miR-221靶向调控QKI-5对卵巢癌SKOV-3细胞上皮-间充质转化影响

miR-221regulates epithelial-mesenchymal transformation of ovarian cancer cell SKOV-3 via targeting QKI-5

目的探讨miR-221与QKI-5的靶向关系及其对卵巢癌SKOV-3细胞上皮间充质转化(EMT)的影响。方法培养人卵巢癌SKOV-3细胞,将miR-221 inhibitor及其阴性对照(inhibitor-NC)分别转染至SKOV-3细胞中,分为miR-221 inhibitor组、inhibitor-NC组和空白对照(blank)组,采用qRT-PCR检测miR-221和QKI-5 mRNA表达水平;蛋白质印迹法检测QKI-5蛋白表达水平;荧光素酶报告实验验证miR-221和QKI-5的靶向调控关系。再将miR-221 inhibitor和si-QKI-5分别或同时共转染至SKOV-3细胞中,分为blank组、inhibitor-NC组、miR-221 inhibitor组、si-NC组、si-QKI-5组和inhibitor+si-QKI-5组,采用MTT法检测细胞增殖活性;Transwell法检测细胞侵袭和迁移能力;蛋白质印迹法检测细胞中QKI-5、基质金属蛋白酶2(MMP-2)、基质金属蛋白酶9(MMP-9)、Snail、N-cadherin和E-cadherin蛋白表达水平。结果QKI-5 mRNA相对表达结果显示,多组间均值差异均有统计学意义(F=964.274,P<0.001);两两多重比较显示,miR-221 inhibitor组miR-221相对表达水平低于blank组和inhibitor-NC组(t值分别为11.090和8.854,均P<0.001),而QKI-5 mRNA相对表达水平高于blank组和inhibitor-NC组(t值分别为9.335和11.293,均P<0.001)。荧光素酶报告结果显示,QKI-5是miR-221的靶基因。与blank组和inhibitor-NC组比较,miR-221 inhibitor组细胞增殖活性降低(F组别=112.881,F时间=1113.609,F组别×时间=10.150,均P<0.001),且细胞迁移和侵袭能力以及MMP-9、MMP-2、Snail和N-cadherin等蛋白表达水平也均降低(均P<0.05),而E-cadherin蛋白水平增高。与miR-221 inhibitor组比较,inhibitor+si-QKI-5组细胞增殖活性增加(F组别=366.545,P<0.001;F时间=1062.321,P<0.001;F组别×时间=68.420,P<0.001),细胞迁移和侵袭能力以及MMP-9、MMP-2、Snail和N-cadherin等蛋白表达水平也均升高(均P<0.05),而E-cadherin蛋白水平降低。结论抑制miR-221表达可以靶向上调QKI-5表达,从而抑制卵巢癌SKOV-3细胞的EMT过程。

Objective To investigate the targeting relationship between miR-221 and QKI-5 and its effect on epithelial-mesenchymal transformation(EMT)of ovarian cancer cell SKOV-3.Methods Human ovarian cancer SKOV-3 cells were transferred with miR-221 inhibitor and its negative control(inhibitor-NC),and were divided into miR-221 inhibitor group,inhibitor-NC and blank group.The expression levels of miR-221 and QKI-5 mRNA were detected by qRT-PCR.The expression level of QKI-5 protein was detected by Western blotting.Luciferase reporting experiment was used to confirm the targeting relationship between miR-221 and QKI-5.The miR-221 inhibitor and si-QKI-5 were respectively or simultaneously transfected into SKOV-3 cells and were divided into blank group,inhibitor-NC group,miR-221 inhibitor group,si-NC group,si-QKI-5 group and inhibitor+si-QKI-5 group.The cell proliferation activity was detected by MTT,and the abilities of invasion and migration were detected by Transwell.The proteins expression of QKI-5,MMP-2,MMP-9,Snail,N-cadherin and E-cadherin were detected by Western blotting.SPSS 22 statistical software was used for statistical analysis of the data.One-way ANOVA analysis or two-way ANOVA analysis was used for comparison between groups,and LSD-t test was used for pair comparison.Results The relative expression results of QKi-5 mRNA showed that the mean difference between the groups was statistically significant(F=964.274,P<0.001).Pairwise comparison showed that the relative expression level of miR-221 in miR-221 inhibitor group was lower than that in blank group and inhibitor NC group(t values were 11.090 and 8.854,respectively,both P<0.001).The relative expression level of QKi-5 mRNA was higher than that in blank group and inhibitor NC group(t values were 9.335 and 11.293,respectively,both P<0.001).Luciferase report results showed that QKI-5 was the target gene of miR-221.Compared with the blank group and inhibitor-NC group,the proliferation activity of miR-221 inhibitor group was decreased(Fgroup=112.881,Ftime=1113.609,Fgroup×time=10.150,both P<0.001).The cell migration and invasion ability as well as the expression levels of MMP-9,MMP-2,Snail and N-cadherin were also decreased(both P<0.05),the levels of E-cadherin protein were increased.Compared with the miR-221 inhibitor group,the cell proliferation activity in the inhibitor+si-QKI-5 group was increased(Fgroup=366.545,P<0.001;Ftime=1062.321,P<0.001;Fgroup×time=68.420,P<0.001),the cell migration and invasion ability as well as the expression levels of MMP-9,MMP-2,Snail and N-cadherin were also increased(both P<0.05),the levels of E-cadherin protein were decreased.Conclusion Inhibition of miR-221 expression can inhibit EMT process in ovarian cancer SKOV-3 cells by targeting upregulation of QKI-5 expression.