m6a甲基转移酶METTL3调控KAI1/CD82表达介导垂体神经内分泌肿瘤细胞生物学行为的机制研究

Mechanism of m6a methyltransferase METTL3 mediating the biological behavior of pituitary neuroendocrine tumor cells by regulating the expression of KAI1/CD82

目的研究m6a甲基转移酶METTL3调控KAI1/CD82表达介导垂体神经内分泌肿瘤细胞增殖、迁移与侵袭的机制。方法通过实时荧光聚合酶链反应(qPCR)和蛋白免疫印迹测定大鼠垂体细胞、大鼠垂体神经内分泌肿瘤细胞系GH3和MMQ中的METTL3与KAI1/CD82表达水平。体外培养GH3细胞,将其随机分为对照组、METTL3过表达质粒组、METTL3空质粒组、METTL3 siRNA组、METTL3 siRNA阴性对照组,经分组转染后,通过qPCR和蛋白免疫印迹检测各组细胞METTL3与KAI1/CD82表达;通过CCK-8实验检测各组细胞活力;通过细胞划痕、Transwell侵袭实验检测各组细胞迁移侵袭情况;通过甲基化RNA免疫共沉淀(MeRIP)实验检测各组细胞KAI1/CD82 m6A甲基化修饰情况。结果相比大鼠垂体细胞,大鼠垂体神经内分泌肿瘤细胞系GH3及MMQ中的METTL3蛋白及mRNA表达水平明显升高,KAI1/CD82蛋白及mRNA表达水平明显降低(P<0.05)。与对照组相比,METTL3空质粒组、METTL3 siRNA阴性对照组细胞各指标差异无统计学意义;与对照组、METTL3空质粒组相比,METTL3过表达质粒组细胞活力、迁移距离、侵袭细胞数、METTL3蛋白及mRNA表达水平、KAI1/CD82 m6A甲基化水平升高(P<0.05),KAI1/CD82蛋白及mRNA表达水平降低(P<0.05);与对照组、METTL3 siRNA阴性对照组相比,METTL3 siRNA组细胞活力、迁移距离、侵袭细胞数、METTL3蛋白及mRNA表达水平、KAI1/CD82 m6A甲基化水平降低(P<0.05),KAI1/CD82蛋白及mRNA表达水平升高(P<0.05)。结论下调METTL3表达可降低KAI1/CD82 m6A甲基化水平,促进KAI1/CD82mRNA转录表达,抑制垂体神经内分泌肿瘤细胞增殖、侵袭及迁移。

Objective To study the mechanism of m6a methyltransferase METL3 mediated proliferation,migration and invasion of pituitary neuroendocrine tumor cells by regulating the expression of KAI1/CD82.Methods The expression levels of METTL3 and KAI1/CD82 in rat pituitary cells,rat pituitary tumor cell lines GH3,and MMQ were determined by qPCR and Western blot assay.GH3 cells were cultured in vitro and randomly divided into the control group,the METL3 overexpression plasmid group,the METL3 empty plasmid group,the METL3 siRNA group and the METL3 siRNA negative control group.After grouping and transfection,the expression levels of METL3 and KAI1/CD82 of each cell group were detected by qPCR and Western blot assay.The cell viability of each group was detected by CCK-8 experiment.The cell migration and invasion in each group were detected by cell scratch and Transwell invasion experiment.The methylation modification of KAI1/CD82 m6A in each group was detected by methylated RNA immunoprecipitation(MeRIP)experiment.Results Compared with rat pituitary cells,the METL3 protein and mRNA expression level in rat pituitary tumor cell lines GH3 and MMQ were significantly increased(P<0.05).KAI1/CD82 protein and mRNA expression levels were significantly reduced(P<0.05).There were no significant differences in cell indicators between the control group,the METTL3 empty plasmid group and the METTL3 siRNA negative control group(P>0.05).Compared with the control group and the METTL3 empty plasmid group,the cell viability,migration distance,number of invaded cells,METL3 protein and mRNA expression level,KAI1/CD82 m6A methylation level increased in the METL3 overexpression plasmid group(P<0.05),and the KAI1/CD82 protein and mRNA expression levels decreased(P<0.05).Compared with the control group and the METTL3 siRNA negative control group,the cell viability,migration distance,number of invaded cells,METTL3 protein and mRNA expression level and KAI1/CD82 m6A methylation level decreased in the METTL3 siRNA group(P<0.05),and KAI1/CD82 protein and mRNA expression levels increased(P<0.05).Conclusion Down-regulating the expression of METTL3 can reduce the level of KAI1/CD82 m6A methylation,promote the transcriptional expression of KAI1/CD82 mRNA,reduce the proliferation,invasion and migration of pituitary tumor cells,and inhibit the occurrence and development of pituitary tumors.