LINC00294调控HPV18^(+)宫颈癌细胞生长的机制

The mechanism of LINC00294 regulating the growth of HPV18+cervical cancer cells

目的探讨LINC00294靶向调控微小RNA-21(miR-21)对人乳头状瘤病毒(HPV)18阳性(+)宫颈癌细胞(HPV18^(+)HeLa)增殖与凋亡的影响。方法收集2018年4月至2021年5月在本院就诊的63例宫颈癌患者的宫颈癌组织标本,其中35例标本为HPV18-,28例标本为HPV18^(+),使用实时荧光定量PCR(RT-qPCR)检测宫颈癌组织中LINC00294、miR-21表达水平;体外培养人宫颈癌细胞株HPV18^(+)HeLa,将其分为对照组、LINC00294-pcDNA组、pcDNA组、miR-21inhibitor组、NCinhibitor组、LINC00294-pcDNA+miR-21mimics组、LINC00294-pcDNA+miRNC组,使用RT-qPCR法检测各组HPV18^(+)HeLa细胞LINC00294、miR-21表达情况;应用双荧光素酶报告基因实验验证LINC00294与miR-21的靶向关系;应用CCK-8法检测各组HPV18^(+)HeLa细胞增殖情况;使用流式细胞术检测各组HPV18^(+)HeLa细胞凋亡情况;应用免疫印迹法(Westernblotting)检测各组HPV18^(+)HeLa细胞CyclinD1、Bax、Bcl-2蛋白表达。结果与HPV18-宫颈癌组织比较,HPV18^(+)宫颈癌组织中LINC00294表达水平显著降低,miR-21表达水平显著升高(均P<0.05)。经生物信息学(starBasev2.0)分析显示,LINC00294与miR-213’UTR区有结合位点,且经双荧光素酶报告基因实验验证,LINC00294与miR-21存在靶向关系。过表达LINC00294或抑制miR-21表达,HPV18^(+)HeLa细胞A值、CyclinD1、Bcl-2蛋白及miR-21表达水平显著下降,细胞凋亡率、Bax蛋白表达水平显著升高(均P<0.05);过表达miR-21可部分逆转过表达LINC00294对HPV18^(+)HeLa细胞的增殖抑制作用和凋亡促进作用。结论HPV18^(+)宫颈癌组织中LINC00294表达水平显著降低,过表达LINC00294可抑制miR-21表达,抑制HPV18^(+)宫颈癌细胞增殖,促进细胞凋亡。

Objective To observe the effects of LINC00294 on the proliferation and apoptosis of human papillomavirus(HPV)18 positive(+)cervical cancer cells(HPV18^(+)HeLa)by targetedly regulating microRNA-21(miR-21).Methods Cervical cancer(CC)tissue specimens were collected from 63 patients with CC in our hospital from April 2018 to May 2021.Among them,35 specimens were HPV18 negative(–),28 specimens were HPV18^(+).Real-time quantification PCR(RT-qPCR)was used to detect the expression levels of LINC00294 and miR-21 in CC tissues.Human CC cell line HPV18^(+)HeLa was cultured in vitro and divided into control group,LINC00294-pcDNA group,pcDNA group,miR-21 inhibitor group,NC inhibitor group,LINC00294-pcDNA+miR-21 mimics group and LINC00294-pcDNA+miR NC group,respectively.RT-qPCR method was used to detect the expressions of LINC00294 and miR-21 in HPV18^(+)HeLa cells;dual luciferase reporter gene experiment to verify the targeting relationship between LINC00294 and miR-21;CCK-8 method to detect the proliferation of HPV18^(+)HeLa cells;flow cytometry to detect the apoptosis of HPV18^(+)HeLa cells;and Western blotting to detect Cyclin D1,Bax and Bcl-2 protein expression in HPV18^(+)HeLa cells.Results Compared with HPV18–CC tissue,the expression level of LINC00294 in HPV18^(+)CC tissue significantly reduced,and that of miR-21 significantly increased(all P<0.05).Bioinformatics(starBase v2.0)analysis showed that LINC00294 had a binding site with miR-213’UTR region,and the dual luciferase reporter gene experiment verified that there was a targeting relationship between LINC00294and miR-21.Overexpression of LINC00294 or inhibition of miR-21 expression,the HPV18^(+)HeLa cells A value,Cyclin D1,Bcl-2 protein and miR-21 expression levels significantly reduced,and the apoptosis rate and Bax protein expression level significantly increased(all P<0.05).Overexpression of miR-21 could partially reverse the proliferation-inhibiting effect and apoptosis-promoting effect of overexpression of LINC00294 on HPV18^(+)HeLa cells.Conclusion The expression level of LINC00294 in HPV18^(+)CC tissues is significantly reduced.Overexpression of LINC00294 can inhibit the expression of miR-21 and the proliferation of HPV18^(+)CC cells,while promote cell apoptosis.