miR-21在肾癌细胞中抗凋亡的机制研究

Anti-apoptosis mechanism of miR-21 in Renal cell carcinoma

目的 探讨miR-21在肾癌细胞中抗凋亡的机制.方法 人类肾癌细胞株Caki-l和OS-RC-2细胞,分别以As-miR-21转染沉默miR-21,转染无关乱码寡核苷酸序列作阴性对照以及不转染寡核苷酸的作空白对照,以AnnexinV FITC凋亡检测试剂盒检测凋亡,用Caspase-Glo 3/7试剂测半胱天冬酶3/7活性,用Western Blot分析测定FASL和TIMP3的表达,用荧光素酶报告实验测定miR-21与FASL和TIMP3的结合,将PGL3-WT-TIMP3-3 UTR质粒中miR-21结合位点“AUAAGCU”剪切后用Lipo-fectamine 2000转染至细胞测定As-miR-21对细胞凋亡的影响.结果 转染AS-miR-21的细胞凋亡率相对于阴性和空白对照细胞显著增加（P＜0.05）.转染AS-miR-21后48h,Caki-l细胞中半胱天冬酶3/7活性显著增加（P＜0.05）.转染PGL3-WT-FASL-3 UTR和PGL3-WT-TIMP3-3UTR质粒的Caki-1和OS-RC-2细胞中沉默miR-21荧光素酶活性显著增加,而转染去除miR-21结合位点的PGL3-MUT-FASL-3UTR和PGL3-MUT-TIMP3-3 LTR质粒的细胞荧光素酶活性不变.将缺乏3＆#39;-UTR的TIMP3质粒转染Caki-1和OS-RC-2细胞,再转染miR-21,TIMP3表达不受影响.而细胞过表达TIMP3则凋亡增加,但转染缺乏3＆#39;-UTR的TIMP3可以拮抗miR-21导致的抗凋亡作用.结论 miR-21通过FASL和TIMP3多条途径增加肾癌细胞的抗凋亡能力.

Objectives To investigate the mechanism of anti-apoptosis effect of miR-21 in renal cell carcinoma.Methods Human Renal cancer carcinoma cell lines Caki-1 and OS-RC-2 were transfected with As-miR-21 or non-special oligonucleotides （as negative control） or nothing （as blank control）.Cell apoptosis was detected by using AnnexinV FITC Apoptosis Dectecting Kit.After reduction of miR-21,Fas ligand and metalloproteinase inhibitor 3 （TIMP3） expression and luciferase activity were detected by Western blot and luciferase reporter assay.The effect of TIMP3 on miR-21-induced anti-apoptosis was determined by transfection with TIMP3 lacking 3＆#39; untranslatedm region and miR-21.Results The reduction of miR-21 by antisense oligonucleotides induced cell apoptosis by activation of caspase pathway in renal cell carcinoma cells.Moreover,bioinformatics analysis revealed that miR-21 has the potential to regulate multiple poptosis-related genes.The reduction of miR-21inhibited Fas ligand and TIMP3 expression by targeting the binding site within the 3＆#39;untranslated region.Finally,the introduction of TIMP3 cDNA without 3 ＆#39; untranslated region abrogated miR-21-induced anti-apoptosis.Conclusions MiR-21 can increase cell anti-apoptosis in renal cancer through FASL and TIMP3 pathways.