Experimental identification of apoptosis-associated miR-21 target genes in Jurkat T-cells using a high-throughput experimental approach

MiR-21 is an important suppressor of T-cell apoptosis that is also widely overexpressed in many types of cancers. The exact mechanisms related to the anti-apoptotic effect of miR-21 is not well understood. In this study, we applied AGO2 RNA Immunoprecipitation followed by gene expression profiling (RIP-Chip) in Jurkat cells to identify apoptosis-associated miR-21 target genes. We showed that expression of miR-21 rapidly increases upon αCD3/αCD28 activation of Jurkat cells. Inhibition of miR-21 resulted in reduced cell growth and induced apoptosis. Upon AGO2-RIP-Chip, we observed an overall increased enrichment of miR-21 target genes in the IP fraction of miR-21-overexpressing Jurkat cells as compared to the IP fraction of empty vector control cells. We noted a systematic decrease in transcript levels of predicted miR-21 target genes compared to EV control. We identified 72 genes that were 2-fold enriched in the AGO2-IP fraction of miR-21-overexpressing cells that contained a predicted miR-21 binding site. Of these, 71 were enriched 2-fold more in the miR-21-overexpressing cells as compared to EV Jurkat cells. The target gene for which the enrichment was most prominently increased upon miR-21 overexpression was the pro-apoptotic protein LATS1. Luciferase reporter assays confirmed direct targeting of the LATS1 3'UTR by miR-21. In line with the luciferase results, Western blot analysis revealed a decrease in LATS1 upon miR-21 inhibition. LATS1 qRT-PCR analysis in primary T-cells showed that LATS1 levels decrease upon T-cell stimulation while the miR-21 levels increase. Collectively, these data identify the miR-21 target LATS1 as a likely candidate whose inhibition contributes to the anti-apoptotic function of miR-21 in T-cells and perhaps also many types of cancers. Gene expression array on Jurkat cells overexpressing miR-21 and empty vector (EV).

miR-21（微小RNA-21）是T细胞凋亡的重要抑制因子，同时在多种癌症中广泛过表达。其抗凋亡作用的确切分子机制尚未完全阐明。本研究采用AGO2 RNA免疫沉淀联合基因表达谱分析（RIP-Chip）技术，在Jurkat细胞中筛选与凋亡相关的miR-21靶基因。我们发现，经αCD3/αCD28激活Jurkat细胞后，miR-21的表达水平迅速升高。抑制miR-21会导致细胞增殖能力下降并诱导细胞凋亡。通过AGO2-RIP-Chip实验，我们观察到：与空载体对照细胞的免疫沉淀组分相比，过表达miR-21的Jurkat细胞的免疫沉淀组分中，miR-21靶基因的整体富集水平显著升高。与空载体（EV）对照组相比，预测的miR-21靶基因的转录本水平呈现系统性下降。我们在过表达miR-21的细胞的AGO2免疫沉淀组分中，筛选得到72个含有预测miR-21结合位点且富集倍数达2倍的基因。其中，与转染空载体的Jurkat细胞相比，71个基因在过表达miR-21的细胞中的富集程度高出2倍。在miR-21过表达后富集程度提升最显著的靶基因为促凋亡蛋白LATS1。双荧光素酶报告基因实验证实，miR-21可直接靶向LATS1的3'非翻译区（3'UTR）。与荧光素酶实验结果一致，蛋白质免疫印迹（Western blot）分析显示，抑制miR-21后LATS1的表达水平下降。对原代T细胞开展LATS1的实时定量聚合酶链反应（qRT-PCR）分析后发现，T细胞活化时LATS1的表达水平下降，而miR-21的表达水平升高。综上，本研究证实miR-21的靶基因LATS1是一个潜在的候选因子，其被miR-21抑制可能是miR-21在T细胞中发挥抗凋亡功能的机制之一，这一机制或许也适用于多种癌症。本数据集包含过表达miR-21与空载体（EV）的Jurkat细胞的基因表达芯片数据。