摘要
目的 基于生物信息数据库分析肝细胞癌(HCC)组织RNF145表达与HCC患者预后、基因突变、临床特征、免疫细胞和免疫检查点间的关系,采用细胞实验验证RNF145对SK-HEP-1细胞增殖、迁移和侵袭的影响,探讨其作用机制。方法 (1)采用TIMER数据库分析33种恶性肿瘤组织中RNF145的表达情况。比较TCGA数据库357例HCC患者癌组织及50份正常组织RNF145 mRNA相对表达量;比较357例HCC患者不同T分期、病理分级、临床分期者癌组织RNF145 mRNA相对表达量。以癌组织RNF145 mRNA相对表达量均数(3.86)为界,将357例HCC患者分为高表达组178例(RNF145 mRNA相对表达量≥3.86)和低表达组179例(RNF145 mRNA相对表达量<3.86),绘制Kaplan-Meier生存曲线,比较RNF145高表达组与低表达组总生存率、无疾病生存率和无进展生存率;采用单因素和多因素Cox回归分析357例HCC患者死亡的影响因素。根据TCGA数据库357例HCC患者癌组织DNA甲基化情况,分析癌组织RNF145表达与DNA甲基化的相关性,鉴定潜在的CpG位点,分析甲基化位点与RNF145表达的相关性。以与RNF145表达具有相关性的特异性甲基化位点(cg02298193、cg06030535、cg11495854、cg01657408)表达的中位数(0.077、0.590、0.075、0.050)为界,以大于等于中位数为高表达、小于中位数为低表达,通过Kaplan-Meier生存曲线比较特异性甲基化位点高、低表者总生存率。应用cBioPortal数据库分析HCC组织RNF145突变频率。应用COSMIC数据库分析RNF145在HCC中常见的突变类型及碱基改变。应用TCGA数据库分析357例HCC患者RNF145高、低表达组癌组织的基因突变情况。应用TCGA数据库分析HCC组织中与RNF145表达呈正、负相关的前5个基因,对其进行KEGG通路富集分析,采用TCGA、ICGC数据库验证与RNF145表达相关的信号通路。应用TIMER数据库分析RNF145表达与免疫细胞和免疫检查点的关系。(2)取2017年3月—2019年1月河南省人民医院3例HCC患者手术切除的癌组织及癌旁组织,采用免疫组织化学法检测RNF145表达。(3)取对数生长期SK-HEP-1细胞,分为敲低组(转染RNF145 siRNA)、空载组(转染RNF145 siRNA-NC)、甲基化组(采用S-腺苷基甲硫氨酸诱导甲基化)和对照组(正常培养不做处理),采用实时荧光定量PCR法检测4组RNF145 mRNA相对表达量。检测敲低组和空载组细胞划痕愈合率、侵袭细胞数和细胞增殖率。结果 (1)TIMER数据库分析结果显示,RNF145在胆管癌、食管癌、头颈鳞状细胞癌、肾透明细胞癌、肾乳头状细胞癌、HCC、胃癌、子宫内膜癌组织中表达均升高。TCGA数据库分析结果显示,HCC患者癌组织RNF145 mRNA相对表达量(2.34±0.70)高于正常组织(1.91±0.50)(t=4.125,P<0.001)。T1~T2期、病理分级G1~G2级、临床分期Ⅰ~Ⅱ期的HCC患者癌组织RNF145 mRNA相对表达量分别低于T3~T4期、G3~G4级、Ⅲ~Ⅴ期者(t=3.879~4.174,P均<0.001)。RNF145高表达组总生存率(42.9%)、无疾病生存率(37.5%)、无进展生存率(48.2%)均低于RNF145低表达组(61.4%、54.4%、65.8%)(χ^(2)=5.914~13.390,P均<0.05)。多因素Cox回归分析结果显示,RNF145表达(HR=1.040,95%CI:1.006~1.074,P=0.020)、T分期(HR=1.675,95%CI:1.288~2.177,P<0.001)是HCC患者死亡的影响因素。HCC患者癌组织RNF145表达与RNF145甲基化程度呈负相关(r=-0.260,P<0.001)。RNF145的启动子区域鉴定出10个潜在的CpG位点,特异性CpG位点cg06030535(r=-0.170,P=0.0014)、cg02298193(r=-0.110,P=0.032)、cg11495854(r=-0.210,P<0.001)、cg01657408(r=-0.270,P<0.001)的甲基化程度与RNF145表达均呈负相关。甲基化位点cg06030535、cg02298193、cg11495854低表达者总生存率(50.88%、55.56%、55.00%)分别高于高表达者(48.49%、38.10%、46.67%)(χ^(2)=5.907~15.870,P均<0.05)。cBioPortal数据库分析结果显示,RNF145体细胞突变总体频率为0.7%,且大多数为错义突变,显著低于TP53和CTNNB1等核心驱动基因。COSMIC数据库分析结果显示,RNF145突变类型主要为错义突变(35.00%),其次为同义突变(12.70%),多数碱基改变替换涉及C>T(23.43%)、A>G(16.86%)、G>A(21.43%)。TCGA数据库分析结果显示,RNF145高、低表达组均有TP53、CTNNB1、TTN和MUC16突变发生,高表达组TP53突变频率(31%)较高。TCGA数据库基因的相关性分析结果显示,与RNF145表达呈正相关的前5个基因为SLC38A1、ACTR3、STK17A、TMEM87B、CDC42SE2,呈负相关的前5个基因为CYP27A1、AGXT、RBP4、SERPINC1、PIPOX,均富集于WNT信号通路;进一步验证结果显示,高表达RNF145可激活WNT信号通路,RNF145表达与WNT信号通路相关。RNF145表达与免疫细胞B细胞(r=0.323,P<0.001)、CD8+T细胞(r=0.249,P<0.001)、CD4+T细胞(r=0.491,P<0.001)、巨噬细胞(r=0.561,P<0.001)、中性粒细胞(r=0.499,P<0.001)、树突状细胞(r=0.431,P<0.001)、CD163(r=0.310,P<0.001)、MS4A4A(r=0.300,P<0.001)、VSIG4(r=0.330,P<0.001)、CD96(r=0.280,P<0.001)、IDO1(r=0.180,P<0.001)、PDCD1(r=0.320,P<0.001)、CD276(r=0.510,P<0.001)、CTLA4(r=0.340,P<0.001)、HAVCR2(r=0.470,P<0.001)、CD274(r=0.270,P<0.001)表达均呈正相关。(2)HCC组织RNF145表达(0.052±0.001)高于癌旁组织(0.030±0.003)(t=8.857,P<0.001)。(3)RNF145mRNA相对表达量在敲低组细胞(0.603±0.056)低于空载组(1.004±0.066)(t=7.997,P<0.001)、甲基化组细胞(0.293±0.183)低于对照组(1.058±0.415)(t=3.378,P=0.015)。敲低组细胞划痕愈合率、侵袭细胞数和细胞增殖率[(46.743±4.911)%、(175.57±62.70)个、(0.882±0.053)%]均低于空载组[(71.680±6.062)%、(322.50±139.70)个、(1.376±0.044)%](t=6.584、10.880、18.710,P均<0.001)。结论 RNF145在HCC组织中呈高表达,其表达与肿瘤恶性程度及不良预后相关;敲低RNF145表达可抑制SK-HEP-1细胞增殖、迁移及侵袭,其机制可能与WNT信号通路激活及肿瘤免疫微环境调控有关。
