miR-214负向调控结肠癌细胞株LoVo中Sema4D的表达

mi R-214 down-regulates Sema4D expression in colon cancer Lo Vo cells

目的:观察miR-214对人结肠癌细胞株LoVo中轴突导向因子4D(semaphorin 4D,Sema4D)表达水平的影响,探讨miR-214对Sema4D的调控作用.方法:设计并合成miR-214模拟物(miR-214mimics),miR-214抑制剂(miR-214 inhibitor),二者对照序列(mi-control,in-control),分别与以脂质体lipofectamine 2000包裹后转染人结肠癌细胞株LoVo.RT-PCR检测转染24 h后各细胞株内miR-214的表达及Sema4D mRNA的表达变化,并用Western blot检测Sema4D蛋白的表达变化.结果:LoVo细胞株转染miR-214 mimics、mi-control,inhibitor、in-control 24 h后miR-214的相对表达量为8.003±0.651,3.464±0.332,0.740±0.088,2.620±0.166,mimics组miR-214表达较其对照组升高,inhibitor组表达较其对照组降低(P<0.05).Sema4D mRNA的相对表达量为:0.420±0.027,0.851±0.062,1.243±0.087,0.660±0.042;Sema4D蛋白/β-actin分别为:0.163±0.037,0.550±0.038,1.137±0.112,0.457±0.046.Sema4D mRNA及蛋白mimics组表达较其对照组降低,inhibitor组较其对照组升高(P<0.05).结论:人结肠癌细胞株LoVo中,Sema4D受miR-214的调控,miR-214可负向调控Sema4D mRNA的表达水平进而影响其蛋白的表达,miR-214可能作为结肠癌治疗的新靶点.

AIM：To observe the effect of miR-214 on Sema4 D expression in human colon cancer cell line LoVo.METHODS：miR-214 mimic,miR-214 inhibitor,and two control sequences（mi-control,in-control） were designed,synthesized and transfected into LoVo cells,respectively.RT-PCR was used to detect the expression changes of miR-214 and Sema4 D mRNA in transfected cells,and the expression change of Sema4 D protein was detected by Western blot.RESULTS：The relative expression levels of miR-214 in LoVo cells transfected with miR-214 mimic,mi-control,miR-214 inhibitor,and in-control were 8.003 ± 0.651,3.464 ± 0.332,0.740 ± 0.088,and 2.620 ± 0.166,respectively.The expression of miR-214 increased significantly in the mimic group compared with the control group,but was significantly lower in the inhibitor group than in the control group（P〈0.05）.The relative expression levels of Sema4 D mRNA in LoVo cells transfected with miR-214 mimic,micontrol,miR-214 inhibitor,and in-control were 0.420 ± 0.027,0.851 ± 0.062,1.243 ±0.087,and 0.660 ± 0.042,respectively,and the relative expression levels of Sema4 D protein were 0.163 ± 0.037,0.550 ± 0.038,1.137 ±0.112,and 0.457 ± 0.046,respectively.Sema4 D mRNA and protein expression in the mimic group was decreased significantly compared with the control group,but was significantly increased in the inhibitor group compared with the control group（P〈0.05）.CONCLUSION：Sema4D expression is regulated by miR-214 in human colon cancer cell line LoVo,and miR-214 can down-regulate the expression of Sema4 D mRNA and thereby influence the protein expression.miR-214 could be used as a new target for treatment of colon carcinoma.