微小RNA-145对卵巢癌细胞迁移和侵袭的影响

Effect of miR- 145 on Migration and Invasion of Ovarian Cancer Cells

目的评估微小RNA-145(miR-145)对卵巢癌细胞迁移和侵袭的影响。方法选取上皮性卵巢癌细胞株SKOV3及3AO,过表达miR-145后,qRT-PCR检测转染前后细胞株miR-145的表达,Transwell小室实验检测转染前后细胞迁移及侵袭能力。采用生物信息学方法特异预测出miR-145靶基因zeb-2后,通过双荧光素酶报告实验进行验证,再敲低zeb-2后检测细胞移动能力。结果过表达miR-145后,SKOV3(t=10.752,P=0.000;t=5.617,P=0.005)及3AO细胞(t=10.111,P=0.001;t=21.746,P=0.000)的迁移及增殖能力均明显下降。双荧光素酶报告实验结果显示,共转染miR-145 mimic和WT 3’UTR表达载体细胞的相对荧光素酶活性明显低于共转染mimic control和WT 3’UTR表达载体的细胞(SKOV3:t=4.572,P=0.010;3AO:t=3.528,P=0.024),共转染miR-145mimic和MUT 3’UTR表达载体细胞的相对荧光素酶活性与共转染mimic control和MUT 3’UTR表达载体细胞差异无统计学意义(SKOV3:t=0.227,P=0.831;3AO:t=0.040,P=0.970)。过表达miR-145 48 h后,实时定量PCR检测结果显示,与阴性对照相比,SKOV3(t=1.490,P=0.211)和3AO细胞(t=0.114,P=0.914)中zeb-2 mRNA表达差异无统计学意义。过表达miR-145 72 h后,Western blot检测结果显示,与阴性对照相比,SKOV3(t=3.769,P=0.020)及3AO细胞(t=4.452,P=0.011)中zeb-2蛋白的表达水平明显下降。转染zeb-2 siRNA 72 h后,Western blot检测结果显示,SKOV3(t=4.660,P=0.010)和3AO细胞(t=4.594,P=0.010)中的zeb-2蛋白表达水平明显下调;Transwell小室实验检测结果显示,SKOV3(t=18.655,P=0.000;t=18.026,P=0.000)及3AO(t=5.500,P=0.005;t=8.780,P=0.001)细胞的迁移和侵袭能力明显下降。结论miR-145可能是通过靶向抑制zeb-2来抑制卵巢癌细胞迁移和侵袭能力。

Objective To evaluate the effect of miR-145 on migration and invasion of ovarian cancer cells. Methods The effect of miR-145 overexpression on the expression levels of miR-145 and zeb-2 were detected with qRT-PCR and Western blotting.The changes of in vitro migration and invasion were examined using Transwell assay.Target genes of miR-145 were predicted by bioinformatics software.Dual-luciferase reporter assay were used to verify zeb-2 as a direct target of miR-145.zeb-2 siRNA was transiently transfected in SKOV3 and 3AO cells,Transwell was used to examine in vitro migration and invasion abilities. Results The migration and proliferation of SKOV3( t =10.752, P =0.000;t =5.617, P =0.005)and 3AO cells( t =10.111, P =0.001;t =21.746, P =0.000)decreased significantly after overexpression of miR-145.The results of dual-luciferase reporter assay showed that the relative luciferase activity of co-transfected miR-145 mimic and WT 3 ’UTR expression vectors was significantly lower than that of co-transfected mimic control and WT 3 ’UTR expression vectors(SKOV3: t =4.572, P =0.010;3AO: t =3.528, P =0.024).There was no significant difference in relative luciferase activity between co-transfected miR-145 mimic/MUT 3 ’UTR expression vector cells and co-transfected mimic control/MUT 3 ’UTR expression vector cells(SKOV3: t =0.227, P =0.831;3AO: t =0.040, P =0.970).Real-time quantitative PCR showed that the zeb-2 expressions in SKOV3( t =1.490, P =0.211)and 3AO cells( t =0.114, P =0.914)were not significantly different from negative control after 48 h of miR-145 overexpression.Western blot analysis showed that the expression of zeb-2 protein in SKOV3( t= 3.769, P =0.020)and 3AO cells( t =4.452, P =0.011)decreased significantly compared with negative control after 72 h of miR-145 overexpression.Seventy-two hours after transfection of zeb-2 siRNA,Western blotting showed that the expression of zeb-2 protein in SKOV3( t =4.660, P =0.010)and 3AO cells( t =4.594, P =0.010)was significantly down-regulated.Transwell assay showed that the migration and invasion abilities of SKOV3( t =18.655, P =0.000;t= 18.026, P =0.000)and 3AO cells( t =5.500, P =0.005;t =8.780, P =0.001)were significantly decreased. Conclusion miR-145 may inhibit the migration and invasion of ovarian cancer cells by targeting zeb-2.