摘要
目的探讨胶质瘤细胞中长非编码RNA 00467(LINC00467)通过竞争性内源RNA(ceRNA)机制调控丝氨酸/苏氨酸蛋白激酶3基因(RIPK3)与肿瘤微环境(TME)及药敏性的关系及其机制。方法对癌症基因组图谱(TCGA)数据库和基因型-组织表达(GTEx)数据库胶质瘤及正常脑组织基因表达进行分析,筛选差异表达基因(DEGs)(P<0.01且表达变化大于2倍的基因),确定有意义的长链非编码RNA(lncRNA)-mRNA关系对,进行生存分析。在Starbase v3.0数据库中筛选微小RNA(miRNA,miR)-lncRNA关系对,确定ceRNA。在metascape、TISCH、CancerSEA数据库进行分析。取25例新鲜胶质瘤组织及25例正常脑组织,应用荧光实时定量反转录聚合酶链反应(qRT-PCR)检测基因表达。应用R软件包及SPSS统计分析软件对结果进行处理。结果对TCGA和GTEx表达谱进行差异表达分析,发现6095个差异表达的lncRNA(3083上调,3012下调)和10164个差异表达的mRNA(6803上调,3361下调)。对胶质瘤中的319795对候选lncRNA-mRNA对进行筛选,确定LINC00467-RIPK3为研究对象,发现LINC00467、RIPK3在胶质瘤组织高表达,且呈正相关(r=0.66,P<0.01)。数据库分析发现RIPK3可能通过海绵吸附miR-3612与LINC00467形成ceRNA。生存分析显示LINC00467-RIPK3高表达的患者预后较差(P<0.05)。胶质瘤单细胞数据集分析发现RIPK3在巨噬细胞中高表达,bulk数据集分析发现RIPK3与巨噬细胞表面分子表达正相关(P<0.01)。应用使用表达数据估计恶性肿瘤中的间质和免疫细胞算法计算胶质瘤患者的免疫得分,分析发现RIPK3与Estimate、基质计分、免疫计分以及肿瘤纯度显著相关(P<0.01)。药敏数据分析显示,RIPK3的表达与多种药物的半数抑制浓度(IC50)值有关(P<0.01)。qRT-PCR结果显示,胶质瘤组织中LINC00467、RIPK3的表达水平高于正常组织(0.74±0.28、0.34±0.20,t=8.94、9.38,P<0.01),而胶质瘤组织miR-3612的表达水平(0.57±0.27)低于正常组织(0.79±0.33,t=-2.17,P<0.01)。结论LINC00467可能通过ceRNA机制促进RIPK3表达而参与胶质瘤TME和药敏性调控。
Objective To investigate the relationship between serine/threonine protein kinase 3(RIPK3)gene regulated by long non-coding RNA 00467(LINC00467)in tumor microenvironment(TME)of glioma cells and drug sensitivity through competing endogenous RNA(ceRNA)mechanism.Methods The Cancer Genome Atlas(TCGA)database and genotype-tissue expression(GTEx)database were analyzed for gene expression in glioma and normal brain tissues,and differentially expressed genes(DEGs,P<0.01 and more than 2-fold change in gene expression)were screened to identify meaningful long non-coding RNA(lncRNA)-mRNA pairs for survival analysis.MicroRNA(miRNA,miR)-lncRNA pairs were screened in the Starbase v3.0 database and ceRNAs were identified for analysis in the metascape,TISCH,CANCERSEA databases.Fluorescence real-time quantitative reverse transcriptase polymerase chain reaction(qRT-PCR)was used to detect gene expression in 25 fresh glioma tissues and 25 normal brain tissues.The results were processed by R software package and SPSS statistical analysis software.Results Differential expression analysis of glioma gene expression profiling in TCGA and GTEx databases showed that 6095 differentially expressed lncRNAs(3083 upregulated,3012 downregulated),10164 differentially expressed mRNAs(6803 upregulated,3361 downregulated)were confirmed.Linc00467-RIPK3 was identified from 319795 pairs of candidate lncRNA-mRNA pairs in glioma.LINC00467 and RIPK3 were found to be highly expressed and positively correlated in glioma tissues(r=0.66,P<0.01).It was revealed that RIPK3 might bind miR-3612 and LINC00467 to form ceRNA via sponge mechanism.Results of survival analysis showed that patients with high expression of LINC00467-RIPK3 had a poorer prognosis(P<0.05).Glioma single-cell data set analysis confirmed that RIPK3 was highly expressed in macrophages,and bulk data set analysis found that RIPK3 was positively correlated with surface molecule expression in macrophage(P<0.01).Estimation of STromal and Immune cells in Malignant Tumor tissues using Expression data(Estimate)algorithm was used to evaluate the immune score of glioma patients,and RIPK3 was significantly correlated with Estimate,stomal score,immune score and tumor purity(P<0.01).Analysis of drug sensitivity data showed that RIPK3 expression was associated with half maximal inhibitory concentration(IC50)values of several target drugs(P<0.01).Results of qRT-PCR showed that the expression levels of LINC00467 and RIPK3 in glioma tissues(3.03±1.25,1.35±0.50)were higher than those in normal tissues(0.74±0.28,0.34±0.20,t=8.94,9.38,P<0.01),while the expression level of miR-3612 in glioma tissues(0.57±0.27)was lower than that in normal tissues(0.79±0.33,t=-2.17,P<0.05).LINC00467 was positively correlated with RIPK3(r=0.85,P<0.01),and LINC00467 and RIPK3 negatively correlated with miR-3612(r=-0.72,-0.83,P<0.01)in glioma tissues.Conclusion Linc00467 might be involved in the regulation of glioma TME and drug sensitivity by promoting RIPK3 expression via ceRNA mechanism.
作者
郗艳国
张靖悦
滕伟
赵永辉
程世翔
董晓林
梁赞
王志峰
李国京
Xi Yanguo;Zhang Jingyue;Teng Wei;Zhao Yonghui;Cheng Shixian;Dong Xiaolin;Liang Zan;Wang Zhifeng;Li Guojing(Department of Neurosurgery,Cangzhou Central Hospital,Cangzhou 061000,China;The Fifth Clinical College of Xinjiang Medical University,Urumqi 830000,China;Department of Medicine,Cangzhou Women&Children’s Healthcare Hospital,Cangzhou 061000,China;Department of Research and Development,Xincheng Hospital of Tianjin University,Tianjin 300450,China;Department of Anesthesiology,Cangzhou Central Hospital,Cangzhou 061000,China)
出处
《中华实验外科杂志》
CAS
北大核心
2023年第9期1711-1714,共4页
Chinese Journal of Experimental Surgery
基金
河北省卫生健康委员会医学重点科技研究计划(20220354)。
