miR181c promotes apoptosis and suppresses proliferation of metanephric mesenchyme cells by targeting Six2 in vitro
miR181c promotes apoptosis and suppresses proliferation of metanephric mesenchyme cells by targeting Six2 in vitro
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DOI：
10.1002/cbf.3052
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发表时间：
2014-10-01
影响因子：
3.6
通讯作者：
Zhou, Qin
中科院分区：
文献类型：
--
作者：
Lv, Xiaoyan;Mao, Zhaomin;Zhou, Qin
microRNA（miRNA）在调节基因表达的微妙平衡中的重要性日益被认识到。在我们的研究中，我们的目的是探讨miR 181 c对Six 2在后肾间充质（MM）细胞中的调节作用。采用生物信息学分析、荧光素酶分析、半定量实时荧光定量PCR、RT PCR、Western blotting、5-乙炔基-2-脱氧尿苷细胞增殖试验、Cell Counting Kit-8试验、免疫荧光和流式细胞术等方法验证miR 181 c对MM细胞系mK 3中Six 2的调控作用。通过生物信息学分析预测miR 181 c结合Six 2的3个非翻译区，随后通过体外荧光素酶报告基因测定验证。miR 181 c模拟物转染mK 3细胞后，Six 2在mRNA和蛋白水平的表达均降低。miR 181 c的异位表达抑制了mK 3细胞的增殖，促进了mK 3细胞的凋亡，甚至使mK 3细胞的表型丧失。这些结果揭示了单个miRNA-miR 181 c能够下调Six 2的表达，抑制增殖并促进凋亡，甚至使肾单位祖细胞表型丧失MM细胞，提示miR 181 c在肾脏发育中的潜在作用。版权所有（c）2014约翰威利父子有限公司
Increasingly recognized importance has been assumed for microRNA (miRNA) in the regulation of the delicate balance of gene expression. In our study, we aimed to explore the regulation role of miR181c towards Six2 in metanephric mesenchyme (MM) cells. Bioinformatics analysis, luciferase assay and semi-quantitative real-time (RT) PCR, subsequently RT PCR, Western blotting, 5-ethynyl-2-deoxyuridine cell proliferation assay, Cell Counting Kit-8 assay, immunofluorescence and flow cytometry, were employed to verify the modulation function of miR181c on Six2 in the mK3 MM cell line that is one kind of MM cells. miR181c was predicted to bind the 3 untranslated region of Six2 by bioinformatics analysis, which was subsequently validated by the in vitro luciferase reporter assay. Moreover, transfection of miR181c mimic can decrease the expression of Six2 both in mRNA and protein levels in mK3 cells. Still, ectopic expression of miR181c inhibits the proliferation, promotes the apoptosis and even makes the nephron progenitor phenotype lose mK3 cells. These results revealed the ability of a single miRNA-miR181c to downregulate the expression of Six2, restrain the proliferation and promote the apoptosis that even makes the nephron progenitor phenotype lose MM cells, suggesting a potential role of miR181c during the kidney development. Copyright (c) 2014 John Wiley & Sons, Ltd.