SNHG3靶向miR-532逆转上皮间质转化对宫颈癌细胞侵袭、迁移及DPP耐药的研究

SNHG3-targeting miR-532 reverses epithelial mesenchymal transformation on invasion,migration and DPP resistance of cervical cancer cells

目的 探讨SNHG3靶向miR-532逆转上皮间质转化对宫颈癌细胞侵袭、迁移及顺铂(DPP)耐药的机制。方法 Real-time PCR法检测正常宫颈上皮细胞及4种宫颈癌细胞系中SNHG3、miR-532表达水平,取宫颈癌细胞将其分为宫颈癌细胞(CC)组、宫颈癌细胞+SNHG3-NC(SN)组、宫颈癌细胞+SNHG3激动剂(SM)组、宫颈癌细胞+SNHG3抑制剂(SI)组、宫颈癌细胞+miR-532-NC(MN)组、宫颈癌细胞+miR-532抑制剂(MI)组、宫颈癌细胞+miR-532激动剂(MM)组、宫颈癌细胞+SNHG3抑制剂+miR-532激动剂(IM)组,免疫印迹法检测上皮间质转化相关蛋白表达,小室法检测细胞侵袭及迁移,CCK-8法检测DPP耐药,双荧光素酶报告实验证实SNHG3和miR-532的相互作用。结果 宫颈癌细胞系中的SNHG3 mRNA表达量高于正常宫颈癌上皮细胞系HUCEC,miR-532 mRNA表达量低于正常宫颈癌上皮细胞系HUCEC(均P<0.05);CC组、SN组、MN组组间比较,细胞E-cadherin、Vimentin蛋白表达、侵袭及迁移数量、DPP抑制率差异无统计学意义(均P>0.05),与SN组、MN组相比,SM组、MI组E-cadherin蛋白表达、DPP抑制率明显降低,Vimentin蛋白表达、侵袭及迁移数量明显升高(均P<0.05),与SM组、MI组相比,SI组、MM组E-cadherin蛋白表达、DPP抑制率明显升高,Vimentin蛋白表达、侵袭及迁移数量明显降低(均P<0.05);SI组与MM组相比,细胞E-cadherin、Vimentin蛋白表达、侵袭及迁移数量、DPP抑制率差异均无统计学意义(P>0.05),与MM组相比,IM组E-cadherin蛋白表达、DPP抑制率明显升高,Vimentin蛋白表达、侵袭及迁移数量明显降低(均P<0.05);双荧光素酶报告结果显示,转染SNHG3后可显著降低miR-532-3′-UTR-WT的荧光素酶活性(P<0.05),但对突变基因无显著影响(P>0.05),且SNHG3与miR-532呈现负相关。结论 抑制SNHG3可有效逆转宫颈癌细胞上皮间质转化,抑制细胞侵袭及迁移,并提高DPP耐药,其作用机制可能与靶向激活miR-532有关。

Objective To investigate the reversal of epithelial mesenchymal transformation by SNHG3 targeting Mir-532 on invasion,migration and DPP resistance of cervical cancer cells.Methods Real-time PCR was used to detect the expression levels of SNHG3 and Mir-532 in normal cervical epithelial cells and 4 cervical cancer cell lines.Cervical cancer cells were divided into cervical cancer cell(CC)group,cervical cancer cell+SNHG3-NC(SN)group,cervical cancer cell+SNHG3 agonist(SM)group,cervical cancer cell+SNHG3 inhibitor(SI)group,cervical cancer cell+Mir-532-NC(MN)group,cervical cancer cell+Mir-532 inhibitor(MI)group,cervical cancer cells+Mir-532 agonist(MM)group,cervical cancer cells+SNHG3 inhibitor+Mir-532 agonist(IM)group.Western blotting was used to detect the expression of epithelial-mesymal transformation related proteins,cell invasion and migration were detected by cell assay,DPP resistance was detected by CCK-8 assay.The interaction between SNHG3 and Mir-532 was confirmed by dual luciferase reporting assay.Results The mRNA expression of SNHG3 in cervical cancer cell lines was higher than that of HUCEC(P<0.05),and the mRNA expression of miR-532 was lower than that of HUCEC(P<0.05).Compared with the CC group,SN group and MN group,the expression of E-cadherin and Vimentin protein,the number of invasion and migration,and the inhibition rate of DPP were basically not different(P>0.05).Compared with the SN group and MN group,the expression of E-cadherin protein and the inhibition rate of DPP in SM group and MI group were significantly decreased(P<0.05).Vimentin protein expression,invasion and migration were significantly increased(P<0.05).Compared with SM and MI groups,E-cadherin protein expression and DPP inhibition rate were significantly increased in SI and MM groups(P<0.05),and Vimentin protein expression,invasion and migration were significantly decreased(P<0.05).Compared with MM group,the expression of E-cadherin and Vimentin protein,the number of invasion and migration,and the inhibition rate of DPP in SI group were almost no difference(P>0.05).Compared with MM group,the expression of E-cadherin protein and the inhibition rate of DPP in IM group were significantly increased(P<0.05).The protein expression,invasion and migration of Vimentin were significantly decreased(P<0.05).Double luciferase reporter results showed that transfection of SNHG3 could significantly reduce the luciferase activity of Mir-532-3′-UTR-WT(P<0.05),but had no significant effect on the mutant gene(P>0.05),and SNHG3 was negatively correlated with miR-532.Conclusion Inhibition of SNHG3 can effectively reverse epithelial mesenchymal transformation of cervical cancer cells,inhibit cell invasion and migration,and improve DPP resistance.The mechanism of action may be related to the targeted activation of Mir-532.