miR-21在肾癌细胞中抗凋亡的机制研究

目的 探讨miR-21对肾癌细胞中抗凋亡的机制。方法 人类肾癌细胞株Caki-1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Lipofectamine 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-1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-TIMP3-3’UTR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-3’UTR 和PGL3-MUT-TIMP3-3’UTR质粒的细胞荧光素酶活性不变。将缺乏3’-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’-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力。

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

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.