摘要
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 regulatory effect of transforming growth factor-β（TGF-β）on miR-200b-200a- 429 expression and the related mechanism. Methods RT-PCR was used to examine the expression of miR-200a, miR-200b and miR-429 in HK-2 cells treated with TGF-β. The miR-200b-200a-429 promoter was amplified by PCR and was cloned into pGL3 basic vector, which was then transfected into HK-2 cells and the luciferase activity of miR-200b-200a-429 gene promoter was detected by luminometer. Results Results of RT-PCR showed that TGF-13 down-regulated miR-200a, miR-200b and miR-429 in HK-2 cells 24 h after TGF-β treatment. Vector of pGL3- miR-200h-200a-429-promoter was constructed successfully and was confirmed by sequencing. Dual-Luciferase reporter gene system was used to verify the promoter activity of the constructed reporter gene vector. TGF-13 was found to significantly inhibit the activities of miR-200b-200a-429 promoter in HK-2 cells （P〈0.05）. Conclusion TGF-β may regulate the activity of miR-200b-200a-429 promoter.
出处
《第二军医大学学报》
CAS
CSCD
北大核心
2014年第6期616-620,共5页
Academic Journal of Second Military Medical University
基金
辽宁省科技攻关课题(2012408002)
全军医学科技青年培育项目(13QNP002)~~
