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目的探讨转化生长因子β(TGF-β)对miR-200b-200a-429簇表达的影响,对其调控机制作初步研究。方法使用RT-PCR方法检测TGF-β对人肾小管上皮细胞HK-2miR-200a、miR-200b和miR-429表达的影响。PCR方法扩增出miR-200b-200a-429簇启动子序列,构建miR-200b-200a-429启动子荧光素酶报告基因表达载体,转染至HK-2细胞,检测其荧光素酶活性。观察TGF-β对miR-200b-200a-429转录的表达调控。结果实时定量PCR检测结果显示TGF-β作用细胞24h后下调了miR-200a、miR-200b和miR-429的表达。成功构建miR-200b-200a-429启动子荧光素酶表达载体,测序正确,然后利用双荧光素酶报告基因系统证实构建的报告基因载体启动子具有活性,发现TGF-β可以抑制miR-200b-200a-429启动子活性(P<0.05)。结论 TGF-β可以调控miR-200b-200a-429启动子的活性。
Objective To investigate the effect of transforming growth factor-β (TGF-β) on the expression of miR-200b-200a-429 cluster and its regulatory mechanism. Methods RT-PCR was used to detect the effect of TGF-β on the expression of HK-2miR-200a, miR-200b and miR-429 in human renal tubular epithelial cells. The promoter of miR-200b-200a-429 cluster was amplified by PCR. The luciferase reporter gene expression vector of miR-200b-200a-429 promoter was constructed and transfected into HK-2 cells to detect the luciferase activity. To observe the regulation of the expression of miR-200b-200a-429 by TGF-β. Results Real-time quantitative PCR results showed that TGF-β-treated cells down-regulated the expression of miR-200a, miR-200b and miR-429 after 24h. The luciferase expression vector of miR-200b-200a-429 promoter was successfully constructed and sequenced correctly. Then, the luciferase reporter gene system was used to confirm the activity of the promoter of the reporter vector and found that TGF-β can inhibit the expression of miR-200b- 200a-429 promoter activity (P <0.05). Conclusion TGF-β can regulate the activity of miR-200b-200a-429 promoter.