Yang Dehui,Chen Xuemei,He Junlin,Wang Yingxiong,Ding Yubin,Li Rong,Liu Xueqing.Effect of hsa-miR-200a on proliferation and apoptosis in human endometrium adenocarcinoma cell line HEC-1B[J].J Amry Med Univ (J Third Mil Med Univ),2012,34(16):1608-1612.
Yang Dehui,Chen Xuemei,He Junlin,Wang Yingxiong,Ding Yubin,Li Rong,Liu Xueqing.Effect of hsa-miR-200a on proliferation and apoptosis in human endometrium adenocarcinoma cell line HEC-1B[J].J Amry Med Univ (J Third Mil Med Univ),2012,34(16):1608-1612.DOI:
Effect of hsa-miR-200a on proliferation and apoptosis in human endometrium adenocarcinoma cell line HEC-1B
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。
Abstract
Objective To investigate the effect of hsa-miR-200a on the proliferation and apoptosis in human endometrium adenocarcinoma cell line HEC-1B and the potential mechanisms. Methods The expression of hsa-miR-200a in HEC-1B cell line was measured by real-time fluorescence quantitative PCR (FQ-PCR). The hsa-miR-200a mimics and inhibitor were synthesized and transferred into HEC-1B cell line by lipofectamine
respectively
and the cell proliferation and apoptosis were detected by methyl-thiazolyl-tetrazolium (MTT) assay and flow cytometry (FCM). Bioinformatics analysis was used to predict the target genes of has-miR-200a on cell proliferation and apoptosis. Western blotting and FQ-PCR were used to determine the protein and mRNA expression of phosphatase and tensin homolog deleted on chromosome ten (PTEN). Results Hsa-miR-200a expression was observed in HEC-1B cells. The hsa-miR-200a mimics and inhibitor were successfully transferred into HEC-1B cells
and the expression of hsa-miR-200a was significantly changed after the transfection. Compared with those of the blank control group and negative control group
HEC-1B cell proliferation was significantly inhibited in the miR-200a inhibitor group. The FCM results showed that the apoptotic rate increased in the hsa-miR-200a inhibitor group. The bioinformatics analysis results showed that SON
Pdcd4 and PTEN were the target genes of has-miR-200a. PTEN protein expression significantly decreased in the HEC-1B cells transfected with hsa-miR-200a mimics
and significantly increased in the HEC-1B cells transfected with hsa-miR-200a inhibitor. SON and Pdcd4 protein expression did not change before and after transfection. Conclusion Hsa-miR-200a is negatively correlated with PTEN protein expression in HEC-1B cell line
indicating that hsa-miR-200a may affect the proliferation and apoptosis in HEC-1B cells by regulating PTEN protein expression.