摘要
目的:探究CD155在宫颈癌中的作用和依赖m^(6)A修饰的调控机制。方法:应用数据库预测宫颈癌细胞中CD155(PVR)的表达情况,CD155对患者预后的影响,运用实时荧光定量PCR(qRT-PCR)法测定宫颈癌细胞HeLa和宫颈永生细胞S12中CD155的表达水平。分别运用MTT、平板克隆、迁移侵袭等实验评估CD155对宫颈癌细胞HeLa生长、增殖、迁移和侵袭的影响。应用Western印迹法测定E-钙黏蛋白(E-cad)和波形蛋白(Vimentin)的表达水平并评价CD155对EMT的影响。MeRIP-RT-qPCR检测CD155是否受m^(6)A调控,Western印迹法和qRT-PCR测定METTL3是否影响CD155蛋白和RNA的表达水平。放线菌素D处理细胞后检测METTL3对CD155RNA半衰期的作用,利用预测网站m^(6)ATaget预测CD155RNA甲基化位点的识别者(Reader),运用Western印迹法和qRT-PCR分析YTHDF1是否影响CD155蛋白和RNA的表达水平,放线菌素D处理细胞后检测YTHDF1对CD155RNA半衰期的作用,RIP-RT-qPCR实验进一步确定YTHDF1和YTHDF1-mut对CD155RNA下拉能力。结果:CD155在宫颈癌组织和细胞中表达较高,体内高表达CD155的患者生存率较低。相比S12细胞,HeLa细胞中CD155的表达增加(t=3.749,P<0.05)。CD155的高表达促进了体外宫颈癌细胞的生长(t=3.152,P<0.05)、增殖(t=3.706,P<0.05)、迁移(t=5.422,P<0.01)和侵袭(t=3.599,P<0.05),抑制E-cad的表达促进了Vimentin的表达。MeRIP-RT-qPCR结果显示CD155RNA受m^(6)A调控,METTL3促进了CD155蛋白和RNA(t=6.725,P<0.05)的表达,敲降METTL3之后CD155RNA半衰期降低(t=5.622、6.063、5.857,均P<0.05)。敲降YTHDF1之后CD155的蛋白水平降低,CD155RNA半衰期降低(t=10.93、5.602、4.359,均P<0.05)。在YTHDF1高表达的细胞中,CD155RNA被有效免疫沉淀(t=4.686,P<0.01),但在YTHDF1-mut转染的细胞中,免疫沉淀结果显示下拉的CD155RNA水平明显降低(t=4.462,P<0.05)。结论:CD155RNA在m^(6)A修饰的情况下,被YTHDF1特异性识别,进而稳定性增强和翻译增加,CD155表达增加促进了�
Objective:To explore the role of CD155 in cervical cancer and its regulatory mechanism dependent on m^(6)A modification.Methods:The database was used to predict the expression of CD155(PVR)in cervical cancer cells,and the effect of CD155 on the prognosis of patients.The expression levels of CD155 in cervical cancer cells HeLa and cervical immortalized cells S12 were determined by real-time quantitative PCR(qRT-PCR).The effects of CD155 on the growth,proliferation,migration,and invasion of cervical cancer cells HeLa were evaluated by MTT,plate cloning,migration and invasion experiments.The expression levels of E-cadherin(E-cad)and vimentin(Vimentin)were determined by Western blotting and the effect of CD155 on EMT process was evaluated.MeRIP-RT-qPCR was used to detect whether CD155 was regulated by m^(6)A,and Western blotting and qRT-PCR were used to determine whether METTL3 affected the expression levels of CD155 protein and RNA.Actinomycin D treated cells to detect the effect of METTL3 on the half-life of CD155 RNA,using the prediction website m^(6)ATaget to predict the recognizer(Reader)of CD155 RNA methylation sites,Western blotting and qRT-PCR were used to analyze whether YTHDF1 affects the expression levels of CD155 protein and RNA,Actinomycin D treated cells to detect the effect of YTHDF1 on the half-life of CD155 RNA,and RIP-RT-qPCR experiment further confirmed the pull-down ability of YTHDF1 and YTHDF1-mut on CD155 RNA.Results:CD155 was highly expressed in cervical cancer tissues and cells,and patients with high CD155 expression in vivo had a lower survival rate.Compared with S12 cells,the expression of CD155 was increased in HeLa cells(t=3.749,P<0.05).High expression of CD155 promoted the growth(t=3.152,P<0.05),proliferation(t=3.706,P<0.05),migration(t=5.422,P<0.01)and invasion(t=3.599,P<0.05)of cervical cancer cells in vitro,inhibited the expression of E-cad and promoted the expression of Vimentin.MeRIP-RT-qPCR results showed that CD155RNA was regulated by m^(6)A,METTL3 promoted the expression of CD155
作者
祁小珍
石立莹
QI Xiao-zhen;SHI Li-ying(Department of Pathogen Biology,School of Basic Medical Sciences,Tianjin Medical University,Tianjin 300070,China)
出处
《天津医科大学学报》
2022年第5期497-502,共6页
Journal of Tianjin Medical University
