NSUN2通过靶基因PRPS2调控核苷酸代谢从而介导肝癌细胞的增殖

NSUN2 regulates nucleotide metabolism through targeting PRPS2 to pro⁃mote liver cancer cell proliferation

目的:探讨NOP2/Sun RNA甲基转移酶家族成员2(NOP2/Sun RNA methyltransferase 2,NSUN2)在肝癌发展中的功能和机制。方法:比较肿瘤基因组图谱(The Cancer Genome Atlas,TCGA)肝癌数据集中肝癌组织与正常组织NSUN2的mRNA表达水平;Western blot检测9对肝癌患者肝癌组织与癌旁组织中NSUN2的蛋白表达水平;Kaplan-Meier法分析肝癌TCGA数据集中NSUN2对肝癌生存预后的影响;活细胞动态成像与分析系统记录经shNSUN2慢病毒感染的Huh7和SNU182细胞的实时生长情况及集落形成实验检测细胞增殖能力;基因富集分析(GSEA)肝癌TCGA数据库中高表达NSUN2组富集的信号通路,探索NSUN2可能甲基化的关键基因和参与的信号通路;Pearson法分析TCGA数据集中NSUN2与预测基因的相关性;在SNU182细胞中敲低NSUN2后,用RT-qPCR和Western blot验证预测基因表达的变化。结果:TCGA数据集分析结果及Western blot实验结果显示NSUN2在肝癌组织中高表达。敲低NSUN2后Huh7细胞和SNU182细胞的增殖能力降低。基因富集分析结果显示嘌呤和嘧啶代谢通路在高表达NSUN2组富集。重亚硫酸盐甲基化测序(Bisulfite-Seq)结果与基因富集分析结果存在共同变化基因有PRPS2、ADSL、POLR2D、CANT1、ENTPD4、TXNRD1和CAD。Pearson相关分析结果显示TCGA数据集中NSUN2与PRPS2的mRNA表达呈正相关(r=0.3283,P<0.05)。RT-qPCR和Western blot结果验证了敲低NSUN2后抑制了SNU182细胞中PRPS2的mRNA和蛋白表达。RT-qPCR结果显示敲低NSUN2后嘌呤从头合成相关基因的表达降低。结论:NSUN2可能通过调控PRPS2的表达参与核苷酸代谢,从而影响肝癌细胞增殖。

AIM:To investigate the function and mechanism of NOP2/Sun RNA methyltransferase 2(NSUN2)in the development of liver cancer.METHODS:The mRNA expression levels of NSUN2 in liver cancer tissues and nor⁃mal tissues in liver cancer TCGA dataset were compared.Western blot was used to detect the protein levels of NSUN2 of patient samples.Kaplan-Meier method was used to analyze the effect of NSUN2 on the survival of liver cancer.Incucyte experiment and colony formation experiment were used to detect the proliferation of Huh7 cells and SNU182 cells after⁃knock-down of NSUN2 expression with shNSUN2 lentivirus.GSEA was used to analyze the signaling pathways enriched in the group with high expression of NSUN2 by TCGA dataset.The published bisulfite sequencing result with the GSEA re⁃sult was overlapped to demonstrate the key genes that may be methylated by NSUN2.Pearson analysis was used to check the correlation between NSUN2 and predicted regulated genes in TCGA dataset.After knock-down of NSUN2 in SNU182 cells.RT-qPCR and Western blot were used to verify the change of predicted genes.RESULTS:The results of TCGA da⁃taset analysis and Western blot showed that NSUN2 was highly expressed in liver cancer tissues.Knock-down of NSUN2 expression inhibited proliferation of Huh7 cells and SNU182 cells.The GSEA result showed that purine and pyrimidine metabolism pathways were enriched in the NSUN2 high expression group.The overlapping candidate genes of bisulfite se⁃quencing result and purine and pyrimidine metabolism genes up-regulated in GSEA were PRPS2,ADSL,POLR2D,CANT1,ENTPD4,TXNRD1 and CAD.The positive correlation of NSUN2 and PRPS2 was found in TCGA dataset by Pear⁃son analysis(r=0.3283,P<0.05).The results of RT-qPCR and Western blot verified that knock-down of NSUN2 expres⁃sion inhibited PRPS2 at both mRNA and protein levels in the SNU182 cells.RT-qPCR result showed that knock-down of NSUN2 inhibited the expression of genes related to purine de novo synthesis.CONCLUSION:NSUN2 may regulate nu⁃cleotide metabolism via regulating the expression of PRPS2,thereby affecting the proliferation of liver cancer cells.