Objective To analyze the relationships of RNF145 expression with the prognosis,gene mutations,clinical features,immune cell,and immune checkpoints in hepatocellular carcinomam(HCC)tissues based on bioinformatic database,to validate the effects of RNF145 on the cell proliferation,migration,and invasion of HCC cells using cellular experiments,and to explore its mechanism.Methods(1)The TIMER database was used to analyze the RNF145 expression in 33 types of malignant tumors.The relative expressions of RNF145 in cancer tissues of 357 HCC patients and 50 normal tissue specimens from the TCGA database were compared.The relative expression of RNF145 mRNA was compared in cancer tissues of HCC patients with different T stages,pathological grades,and clinical stages.All 357 HCC patients were divided into the high-expression group(n=178,RNF145 mRNA relative expression≥3.86)and the low-expression group(n=179,RNF145 mRNA relative expression<3.86)based on the mean relative expression of RNF145 mRNA in cancer tissues(3.86).Kaplan-Meier survival curves were plotted,and the overall survival rate,disease-free survival rate,and progression-free survival rate were compared between the high-and low-expression groups.Univariate and multivariate Cox regression analyses were used to identify the influencing factors of death in 357 HCC patients.Based on the DNA methylation status of cancer tissues in 357 HCC patients from the TCGA database,the correlation between RNF145 expression and DNA methylation in cancer tissues was analyzed,the potential CpG sites were identified,and the correlation between methylation sites and RNF145 was analyzed.The median values(0.077,0.590,0.075,0.050)of RNF145 related specific methylation sites(cg02298193,cg06030535,cg11495854,cg01657408)were used as the thresholds.Those greater than or equal to the thresholds were considered as high expression,and those less than the thresholds were considered as low expression.The overall survival rates were compared between those with high and low expressions using Kaplan-Meier survival curves.The frequency of RNF145 mutation in HCC tissues was analyzed using cBioPortal database,and the common mutation types and base alterations of RNF145 in HCC tissues were analyzed using COSMIC database.The gene mutation status in the cancer tissues of 357 HCC patients in the high-and low-expression groups was analyzed using TCGA database.The top 5 genes positively and negatively correlated with RNF145 expression in HCC tissues were analyzed using TCGA database,and the enrichment analysis of KEGG was conducted on them.The signaling pathways related to RNF145 expression were validated using TCGA and ICGC databases.The relationships of the RNF145 with the immune cells and immune checkpoints were analyzed using TIMER database.