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目的探讨hsa-miR-200a对人子宫内膜腺癌HEC-1B细胞增殖与凋亡的影响及其作用机制。方法应用荧光实时定量PCR(FQ-PCR)法检测hsa-miR-200a在人子宫内膜腺癌细胞HEC-1B中的表达水平;人工合成hsa-miR-200a模拟物、抑制剂,分别转染HEC-1B细胞;采用MTT法、流式细胞仪检测上调及抑制hsa-miR-200a的表达对HEC-1B细胞增殖活性与凋亡的影响;运用生物信息学分析hsa-miR-200a指向的与增殖和凋亡相关的靶基因;Western blot、FQ-PCR检测转染前后靶蛋白及其mRNA的表达水平。结果子宫内膜腺癌细胞HEC-1B表达hsa-miR-200a;FQ-PCR检测hsa-miR-200a模拟物、抑制剂转染HEC-1B细胞成功;MTT法检测结果表明hsa-miR-200a抑制剂转染组中细胞增殖抑制明显;流式细胞仪检测结果表明hsa-miR-200a抑制剂转染组中细胞凋亡增加;生物信息学分析结果表明hsa-miR-200a调控的靶基因是SON、Pdcd4、磷酸酶-张力蛋白同源基因(phosphatase and tensin homolog deleted on chromosome ten,PTEN);靶蛋白PTEN在转染模拟物组表达下调,抑制剂组中表达上调,且转染前后靶基因mRNA水平无显著变化,而蛋白SON、Pdcd4的表达量没有显著变化。结论 HEC-1B细胞中hsa-miR-200a表达与PTEN蛋白表达呈负相关,表明hsa-miR-200a可能通过调节PTEN蛋白表达水平从而影响HEC-1B细胞的增殖与凋亡。
Objective To investigate the effect and mechanism of hsa-miR-200a on proliferation and apoptosis of human endometrial adenocarcinoma HEC-1B cells. Methods The expression of hsa-miR-200a in human endometrial adenocarcinoma HEC-1B cells was detected by real-time fluorescence quantitative PCR (FQ-PCR). The hsa-miR-200a mimics and inhibitors were transfected HEC-1B cells. MTT assay was used to detect the effect of hsa-miR-200a on the proliferation and apoptosis of HEC-1B cells by flow cytometry. The level of hsa-miR- Proliferation and apoptosis. Western blot and FQ-PCR were used to detect the expression of target protein and its mRNA before and after transfection. Results Hsa-miR-200a was expressed in endometrial adenocarcinoma cell line Hsa-miR-200a. Hsa-miR-200a mimics were detected by FQ-PCR and HEC-1B cells were transfected successfully. The results of MTT assay showed that hsa-miR- The results of flow cytometry showed that apoptosis increased in hsa-miR-200a inhibitor-transfected group. Bioinformatics analysis showed that the target gene regulated by hsa-miR-200a was SON , Pdcd4, and PTEN. The target protein PTEN was down-regulated in transfection mock group and up-regulated in inhibitor group, and the mRNA expression of target gene before and after transfection No significant changes in the level of protein SON, Pdcd4 expression did not change significantly. Conclusions The expression of hsa-miR-200a in HEC-1B cells is negatively correlated with the expression of PTEN protein, indicating that hsa-miR-200a may affect the proliferation and apoptosis of HEC-1B cells by regulating the expression of PTEN protein.