hsa-microRNA-218对颗粒溶素表达影响的探讨

Effect of hsa-microRNA-218 on granulysin expression

目的:探讨hsa-microRNA-218(hsa-mir-218)对颗粒溶素(Granulysin,GLS)表达的影响。方法:抽提佛波酯(Phorbol 12-myristate 13-acetatefor,PMA)激活的THP-1细胞的总RNA,逆转录后PCR扩增GLS基因,将其克隆至pDsRed-Express-C1,构建GLS表达质粒pDsRed-GLS。将pDsRed-GLS与pGenesil-mir-218(pGenesil-mir-control)共转染293T细胞,36 h后激光共聚焦显微镜观察两种质粒在细胞中的表达情况,48 h后提取细胞总RNA和蛋白,RT-PCR和Western blot检测hsa-mir-218对GLS表达的影响。结果:酶切和测序结果证实重组质粒pDsRed-GLS构建成功,且能与microRNA干扰质粒在293T细胞中共表达。与转染pGenesil-mir-control组细胞相比,Western blot结果显示转染pGenesil-mir-218组细胞GLS蛋白表达下降约50%,而GLS mRNA表达无明显变化。结论:hsa-mir-218在转录后水平干扰了GLS的表达,为进一步探讨mir-218干扰GLS表达的机制打下基础。

Objective：To elucidate the effect of hsa-microRNA-218（hsa-mir-218）on exogenous granulysin （GLS） expression in 293T cells.Methods：Total RNA was extracted from THP-1 cells induced by phorbol 12-myristate 13-acetatefor （PMA）, and GLS gene was amplified by RT-PCR, and then cloned into pDsRed-Express-C1 to construct the GLS expression vector pDsRed-GLS.Then 293T cells were co-transfected with pDsRed-GLS and pGenesil-mir-218 （pGenesil-mir-control） and laser confocal microscopy was per-formed 36 h later to detect their co-expression .Total RNA and protein were extracted 48 h post transfection , and RT-PCR and Western blot were performed to detect the effect of hsa-mir-218 on exogenous GLS expression .Results：The GLS expression vector pDsRed-GLS was constructed successfully and laser confocal microscopy indicated that it was co -expressed with the interference vector .Compared with that of cells transfected with pGenesil-mir-control, Western blot showed a markedly decrease of GLS protein expression （50%） in the cells transfected with pGenesil-mir-218.However, GLS mRNA expression remained unchanged .Conclusion： hsa-mir-218 nega-tively regulates GLS expression at a post-transcriptional level , and this provides an experimental basis for future study of mechanism of GLS expression regulated by mir-218 .