of migrating cells was 15.7±1.6, significantly lower than 94.1±1.8 in the control group (P < 0.05). In the M101 group, the quantity of invasive cells was 9.1±0.4, significantly lower compared with 51.6±0.9 in the control group (P < 0.05). In the M101 group, the expression levels of vimentin and USP22 protein were significantly down-regulated, whereas the expression levels of -catenin and E-cadherin protein were significantly up-regulated. Dual-luciferase assay revealed that USP22 was the target gene of the downstream miR-101 signaling pathway.
      Conclusion   miR-101 regulates the expression of epithelial-mesenchymal transition-related proteins and suppresses the epithelial-mesenchymal transition of hepatocellular carcinoma MHCC97H cells probably through down-regulating the expression level of USP22.

     

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