m6A甲基化修饰调控结直肠癌细胞中PD-L1表达

Effects of RNA m6A modification on PD-L1 expression in colorectal cancer

目的检测程序性死亡配体-1(PD-L1)mRNA在结直肠癌细胞中是否存在m6A甲基化修饰以及m6A对PD-L1表达的影响,为结直肠癌的免疫治疗提供新的理论依据。方法利用实时荧光定量聚合酶链式反应技术(qPCR)及蛋白质印迹法检测m6A相关蛋白表达变化对PD-L1mRNA和蛋白表达的影响,通过双荧光素酶报告基因实验验证YTHDF2对PD-L1mRNA的调控作用,采用甲基化RNA免疫共沉淀结合qPCR技术(MeRIP-qPCR)确认YTHDF2对PD-L1m6A水平的影响,通过qPCR比较PD-L1和YTHDF2在结直肠癌组织中表达的相关性。结果qPCR和蛋白质印迹检测结果显示m6A阅读蛋白YTHDF2可负向调控PD-L1mRNA和蛋白表达水平,双荧光素酶实验结果说明YTHDF2可识别并作用于PD-L1基因上的潜在m6A修饰位点,MeRIP-qPCR分析发现YTHDF2可以抑制PD-L1mRNA的m6A水平,qPCR进一步发现YTHDF2与PD-L1在癌组织中的表达量呈负相关关系。结论PD-L1在结直肠癌细胞中存在m6A修饰,且YTHDF2可抑制其在结直肠癌细胞中的m6A水平及表达量。

Objective To investigate whether PD-L1 mRNA is modified by m6A modification and the effects of m6A modification on PD-L1 expression in colorectal cancer,to provide novel insights into immunotherapy of colorectal cancer.Methods qPCR analysis and Western blotting were used to detect whether PD-L1 mRNA and protein expression were regulated by specific proteins related with m6A modification,dual-luciferase reporter experiments were performed to explore the regulation of YTHDF2 on PD-L1,MeRIP-qPCR analysis was employed to verify the effects of YTHDF2 on m6A level of PD-L1 mRNA in colorectal cancer cells,and the correlation of YTHDF2 expression and PD-L1 expression in tumor tissues were compared by qPCR.Results The results of qPCR and Western blotting showed that YTHDF2,a m6A reader,could negatively regulate PD-L1 mRNA and protein expression,the results of dual-luciferase reporter experiments found that YTHDF2 recognized and combined with potential m6A modification sites of PD-L1,MeRIP-qPCR analysis demonstrated that YTHDF2 suppressed the m6A level of PD-L1 in colorectal cancer cells,and results of qPCR analysis further confirmed that YTHDF2 expression was negatively correlated with PD-L1 expression in colorectal cancer tissues.Conclusion PD-L1 mRNA was regulated by m6A modification in colorectal cancer cells,and YTHDF2 could regulate the m6A level and expression of PD-L1 in colorectal cancer cell.