miR-20a通过靶向抑制PTEN诱导肝癌细胞放射抵抗

miR-20a induces cell radioresistance by targeting PTEN in hepatocellular carcinoma

目的 探讨miR-20a在肝癌放射敏感性中的作用及机制.方法 荧光定量PCR检测肝癌细胞系和组织标本中miR-20a的表达;构建稳定过表达miR-20a肝癌细胞系,通过克隆实验探讨miR-20a对肝癌细胞放射敏感性影响;蛋白印记法检测Bcl-2、Caspase-3以及 γ-H2AX的表达;生物信息学预测miR-20a下游调控的靶基因,双荧光素酶报告系统、荧光定量PCR及蛋白印记法进一步验证;在稳定过表达miR-20a的肝癌细胞系中转染pCDNA3.0-PTEN探讨细胞放射敏感性的变化,明确PTEN是否为miR-20a诱导肝癌放射抵抗的一个功能性靶基因.结果 miR-20a在肝癌细胞系和组织标本中表达上调（P〈0.05）;经过同等条件的放射处理后,与空白及对照组（WT组、LV-con组）相比,过表达miR-20a组（LV-miR-20a组）细胞存活分数升高,放射抵抗增强（P〈0.05）,Bcl-2表达上调,Caspase-3及 γ-H2AX表达下调;过表达PTEN能够逆转miR-20a引起的放射抵抗.结论 miR-20a在肝癌细胞系及组织标本中表达上调;过表达miR-20a可以促进肝癌细胞放射抵抗,PTEN是miR-20a诱导肝癌放射抵抗的一个功能性靶基因,预示着miR-20a/PTEN位点可能是是肝癌临床放疗相关的有效分子靶点.

Objective To explore the role and mechanism of miR-20a in the radiosensitivity of hepatocellular carcinoma （HCC）. Methods The expression level of miR-20a in HCC cell lines and tissue specimens was detected by real-time fluorescent quantitative PCR.HCC cell line stably over-expressing miR-20a was constructed. The effect of miR-20a on HCC cell radiosensitivity was evaluate by cloning assay. The expression levels of Bcl-2,Caspase-3 and γ-H2AX proteins were quantitatively detected by Western blot. The target gene of the downstream regulation of miR-20a was predicted by bioinformatics analysis,which was further verified by dual luciferase reporter assay,real-time fluorescent quantitative PCR and Western blot. HCC cell line stably overexpressing miR-20a was transfected with pCDNA3. 0-PTEN to investigate the changes in the radiosensitivity of cells and to determine whether PTEN is a functional target gene for miR-20a-induced radioresistance of HCC. Results The expression levels of miR-20a was significantly up-regulated in HCC cell line and tissue specimens （ both P〈 0. 05）. After identical radiotherapy, the cell survival rate,the radioresistance was strengthened （ P〈 0. 05）,the expression of Bcl-2 was up-regulated, whereas the expression levels of Caspase-3 and γ-H2AX were down-regulated in the LV-miR-20a group compared with those in the blank and control groups （ WT and LV-con groups）. Overexpression of PTEN could reverse the miR-20a-induced radioresistance. Conclusion miR-20a is up-regulated in the HCC cell lines and tissue specimens. Overexpression of miR-20a can promote the radioresistance of HCC cells. PTEN is a functional target gene for miR-20a-induced radioresistance of HCC,indicating that miR-20a/ PTEN site may be an effective molecular target associated with clinical radiotherapy for liver cancer.