蛋白质精氨酸残基化学修饰酶PADs与PRMTs在人肝癌细胞系中的表达

Expression of chemical modification enzymes PADs and PRMTs targeting protein arginine residues in human hepatocellular carcinoma cell lines

目的探究蛋白质精氨酸脱亚胺酶(PADs)和蛋白质精氨酸甲基转移酶(PRMTs)在肝癌细胞系中的表达水平,并分析PADs催化的蛋白瓜氨酸化水平是否与PRMTs表达有关。方法通过RT-qPCR检测肝癌细胞系中PADs和PRMTs的mRNA转录水平,Western blot检测PADs、瓜氨酸化蛋白和PRMTs蛋白表达水平。利用生物信息学分析TCGA数据库中肝癌组织PADs和PRMTs的mRNA相关性,免疫组化检测肝癌组织PADs、瓜氨酸化蛋白和PRMTs表达。结果与正常肝细胞LX-2相比,PAD2和PRMT1在Huh7细胞中mRNA转录水平升高(P<0.001),PAD4、PRMT1、PRMT5和PRMT7在HepG2细胞中mRNA转录水平升高(P<0.01)。PAD2和PAD4在HepG2和Huh7中的蛋白水平显著高于LX-2(P<0.01)。瓜氨酸化蛋白在HepG2和Huh7中的水平低于LX-2。肝癌组织中PADs和PRMTs的表达强于癌旁组织,但瓜氨酸化蛋白几乎呈阴性。PAD2和所有PRMTs家族成员在临床大样本中呈显著正相关性(P<0.05)。结论肝癌细胞中PADs有较高的表达水平,而PADs催化的蛋白瓜氨酸化水平较低,可能是受胞内高水平表达的PRMTs酶竞争性影响。

Objective To explore the expression of protein arginine deiminases(PADs)and protein arginine methyltransferases(PRMTs)in hepatocellular carcinoma,and to analyze whether the level of protein citrullination catalyzed by PADs is related to the expression of PRMTs.Methods The mRNA transcription of PADs and PRMTs in hepatoma cells was detected by quantitative PCR and the protein expression of PADs,citrullinated proteins and PRMTs was detected by Western blot.The mRNA correlation between PADs and PRMTs in liver cancer tissues in TCGA database was analyzed by bioinformatics,and the expressions of PADs,citrullinated proteins and PRMTs in liver cancer tissues was detected by immunohistochemistry.Results Compared with normal liver cell LX-2,the mRNA transcription of PAD2 and PRMT1 in Huh7 cells was higher(P<0.001),and that of PAD4,PRMT1,PRMT5 and PRMT7 in HepG2 cells was higher(P<0.01).The protein level of PAD2 and PAD4 in HepG2 and Huh7 was significantly higher than those in LX-2(P<0.01).The expression level of citrullinated protein in HepG2 and Huh7 was lower than that in LX-2.The expression of PADs and PRMTs in hepatocellular carcinoma was stronger than that in adjacent tissues,and the expression of citrullinated proteins was almost negative.There were positive correlation between PAD2 and all PRMTs in clinical sample database(P<0.05).Conclusions The expression of PADs is high in hepatocellular carcinoma,whereas the expression of citrullinated proteins is low,which may be affected by the competition of PRMTs enzyme highly expressed in cells.