miR-369-3p靶向CTHRC1对宫颈癌细胞顺铂耐药性的影响

Effect of miR-369-3p targeting CTHRC1 on cisplatin resistance of cervical cancer cells

目的:探讨微小RNA-369-3p(miR-369-3p)是否通过靶向胶原三股螺旋重复蛋白1(CTHRC1)影响宫颈癌细胞顺铂(DDP)耐药性。方法:体外培养人宫颈癌细胞HeLa,采用顺铂梯度暴露法构建顺铂耐药宫颈癌细胞株HeLa/DDP;实验分组:DDP+miR-NC组、DDP+miR-369-3p组、DDP+si-NC组、DDP+si-CTHRC1组、DDP+miR-369-3p+pcDNA组、DDP+miR-369-3p+pcDNA-CTHRC1组;采用MTT法检测细胞增殖抑制率及计算IC_(50)值;qRT-PCR检测miR-369-3p、CTHRC1表达量;Transwell小室实验检测细胞迁移及侵袭;双荧光素酶报告实验验证miR-369-3p、CTHRC1的靶向关系;Western blot检测CTHRC1、CyclinD1、MMP-2、MMP-9、p21表达。结果:与HeLa组比较,HeLa/DDP组细胞增殖抑制率、miR-369-3p的表达水平显著降低(P<0.05),IC_(50)值、CTHRC1 mRNA及蛋白水平显著升高(P<0.05);HeLa/DDP细胞转染miR-369-3p mimics或转染si-CTHRC1后,细胞增殖抑制率、p21蛋白水平显著升高(P<0.05),迁移细胞数、侵袭细胞数显著减少(P<0.05),CyclinD1、MMP-2、MMP-9蛋白水平显著降低(P<0.05);双荧光素酶报告实验证实miR-369-3p可靶向结合CTHRC1;CTHRC1过表达可明显逆转miR-369-3p过表达对细胞增殖、迁移、侵袭及顺铂耐药性的作用。结论:miR-369-3p过表达可能通过下调CTHRC1表达抑制HeLa/DDP细胞增殖、迁移及侵袭从而逆转宫颈癌细胞顺铂耐药性。

Objective:To investigate whether miR-369-3p affects the resistance of cervical cancer cell cisplatin(DDP)by targeting CTHRC1.Methods:Human cervical cancer cell HeLa was cultured in vitro,and cisplatin-resistant cervical cancer cell line HeLa/DDP was constructed by cisplatin gradient exposure method.Experimental grouping:DDP miR-NC group,DDP miR-369-3p group,DDP si-NC group,DDP si-CTHRC1 group,DDP miR-369-3p pcDNA group,DDP miR-369-3p pcDNA-CTHRC1 group.MTT method was used to detect cell proliferation inhibition rate and calculated cisplatin IC50 value.qRT-PCR was used to detect expression levels of miR-369-3p and CTHRC1.Transwell cell test was used to detect cell migration and invasion.Dual luciferase report experiment verified the targeting relationship between miR-369-3p and CTHRC1.Western blot was used to detect expression levels of CTHRC1,CyclinD1,MMP-2,MMP-9 and p21.Results:Compared with HeLa group,cell proliferation inhibition rate and expression level of miR-369-3p in HeLa/DDP group were significantly reduced(P<0.05),and the IC_(50) value,expression of CTHRC1 mRNA and protein level of CTHRC1 were significantly increased(P<0.05).After HeLa/DDP cells were transfected with miR-369-3p mimics or si-CTHRC1,the cell proliferation inhibition rate and protein level of p21 were significantly increased(P<0.05),and the number of migrating cells and invading cells were significantly reduced(P<0.05),protein levels of CyclinD1,MMP-2,MMP-9 were significantly reduced(P<0.05).Dual luciferase report experiment confirmed that miR-369-3p could target to bind to CTHRC1.CTHRC1 overexpression could significantly reverse the effects of miR-369-3p overexpression on cell proliferation,migration,invasion and cisplatin resistance.Conclusion:Overexpression of miR-369-3p may reverse the proliferation,migration and invasion of HeLa/DDP cells by down-regulating the expression of CTHRC1 to reverse the resistance of cervical cancer cells to cisplatin.