M6A甲基化调控因子对结直肠癌预后及细胞生物学行为的影响

Effects of N6-methyladenosine methylation regulators on prognosis and cell biological behaviors of colorectal cancer

目的探讨N6-甲基腺苷(N6-methyladenosine,m6A)相关基因对结直肠癌预后及细胞生物学行为的影响。方法从肿瘤基因组图谱(the cancer genome atlas,TCGA)下载长链非编码RNA(long noncoding RNA,lncRNA)和21个M6A相关基因的表达谱数据,R软件分析差异基因;聚类分析和主成分分析(principal component analysis,PCA)分析M6A相关基因;单因素Cox回归分析筛选预后相关M6A基因,LASSO算法建立风险预测模型;筛选结直肠癌相关差异lncRNA(differentially expressed lncRNA,DElncRNA),与M6A相关基因建立共表达网络。使用实时荧光定量PCR(real time fluorescence quantitative PCR,RT-qPCR)检测M6A reader脆性X智力低下1(fragile X mental retardation 1,FMR1)在结直肠癌组织中的表达,在结直肠癌细胞中敲除FMR1基因,通过CCK-8、划痕实验、Transwell和细胞凋亡实验检测细胞的增殖、迁移、侵袭和凋亡情况。结果21个M6A相关基因中有15个基因在结直肠癌中差异表达。聚类分析显示,结直肠癌肿瘤分成2种亚型;从建立的风险模型得出烷基化修复同源蛋白5(a-ketoglutarate-dependent dioxygenase alk B homolog 5,ALKBH5)、YTH结构域蛋白2(YTH domaincontaining protein 2,YTHDC2)和FMR1可能是独立的预后因子。随后,筛选出1784个DElncRNA,构建包含3个预后因子和18个DElncRNAs的共表达网络。网络图中发现FMR1基因占主导地位,RT-qPCR实验发现FMR1在结直肠癌组织中上调;FMR1基因的敲除能抑制结直肠癌HT-29细胞的增殖,迁移和侵袭能力,促进结直肠癌细胞的凋亡。结论M6A甲基化调控因子ALKBH5、YTHDC2和FMR1可能是结直肠癌中的独立预后因子,FMR1基因的敲除能抑制结直肠癌细胞的增殖、迁移、侵袭,促进结直肠癌细胞的凋亡。

Objective To investigate the effects of N6-methyladenosine(M6 A)related genes on the prognosis and cell biological behaviors of colorectal cancer.Methods The long non-coding RNA(lncRNA)for colorectal cancer and the expression profile data of 21 M6 A-related genes were downloaded from The Cancer Genome Atlas(TCGA),and the differential genes were analyzed with R software.Cluster analysis and principal component analysis(PCA)were further conducted,and prognostic related M6 A genes were screened by univariate Cox regression analysis.The risk prediction model was then established with LASSO algorithm.In addition,differentially expressed lncRNA associated with colorectal cancer(DElncRNA)were screened out,and a co-expression network with M6 A-related genes was subsequently established.Moreover,RT-qPCR was performed to determine the gene expression of fragile X mental retardation 1(FMR1)in colorectal cancer tissues.After FMR1 gene was knocked out in colorectal cancer cells,the cell proliferation,migration,invasion and apoptosis were detected by CCK8 assay,cell scratch test,Transwell assay and flow cytometry,respectively.Results Fifteen of the 21 M6 A-related genes were differentially expressed in colorectal cancer,and cluster analysis indicated 2 subtypes of colorectal cancer tumors.According to the risk model,ALKBH5(a-ketoglutarate-dependent dioxygenase alk B homolog 5),YTHDC2(YTH domain-containing protein 2)and FMR1 may be the independent prognostic factors.With a total of 1784 DElncRNAs screened out,a co-expression network was successfully constructed containing the 3 prognostic factors and 18 DElncRNAs.FMR1 was found to be the hub gene in the network diagram.RT-qPCR showed that FMR1 was up-regulated in colorectal cancer tissues.Knockout of FMR1 gene inhibited the proliferation,migration and invasion,and promoted cell apoptosis in colorectal cancer HT-29 cells.Conclusion M6 A methylation regulators ALKBH5,YTHDC2 and FMR1 may be the independent prognostic factors in colorectal cancer.FMR1 knockout can inhibit the proliferation,migration and invasion,and promote the apoptosis of colorectal cancer cells.