Cheng Yang, Yin Baobing, Hou Tianlu, Chen Tianyang, Ping Jian
Institute of Liver Disease, Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai, China.
Department of General Surgery, Huashan Hospital Affiliated to Fudan University, Shanghai, China.
J Cell Physiol. 2019 Sep;234(9):16215-16225. doi: 10.1002/jcp.28285. Epub 2019 Feb 19.
This study aimed to validate the methylation of key genes in hepatocellular carcinoma (HCC) screened by bioinformatics analysis and explore whether they affected HCC cell proliferation, migration, and invasion. Using The Cancer Genome Atlas (TCGA) database, HCC-related differentially methylated positions (DMPs) were screened, genes corresponding to DMPs were selected, and prognosis-related genes were identified. A representative DMP was used to divide the DMPs into hyper- and hypomethylated groups. Expression of key genes in cell lines was detected using quantitative real-time polymerase chain reaction and western blot analysis. After treatment of HepG2 cells with 5-Aza-2'-deoxycytidine (5-Aza-DC), gene expression was observed. Bisulfite sequencing PCR assay was used to detect methylation frequency. Overexpressed GRASP lentiviral vectors were constructed to analyze their influence on cell proliferation, migration, and invasion using cell counting kit-8 and transwell assays. Forty-three HCC prognosis-related genes were screened using the TCGA database. cg00249511 (SCT) was used to divide the DMPs into hyper- and hypomethylated groups, distinguishing between high- and low-risk samples. The prognosis survival model constructed using 12 genes revealed the prognosis type. GRASP messenger RNA was downregulated in HepG2 and upregulated after 5-Aza-DC treatment. In HCC tissues, methylation frequency of GRASP was upregulated. GRASP overexpression inhibited HepG2 cell proliferation, invasion, and G-CSFR expression. Thus, GRASP might be a prognosis-related gene controlled by methylation.
本研究旨在验证通过生物信息学分析筛选出的肝细胞癌（HCC）关键基因的甲基化情况，并探讨它们是否影响HCC细胞的增殖、迁移和侵袭。利用癌症基因组图谱（TCGA）数据库，筛选出与HCC相关的差异甲基化位点（DMP），选择与DMP对应的基因，并鉴定出与预后相关的基因。使用一个代表性的DMP将DMP分为高甲基化组和低甲基化组。通过定量实时聚合酶链反应和蛋白质免疫印迹分析检测细胞系中关键基因的表达。用5-氮杂-2'-脱氧胞苷（5-Aza-DC）处理HepG2细胞后，观察基因表达情况。采用亚硫酸氢盐测序PCR法检测甲基化频率。构建过表达GRASP的慢病毒载体，使用细胞计数试剂盒-8和Transwell实验分析其对细胞增殖、迁移和侵袭的影响。利用TCGA数据库筛选出43个与HCC预后相关的基因。使用cg00249511（SCT）将DMP分为高甲基化组和低甲基化组，区分高风险和低风险样本。使用12个基因构建的预后生存模型揭示了预后类型。GRASP信使核糖核酸在HepG2中表达下调，5-Aza-DC处理后上调。在HCC组织中，GRASP的甲基化频率上调。GRASP过表达抑制HepG2细胞增殖、侵袭和G-CSFR表达。因此，GRASP可能是一个受甲基化调控的预后相关基因。
