miR-214负向调控卵巢癌细胞株SKOV-3中Sema4D的表达

Negative regulation of Sema4D expression in ovarian cancer SKOV-3 cell line by miR-214

目的:观察miR-214对人卵巢癌细胞株SKOV-3中Sema4D表达水平的影响,探讨miR-214对Sema4D的调控作用。方法:运用RT-PCR法检测卵巢癌细胞株SKOV-3中miR-214及Sema4D mRNA的表达情况,采用West Blot法检测Sema4D蛋白的表达情况;设计并合成miR-214模拟物和miR-214抑制剂,二者空白对照序列(模拟物对照、抑制剂对照),分别以脂质体liposome 2000包裹后转染人卵巢癌细胞株SKOV-3,并设立空转liposome 2000(lipo 2000)为对照。转染后24 h检测Sema4D mRNA及蛋白的表达变化。实验数据采用单因素方差分析和t检验进行统计学分析。结果:SKOV-3细胞株miR-214及Sema4D表达阳性;转染后24 h模拟物组、模拟物对照组、抑制剂组、抑制剂对照组、脂质体组Sema4D mRNA相对表达水平分别为0.20±0.30、0.39±0.16、1.02±0.36、0.53±0.23和0.28±0.13;蛋白相对表达水平分别为0.10±0.15、0.30±0.15、1.15±0.57、0.51±0.12和0.52±0.27;模拟物组Sema4D mRNA及蛋白相对表达水平均明显低于模拟物对照组及脂质体组(P<0.05);抑制剂组Sema4D mRNA及蛋白相对表达水平均明显高于抑制剂对照组及脂质体组(P<0.05)。miR-214负向调控Sema4D。结论:人卵巢癌细胞株SKOV-3中,Sema4D是miR-214的靶基因,miR-214可通过下调Sema4D mRNA和蛋白表达水平从而发挥该靶基因转录和翻译水平的负性调控作用。

Objective： To observe the effect of miR- 214 on Sema4 D expression in human ovarian cancer SKOV- 3 cell line,explore the regulatory effect of miR- 214 on Sema4 D. Methods： RT- PCR was used to detect the expressions of miR- 214 and Sema4 D mRNA in human ovarian cancer SKOV- 3 cell line,West Blot was used to detect the expression of Sema4 D protein; miR- 214 mimics,miR-214 inhibitor,mimics control and inhibitor control were designed and synthesized,then they were transfected into human ovarian cancer SKOV- 3 cell line after parceled by liposome 2000,blank liposome 2000 was designed as control group. The changes of expressions of Sema4 D mRNA and protein at 24 hours after transfection were detected. The data were analyzed by univariate analysis of variance and t test. Results：miR- 214 and Sema4 D were positive in human ovarian cancer SKOV- 3 cell line; the relative expression levels of Sema4 D mRNA in mimics group,mimics control group,inhibitor group,inhibitor control group and liposome group at 24 hours after transfection were 0. 20 ± 0. 30,0. 39 ± 0. 16,1. 02 ± 0. 36,0. 53 ± 0. 23 and 0. 28 ± 0. 13,respectively; the relative expression levels of Sema4 D protein were 0. 10 ± 0. 15,0. 30 ± 0. 15,1. 15 ± 0. 57,0. 51 ± 0. 12 and 0. 52 ± 0. 27,respectively; the relative expression levels of Sema4 D mRNA and protein in mimics group were statistically significantly lower than those in mimics control group and liposome group（ P〈0. 05）; the relative expression levels of Sema4 D mRNA and protein in inhibitor group were statistically significantly higher than those in inhibitor control group and liposome group（ P〈0. 05）. miR- 214 regulated Sema4 D negatively. Conclusion： In human ovarian cancer SKOV- 3 cell line,Sema4 D is the target gene of miR- 214,miR- 214 plays a role in negative regulation of the target gene at transcription and translation levels by down- regulating the expression levels of Sema4 D mRNA and protein.