miR-302d的载体构建及其与Tle4基因的靶向关系验证

Vector Construction of miR-302d and Targeting Verification Between miR-302d and Tle4 Gene

试验旨在探究精原干细胞(SSCs)形成过程中的关键miR-302d行使功能的分子机制。试验克隆mi R-302d前体序列并连入PGMLV-CMV-MCS-EF1-Zs Green1-T2A-Puro构建mi R-302d过表达载体(mi R-302d mimics);合成mi R-302d的干扰靶点并连入PGMLV-SC5载体构建mi R-302d干扰载体(mi R-302d inhibitor)。通过生物信息预测获得mi R-302d靶基因并构建其3′UTR的野生型(WT)和突变型载体(MT);将mi R-302d过表达载体分别与WT和MT载体共转染DF-1和293T细胞,检测靶基因3′UTR的活性。同时检测在DF-1细胞中过表达或干扰mi R-302d后靶基因的表达变化。结果显示:成功构建mi R-302d的过表达和干扰载体;预测结果显示Tle4为mi R-302d的潜在靶基因,且该基因为ESCs分化为SSCs过程的重要基因;双荧光素酶活性检测结果显示不论是在DF-1还是293T细胞中,与转染WT+mi R-NC组相比,转染WT+mi R-302d组的双荧光素酶活性显著下降(P<0.05),而共转染MT+mi R-302d组的荧光素酶活性差异不显著(P>0.05),且过表达mi R-302d后下调Tle4的表达,而干扰mi R-302d后上调Tle4的表达。结果表明,miR-302d可直接调节靶基因Tle4的表达。

The experiment aimed to investigate the molecular mechanism of the key miR-302d driving function during the formation of SSCs.mi R-302d precursor sequence was cloned and linkaged to PGMLV-CMV-MCS-EF1-Zs Green1-T2A-Puro to construct miR-302d overexpression vector(miR-302d mimics);miR-302d interference target was synthesized and linkaged to PGMLV-SC5 vector to construct mi R-302d interference vector(mi R-302d inhibitor).mi R-302d target genes was obtained by bioinformatic prediction and wild type vector(WT)and mutant vector(MT)of their 3′UTR was constructed.The mi R-302d overexpression vector was cotransfected with WT and MT vectors in DF-1 and 293T cells respectively to detect the activity of the target gene 3′UTR.The changes of target gene expression after overexpression or interference with mi R-302d in DF-1 cells were also detected.The results showed that the overexpression and interference vectors of mi R-302d were successfully constructed.The prediction results showed that Tle4 was a potential target gene of mi R-302d and it was an important gene in the process of differentiation of ESCs into SSCs;The results of dual luciferase activity assay showed that both in DF-1 and 293T cells,compared with the transfected WT+mi R-NC group,the dual luciferase activity of transfected WT+mi R-302d group showed a significant decrease in dual luciferase activity(P<0.05)while the difference in luciferase activity between WT+mi R-NC and MT+mi R-302d group was not significant(P>0.05),and the expression of Tle4 was down-regulated after overexpression of mi R-302d,while the expression of Tle4 was up-regulated after interference with mi R-302d.The result indicated that mi R-302d could directly regulate the expression of the target gene Tle4.