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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 the expression of Sema4D in human ovarian cancer cell line SKOV-3 and to explore the regulatory effect of miR-214 on Sema4D. Methods: The expression of miR-214 and Sema4D mRNA in ovarian cancer cell line SKOV-3 was detected by RT-PCR. The expression of Sema4D protein was detected by West Blot assay. The miR-214 mimics and miR-214 inhibitor (Blank control). The recombinant plasmid was encapsulated in liposome 2000 and transfected into human ovarian cancer cell line SKOV-3. Liposome 2000 (lipo 2000) was used as a control. The expression of Sema4D mRNA and protein was detected 24 h after transfection. The experimental data using one-way ANOVA and t test for statistical analysis. Results: The expression of miR-214 and Sema4D in SKOV-3 cell line was positive. The relative expression levels of Sema4D mRNA in mock, mock, inhibitor, liposome and control group at 24 h 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 proteins were 0.10 ± 0.15, 0.30 ± 0.15, 1.15 ± 0.57, 0.51 ± 0.12 and 0.52 ± 0.27, respectively; (P <0.05). The relative expression levels of Sema4D mRNA and protein in inhibitor group were significantly higher than that in inhibitor control group and liposome group (P < 0.05). miR-214 negatively regulates Sema4D. Conclusion: Sema4D is the target gene of miR-214 in human ovarian cancer cell line SKOV-3. MiR-214 can negatively regulate the transcription and translation of the target gene by down-regulating the expression of Sema4D mRNA and protein.