(2)The surgically resected cancer tissues and adjacent tissues of 3 HCC patients in Henan Provincial People's Hospital from March 2017 to January 2019 were obtained,and the RNF145 expression was detected using immunohistochemistry.(3)The SK-HEP-1 cells in logarithmic growth phase were taken and were divided into a knockdown group(transfected with RNF145 siRNA),an empty vector group(transfected with RNF145 sirNA-NC),a methylation group(treated with S-adenosylmethionine disulfate to induce methylation),and a control group(normal culture without treatment).The relative expression of RNF145 mRNA was detected by real-time fluorescence quantitative PCR in four groups.The cell scratch wound healing rate,the cell migration number and the cell proligeration rate were detected in the knockdown group and the empty vector group.Results(1)The results of TIMER database analysis showed that the RNF145 expression was elevated in tissues of cholangiocarcinoma,esophageal carcinoma,head and neck squamous cell carcinoma,clear cell renal cell carcinoma,papillary renal cell carcinoma,HCC,gastric cancer,and endometrial cancer.The results of TCGA database analysis showed that the relative expression of RNF145 mRNA was higher in cancer tissues of HCC patients(2.34±0.70)than that in normal tissues(1.91±0.50)(t=4.125,P<0.001).The relative expressions of RNF145 mRNA of cancer tissues were lower in HCC patients with stage T_1-T_2,pathological grade G_1-G_2,and clinical stageⅠ-Ⅱthan those in patients with stage T_3-T_4,pathological grade G_3-G_4,and clinical stageⅢ-Ⅵ(t=3.879-4.174,all P values<0.001).The overall survival rate,disease-free survival rate,and progression-free survival rate were lower in the high-expression group(42.9%,37.5%,48.2%)than those in the low-expression group(61.4%,54.4%,65.8%)(χ^(2)=5.914-13.390,all P values<0.05).The results of multivariate Cox regression analysis showed that the RNF145 expression(HR=1.040,95%CI:1.006-1.074,P=0.020)and stage T(HR=1.675,95%CI:1.288-2.177,P<0.001)were the influencing factors of death of HCC patients.The RNF145 expression in cancer tissues of HCC patients was negatively correlated with the RNF145 methylation degree(r=-0.260,P<0.001).Ten potential CpG sites were identified in the driver region of RNF145,and the methylation degrees of specific CpG sites cg06030535(r=-0.170,P=0.0014),cg02298193(r=-0.110,P=0.032),cg11495854(r=-0.210,P<0.001)and cg01657408(r=-0.270,P<0.001)were negatively correlated with RNF145 expression.The overall survival rates were higher in patients with low expression of methylation sites cg06030535,cg02298193 and cg11495854(50.88%,55.56%,55.00%)than these in patients with high expression of those methylation sites(48.49%,38.10%,46.67%)(χ^(2)=5.907-15.870,all P values<0.05).The results of the cBioPortal database analysis showed that the overall frequency of RNF145 somatic mutations was 0.7%,and most of them was missense mutations,which was significantly lower than that of the core driver genes such as TP53 and CTNNB1.The results of COSMIC database analysis showed that the main mutation type of