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miR-143对肾癌GRC-1细胞增殖与凋亡及其靶点HIF-1α的影响

李栋1,刘希胜2,王新明1,王帅1,陶肖馨3   

  1. 1 溧阳市人民医院放射科 2南京医科大学第一附属医院放射科 3 溧阳市人民医院放射科
  • 收稿日期:2016-07-31 修回日期:2016-09-19 出版日期:2016-11-30 发布日期:2016-11-30
  • 通讯作者: 刘希胜

Effects of miR-143 on proliferation and apoptosis of renal cell carcinoma GRC-1 cells and its target HIF-1α

LI Dong, LIU Xisheng, WANG Xinming, WANG Shuai, TAO Xiaoxin.   

  1. Department of Radiology, Liyang Municipal People's Hospital
  • Received:2016-07-31 Revised:2016-09-19 Online:2016-11-30 Published:2016-11-30
  • Contact: LIU Xisheng

摘要: 目的 探讨miR-143对肾癌GRC-1细胞增殖凋亡及其靶点缺氧诱导因子1α(HIF-1α)的影响。
方法 转染miR-143的模拟物于肾癌细胞株GRC-1细胞,采用实时定量PCR(qPCR)法检测转染的效果,根据实验将GRC-1细胞分为3组:未转染组、miR-143对照组和miR-143转染组,采用MTT法检测各组细胞的增殖情况,流式细胞术检测各组细胞凋亡情况;qPCR及Western blotting检测各组细胞的HIF-1α mRNA和蛋白水平;双荧光素酶报告基因实验验证miR-143与HIF-1α基因间的靶向关系。
结果 miR-143转染组的miR-143水平高于未转染组和miR-143对照组,差异有统计学意义(P<0.05);与其余两组比较,miR-143转染组的增殖率降低而凋亡率升高,差异有统计学意义(P<0.05);瞬时过表达miR-143可降低GRC-1细胞的HIF-1α mRNA和蛋白水平(P<0.01),双荧光素酶报告基因实验证明HIF-1α是miR-143的直接作用靶点。结论 miR-143可以调控肾癌GRC-1细胞的增殖凋亡,而HIF-1α是miR-143的直接靶点。

Abstract: Objective To investigate the effects of miR-143 on proliferation and apoptosis of renal cell carcinoma GRC-1 cells and its target hypoxia inducible factor-1α (HIF-1α).
MethodsRenal cell carcinoma cell line GRC-1 cells were transfected with miR-143 mimics, and real time quantitative polymerase chain reaction (qPCR) was performed to evaluate the efficiency of transfection. According to the experiment, the GRC-1 cells were divided into 3 groups: non-transfection group, miR-143 control group and miR-143 transfection group. MTT method was used to detect the proliferation of cells in each group. Flow cytometry was used to detect the apoptosis of each group. The qPCR and Western blotting were employed to measure the mRNA and protein level of HIF-1α. Dual luciferase reporter gene was applied to verify the relationship between miR-143 and HIF-1α. Results The level of miR-143 in miR-143 transfection group was higher than those in non-transfection group and miR-143 control group, and the difference was statistically significant (P<0.05). Compared with other two groups, there were decreased proliferation rates but increased apoptotic rates in miR-143 transfection group (P<0.05). Transient overexpression of miR-143 in GRC-1 cells decreased the expression of HIF-1α on both mRNA and protein levels. Moreover, dual-luciferase reporter assay confirmed that HIF-1α was a direct target of miR-143 in GRC-1 cells.
ConclusionmiR-143 may regulate the proliferation of GRC-1 cells, and HIF-1α is a direct target of miR-143.

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