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研究在HeLaS3细胞中过表达Lin28a/Lin28b对let-7家族miRNA表达水平和活性的影响。首先,构建Lin28a和Lin28b的表达载体pAAV2neo-Lin28a和pAAV2neo-Lin28b,分别转染HeLaS3细胞并筛选获得Lin28a和Lin28b的稳定表达细胞株HeLaS3/pAAV2neo-Lin28a和HeLaS3/pAAV2neo-Lin28b。然后,以pAAV2neo-Gluc-(Fluc)为基本骨架,构建并获得检测let-7家族miRNA活性的8种质粒型载体,并包装为相应的重组腺相关病毒(Recombinant adeno-associated virus,rAAV),作为检测miRNA靶序列介导的转录后抑制活性的传感器,命名为Asensor。在此基础上,以HeLaS3细胞为对照,用Western blot检测HeLaS3/pAAV2neo-Lin28a和HeLaS3/pAAV2neo-Lin28b细胞中Lin28a和Lin28b表达水平,用QRT-PCR测定let-7家族各成员表达水平,用Asensor检测let-7家族各成员活性。Western blot结果显示,HeLaS3/pAAV2neo-Lin28a和HeLaS3/pAAV2neo-Lin28b均能有效地表达Lin28a和Lin28b蛋白;QRT-PCR检测结果显示,相比于HeLaS3细胞,HeLaS3/pAAV2neo-Lin28a细胞中let-7家族各成员表达水平都下降(除let-7e外),但不同成员下降幅度存在差异;Asensor检测结果显示,let-7家族所有成员活性水平都下降,但不同成员下降幅度也存在差异,且同一成员活性水平与表达水平及其下降趋势也不一致。相比于HeLaS3细胞,HeLaS3/pAAV2neo-Lin28b细胞中let-7家族成员的表达和活性水平均明显下降,但表达水平的下降幅度比HeLaS3/pAAV2neo-Lin28a细胞大,而活性的下降幅度却与之相近。本研究建立了一种检测和比较miRNA靶序列介导的转录后抑制活性的方法,首次研究了过表达Lin28a和Lin28b对细胞中的let-7家族miRNA活性影响,并发现Lin28a和Lin28b对let-7家族miRNA表达水平的影响和对其相应活性的影响不一致性,说明在检测miRNA表达水平的同时检测miRNA活性能更全面揭示miRNA的调节功能,为进一步研究let-7家族的调控机制奠定了基础。
To investigate the effect of overexpression of Lin28a / Lin28b on let-7 family miRNA expression and activity in HeLaS3 cells. Firstly, the expression vectors pAAV2neo-Lin28a and pAAV2neo-Lin28b of Lin28a and Lin28b were constructed and transfected into HeLaS3 cells respectively. Stable expression cell lines HeLaS3 / pAAV2neo-Lin28a and HeLaS3 / pAAV2neo-Lin28b of Lin28a and Lin28b were obtained. Then, based on pAAV2neo-Gluc- (Fluc) as the basic framework, eight plasmid vectors for detecting the let-7 family miRNA activity were constructed and packaged into the corresponding recombinant adeno-associated virus (rAAV) As a sensor to detect miRNA target sequence-mediated post-transcriptional inhibitory activity, it was named Asensor. On the basis of this, the expression levels of Lin28a and Lin28b in HeLaS3 / pAAV2neo-Lin28a and HeLaS3 / pAAV2neo-Lin28b cells were detected by Western blot using HeLaS3 cells as a control, and the expression levels of let-7 family members were determined by QRT-PCR. Detection of let-7 family member activity. The results of Western blot showed that both HeLaS3 / pAAV2neo-Lin28a and HeLaS3 / pAAV2neo-Lin28b could effectively express Lin28a and Lin28b proteins. QRT-PCR results showed that the let-7 family of HeLaS3 / pAAV2neo-Lin28a cells The expression levels of all the members decreased (except let-7e), but there were differences among the members. The Asensor test showed that the activity of all members of let-7 family decreased, but the differences among different members also decreased. Activity levels and expression levels and the downward trend is also inconsistent. Compared with HeLaS3 cells, the expression and activity of let-7 family members in HeLaS3 / pAAV2neo-Lin28b cells decreased significantly, but the expression level decreased more significantly than that of HeLaS3 / pAAV2neo-Lin28a cells similar. This study established a method for the detection and comparison of miRNA target sequence-mediated post-transcriptional inhibitory activity. We first investigated the effect of overexpression of Lin28a and Lin28b on the let-7 family miRNA activity in cells and found that the effect of Lin28a and Lin28b on let- 7 family miRNA expression and its impact on the corresponding activity of inconsistency, indicating that the detection of miRNA expression levels while detecting miRNA activity can more fully reveal the regulatory function of miRNA, lay the foundation for further study of let-7 family regulation mechanism .