RNF145 was missense mutations(35.00%),followed by synonymous mutations(12.70%),and most base alteration substitutions involved C>T(23.43%),A>G(16.86%),and G>A(21.43%).The results of TCGA database analysis showed that TP53,CTNNBl,TTN and MUC16 mutations occurred in both high-and low-expression groups,and the TP53 mutation frequency was higher in high-expression group(31%).The correlation analysis results of genes in TCGA database showed that the top 5 genes positively correlated with RNF145 expression were SLC38A1,ACTR3,STK17A,TMEM87B and CDC42SE2,and the top 5 genes negatively correlated with RNF145 expression were CYP27A1,AGXT,RBP4,SERPINCI and PIPOX,which were all enriched in the WNT signaling pathway.The further validation results showed that RNF145 high expression activated the WNT signaling pathway,and RNF145 expression was associated with the WNT signaling pathway.The RNF145 expression was positively correlated with the expressions of immune B cells(r=0.323,P<0.001),CD8+T cells(r=0.249,P<0.001),CD4+T cells(r=0.491,P<0.001),macrophages(r=0.561,P<0.001),neutrophils(r=0.499,P<0.001),dendritic cells(r=0.431,P<0.001),CD163(r=0.310,P<0.001),MS4A4A(r=0.300,P<0.001),VSIG4(r=0.330,P<0.001),CD96(r=0.280,P<0.001),IDO1(r=0.180,P<0.001),PDCD1(r=0.320,P<0.001),CD276(r=0.510,P<0.001),CTLA4(r=0.340,P<0.001),HAVCR2(r=0.470,P<0.001)and CD274(r=0.270,P<0.001).(2)The RNF145 expression was higher in HCC tissues(0.052±0.001)than that in adjacent tissues(0.030±0.003)(t=8.857,P<0.001).(3)The relative expression of RNF145 mRNA was lower in the knockdown group(0.603±0.056)than that in the empty vector group(1.004±0.066)(t=7.997,P<0.001),and in the methylation group(0.293±0.183)than that in the control group(1.058±0.415)(t=3.378,P=0.015).The cell scratch wound healing rate,the cell migration number and the cell proliferation rate were lower in the knockdown group[(46.743±4.911)%,175.57±62.70,(0.882±0.053)%]than those in the empty vector group[(71.680±6.062)%,322.50±139.70,(1.376±0.044)%](t=6.584,10.880,18.710,all P values<0.001).Conclusions RNF145 is highly expressed in HCC tissues,and its expression is associated with the degree of tumor malignancy and poor prognosis.To knockdown RNF145 expression can inhibit the proliferation,migration and invasion of SK-HEP-1 cells,and the mechanism may be related to the activation of WNT signaling pathway and the regulation of tumor immune microenvironment.
作者
杨志钊
陈家兴
崔永强
赵志磊
张晓
YANG Zhizhao;CHEN Jiaxing;CUI Yongqiang;ZHAO Zhilei;ZHANG Xiao(Department of Hepatobiliary and Pancreatic Surgery,Zhengzhou University People's Hospital,Henan Provincial People's Hospital,Zhengzhou,Henan 450003,China;Department of Hepatobiliary and Pancreatic Surgery,Henan Provincial People's Hospital,Zhengzhou,Henan 450o03,China)
出处
《中华实用诊断与治疗杂志》
2025年第7期597-610,共14页
Journal of Chinese Practical Diagnosis and Therapy
基金
河南省青年健康科技创新人才培训项目(CYQ20220080)
河南省医学科技研究与发展计划联合建设项目(LHGJ20240041)。
关键词
肝细胞癌
RNF145
细胞增殖
迁移
侵袭
WNT信号通路
hepatocellular carcinoma
RNF145
cell proliferation
migration
invasion
WNT signaling pathway
