miR-519d在结肠癌细胞系中的表达及对其增殖、凋亡和侵袭的影响

Expression of miR-519d in Colon Cancer Cell Lines and Its Effect on Proliferation,Apoptosis and Invasion

目的观察miR-519d在结肠癌细胞系中的表达,及对结肠癌细胞系增殖、凋亡及侵袭的影响,并探索其机制。方法通过实时荧光定量PCR技术(qRT-PCR)检测人正常结肠黏膜上皮细胞系NCM460和结肠癌细胞系HCT116、SW620、LOVO、HT29中miR-519d的表达差异。将结肠癌细胞系SW620分为3组,即miR-519d过表达组(miR-519d组)、阴性对照组(miR-NC组)及空白对照组(Blank组),采用Lipofetamine^(TM) 3000分别转染miR-519d mimics及阴性对照序列(Scramble),Blank组仅给予空白对照。采用MTT法测定细胞增殖能力,采用PI/AnnexinⅤ-FITC双染和流式细胞术测定细胞凋亡能力,采用Transwell法测定细胞侵袭能力。蛋白印迹法测定信号转导及转录激活因子3(STAT3)和X连锁凋亡抑制蛋白(XIAP)的表达变化。结果结肠癌细胞系HCT116、SW620、LOVO和HT29的miR-519d相对表达量均低于正常结肠黏膜上皮细胞系NCM460(均P<0.05)。转染24 h后,miR-519d组miR-519d相对表达量高于miR-NC组(t=43.060,P<0.01),Blank组与miR-NC组差异无统计学意义。MTT实验显示,细胞铺板72 h及96 h时miR-519d组细胞增殖能力明显低于miR-NC组(均P<0.05),Blank组与miR-NC组差异无统计学意义。miR-519d组细胞凋亡率高于miR-NC组[(23.65±1.23)%vs.(4.96±0.63)%,P=0.002],miR-NC组与Blank组细胞凋亡率差异无统计学意义。miR-519d组侵袭细胞数少于miR-NC组[(55±8)vs.(137±14),P=0.003],Blank组与miR-NC组侵袭细胞数差异无统计学意义。miR-519d组XIAP蛋白相对表达量低于miR-NC组[(0.32±0.04)vs.(1.0±0.05),P<0.01],Blank组与miR-NC组XIAP蛋白表达量差异无统计学意义。miR-519d组STAT3蛋白相对表达量低于miR-NC组[(0.44±0.04)vs.(1.05±0.07),P<0.01],Blank组与miR-NC组STAT3蛋白表达量差异无统计学意义。结论miR-519d过表达可抑制结肠癌细胞增殖及侵袭,并促进其凋亡,其作用机制可能与STAT 3和XIAP蛋白表达下调有关。

Objective To observe the expression of miR-519d in colon cancer cell line and its effect on proliferation,apoptosis and invasion,and explore its mechanism.Methods The expression levels of miR-519d in human normal colon epithelial cell line NCM460 and colon cancer cell line HCT116,SW620,LOVO and HT29 were detected by quantitative real-time PCR(qRT-PCR).Colon cancer cell line SW620 was divided into three groups:miR-519d overexpression group(miR-519d group),negative control group(miR-NC group)and blank control group(Blank group).miR-519d mimics and negative control sequence(Scramble)were transfected with Lipofectamine^(TM) 3000.Blank group was given blank control only.Cell proliferation was measured by MTT assay.Apoptosis ability was measured by PI/AnnexinⅤ-FITC double staining and flow cytometry.Cell invasion ability was measured by transwell assay.The expression levels of signal transducer and activator of transcription 3(STAT3)and X-linked inhibitor of apoptosis protein(XIAP)were detected by Western blotting.Results The relative expression levels of miR-519d in HCT116,SW620,LoVo and HT29 cell lines were lower than that in normal colon epithelial cell line NCM460(all P<0.05).24 h after transfection,the relative expression of miR-519d in miR-519d group was higher than that in miR-NC group(t=43.060,P<0.01).There was no significant difference between Blank group and miR-NC group.According to result of MTT assay,at 72 h and 96 h after cell planing,the proliferation of miR-519d group was much more slow than miR-NC group,while there was no significant difference between the Blank group and the negative control group.The apoptosis rate of miR-519d group was higher than that of miR-NC group[(23.65±1.23)%vs.(4.96±0.63)%,P=0.002].There was no significant difference between miR-NC group and Blank group.The number of invasive cells in miR-519d group was less than that in miR-NC group[(55±8)vs.(137±14),P=0.003].There was no significant difference between Blank group and miR-NC group.The relative expression of XIAP protein in miR-519d group was lower than that in miR-NC group[(0.32±0.04)vs.(1.0±0.05),P<0.01].There was no significant difference between Blank group and miR-NC group in XIAP protein level.The relative expression of STAT3 protein in miR-519d group was lower than that in miR-NC group[(0.44±0.04)vs.(1.05±0.07),P<0.01)].Conclusion Overexpression of miR-519d can inhibit the proliferation and invasion of colon cancer cells,and promote apoptosis.The mechanism may be related to downregulation of STAT3 and XIAP proteins.