肝细胞癌PD-1免疫治疗敏感性的靶基因分析  

Target gene analysis of PD-1 immunotherapy sensitivity in hepatocellular carcinoma

作　　者：史宇泽 丁可 孙倍成 Shi Yuze;Ding Ke;Sun Beicheng(Dept of Hepatobiliary Surgery,The Affiliated Drum Tower Hospital of Nanjing University Medical School,Nanjing 210008;Dept of Hepatobiliary,Pancreatic and Transplantation Surgery,The First Affiliated Hospital of Anhui Medical University,Hefei 230022;Dept of Hepatobiliary Surgery,Nanjing Drum Tower Hospital Clinical College of Nanjing Medical University,Nanjing 210008)

机构地区：[1]南京大学医学院附属鼓楼医院肝胆外科,南京210008 [2]安徽医科大学第一附属医院肝胆胰及移植外科,合肥230022 [3]南京医科大学鼓楼临床医学院肝胆外科,南京210008

出　　处：《安徽医科大学学报》2024年第8期1323-1329,1338,共8页Acta Universitatis Medicinalis Anhui

基　　金：国家自然科学基金(编号:81930086、82120108012);安徽省高校科研项目(编号:2022AH010070);安徽省临床医学研究转化专项(编号:202204295107020008)。

摘　　要：目的探究肝细胞癌(HCC)患者程序性死亡受体1(PD-1)免疫治疗敏感性的特征基因。方法通过加权基因共表达网络分析(WGCNA)及差异性分析筛选与PD-1免疫治疗敏感性相关数据集GSE202069及ERP117672中共同差异基因,将上述共同差异基因通过Lasso回归筛选PD-1免疫治疗敏感性的特征基因;通过GEPIA及Ualcan数据库预测特征基因在HCC的表达水平,通过实时荧光定量反转录聚合酶链反应(RT-qPCR)、蛋白质印迹(WB)及免疫组织化学染色(IHC)实验验证其表达。构建过表达3-羟基丁酸脱氢酶1(BDH1)细胞系,通过肿瘤功能学实验细胞计数试剂盒8(CCK-8)、平板克隆、EdU染色、细胞划痕、Transwell实验探究其对肿瘤细胞增殖、迁移及侵袭能力的影响。结果通过WGCNA及差异性分析筛选出两项数据集共有118个共同差异基因,Lasso回归筛选出共同差异基因中与PD-1免疫治疗敏感性特征基因(含黄素二甲基苯胺单加氧酶3(FMO3),过氧化物酶体反式-2-烯酰辅酶A还原酶(PECR),BDH1,溶质载体家族7成员1(SLC7A1),细胞色素b5 A型(CYB5A)和磷酸烯醇丙酮酸羧激酶1(PCK1));生存分析表明BDH1与HCC最为相关(总体生存率:P<0.001;复发:P=0.007)。GEPIA及Ualcan数据库分析显示BDH1在HCC组织中低表达,通过RT-qPCR、WB及IHC对本中心收集的HCC样本进一步证实BDH1在HCC中低表达。CCK-8、平板克隆、EdU染色、细胞划痕、Transwell实验结果显示,与Hep3B pCDH组相比,过表达BDH1使得HCC细胞吸光度降低(t=4.766,P<0.01),克隆形成数目减少(t=16.02,P<0.0001),增殖细胞比例下降(t=23.13,P<0.0001),细胞迁移率减慢(t=25.28,P<0.0001),穿过小室数目减少(t=10.78,P=0.004)。结论BDH1是观察HCC患者PD-1免疫治疗敏感性的特征基因;BDH1具有抑制HCC细胞增殖、迁移和侵袭的能力。Objective To investigate the characteristic genes of Programmed cell death protein 1(PD-1)immunotherapy sensitivity in Hepatocellular carcinoma(HCC).Methods The common differential genes in GSE202069 and ERP117672 data sets were investigated by Weighted Gene Co-expression Network Analysis(WGCNA)and Difference analysis,and the characteristic genes of PD-1 immunotherapy sensitivity were screened through Lasso regression.The expression levels of characteristic genes in HCC were predicted by GEPIA and Ualcan databases,and their expression was verified by real-time quantitative reverse transcription polymerase chain reaction(RT-qPCR),Western blot(WB)and Immunohistochemistry(IHC).3-hydroxybutyrate dehydrogenase 1(BDH1)overexpressed cell line was constructed,followed by cell counting kit-8(CCK-8),EdU,cell scratches and Transwell experiment to investigate the effects of BDH1 on the proliferation,migration and invasion of HCC cells.Results Total of 118 common differentially expressed genes were identified in two datasets by WGCNA and differential analysis.The characteristic genes associated with PD-1 immunotherapy sensitivity screened through Lasso regression including Flavin containing dimethylaniline monoxygenase 3(FMO3),Peroxisomal trans-2-enoyl-CoA reductase(PECR),BDH1,Solute carrier family 7 member 1(SLC7A1),Cytochrome b5 type A(CYB5A)and Phosphoenolpyruvate carboxykinase 1(PCK1).Survival analysis showed that BDH1 was most associated with HCC(Overall survival:P<0.001,Recurrence:P=0.007).GEPIA and Ualcan databases showed low expression of BDH1 in HCC tissues,while RT-qPCR,WB,and IHC further confirmed this.CCK-8,plate cloning assay,EdU staining,cell scratch,and Transwell experiments showed that compared with the Hep3B pCDH group,overexpression of BDH1 resulted in a decrease in the absorbance of HCC cells(t=4.766,P<0.01),a decrease in the number of clone formation(t=16.02,P<0.0001),a decrease in the proportion of proliferating cells(t=23.13,P<0.0001),a decrease in cell migration rate(t=25.28,P<0.0001),and a decrease in

关 键 词：肝细胞癌 PD-1免疫治疗 BDH1 肿瘤功能学实验 

分 类 号：R657.3[医药卫生—外科学]