Wilms肿瘤1相关蛋白促进宫颈癌细胞增殖的作用及其机制

Promotion effect of Wilms’tumor 1 associated protein on the proliferation of cervical cancer cells and its mechanism

目的探讨Wilms肿瘤1相关蛋白(WTAP)对宫颈癌细胞增殖的影响及其机制。方法结合OncoLnc数据库和Cancer RNA-Seq Nexus(CRN)数据库分析WTAP基因与宫颈癌患者预后的相关性;通过Western blot分析人正常宫颈上皮细胞(HUCEC)、宫颈癌SiHa和HeLa细胞之间WTAP蛋白表达的差异;利用小干扰RNA(siRNA)和质粒转染分别调控HeLa细胞中WTAP的表达。HeLa细胞分为对照siRNA组(NC siRNA)、WTAP-siRNA组(WTAP siRNA)、对照质粒组(Empty vector)、WTAP过表达质粒组(WTAP OE vector)。通过Western blot验证转染效果,CCK8和克隆形成实验检测细胞增殖,Western blot检测丝氨酸/苏氨酸蛋白激酶(AKT)活性和G蛋白信号调节物5(RGS5)的表达,m6A-IP-qPCR检测RGS5的N6-甲基腺嘌呤(m6A)修饰水平。结果WTAP基因高表达组宫颈癌患者的总生存期显著低于WTAP基因低表达组患者[Log-rank检验P=0.0412(OncoLnc)和0.0078(CRN)]。与HUCEC相比,宫颈癌细胞SiHa和HeLa细胞WTAP蛋白表达显著升高,HeLa细胞升高更加显著(P<0.05)。与对照siRNA组相比,WTAP-siRNA组HeLa细胞增殖能力显著升高(P<0.05),AKT磷酸化水平显著降低、RGS5表达显著增多(P<0.05),RGS5 m6A水平显著降低(P<0.05);与对照质粒组相比,WTAP过表达质粒组HeLa细胞增殖能力显著降低(P<0.05),AKT磷酸化水平显著升高、RGS5表达显著减少(P<0.05),RGS5 m6A水平显著升高(P<0.05)。结论WTAP促进宫颈癌HeLa细胞增殖,其机制与调节RGS5的m6A修饰,从而影响AKT信号通路有关。

Objective To determine the effect of Wilms’tumor 1 associated protein(WTAP)on the proliferation of cervical cancer cells and its mechanism.Methods OncoLnc and Cancer RNASeq Nexus(CRN)were used to analyze the correlation between WTAP expression and prognosis of cervical cancer patients.Western blot was used to analyze the differential WTAP protein expression between human cervical epithelial cells(HUCEC)and cervical cancer cells(SiHa and HeLa).Small interfering RNA(siRNA)and plasmid transfection were used to regulate the expression of WTAP in HeLa cells.HeLa cells were divided into a negative control(NC)siRNA group,a WTAP siRNA group,an empty vector group,and a WTAP OE vector group.The transfection effect was verified by Western blot.The proliferation of HeLa cells was detected by CCK8 and clone formation assay.AKT serine/threonine kinase(AKT)activity and regulator of G protein signaling 5(RGS5)expressions were measured by Western blot,and the level of RGS5 N6-methyladenosine(m6A)was detected by m6AIP-qPCR.Results The overall survival of patients with cervical cancer in WTAP gene high expression group was significantly lower than that of patients with low expression of WTAP gene,the Log-rank test P=0.0412(oncolnc)and 0.0078(CRN).Compared with the HUCEC,the protein expression of WTAP in SiHa and HeLa cells was significantly higher,the increased expression was more significant in HeLa cells(P<0.05).Compared with the scrambled siRNA group,the proliferation of HeLa cells in the WTAP siRNA group increased significantly(P<0.05),AKT phosphorylation decreased significantly,RGS5 expression increased significantly(P<0.05),and RGS5 m6A level decreased significantly(P<0.05).Compared with the empty vector group,the proliferation of HeLa cells in the WTAP OE vector group decreased significantly(P<0.05),AKT phosphorylation increased significantly,RGS5 expression decreased significantly(P<0.05),and RGS5 m6A level increased significantly(P<0.05).Conclusion WTAP promotes the proliferation of cervical cancer HeLa cells,whose mechanism is related to m6A modification of RGS5 and AKT signaling pathway.