长链非编码RNA-微小RNA-mRNA调控网络在晚期膀胱癌的发展中的作用  

Critical role of long non-coding RNA-microRNA-mRNA regulatory network in the development of advanced bladder cancer

作　　者：庞明睿 杜洋[1] 喻希 王磊[1] 袁静萍[2] 刘雯[2] 刘修恒[1] Pang Mingrui;Du Yang;Yu Xi;Wang Lei;Yuan Jingping;Liu Wen;Liu Xiuheng(Department of Urology,Renmin Hospital of Wuhan University,Wuhan 430060,China;Department of Pathology,Renmin Hospital of Wuhan University,Wuhan 430060,China)

机构地区：[1]武汉大学人民医院泌尿外科,430060 [2]武汉大学人民医院病理科,430060

出　　处：《中华实验外科杂志》2021年第6期1018-1022,共5页Chinese Journal of Experimental Surgery

基　　金：武汉市应用与基础前沿项目(2018060401011321)。

摘　　要：目的:探索晚期膀胱癌发展过程中与差预后相关的RNA,构建一个长链非编码RNA-微小RNA-mRNA(lncRNA-miRNA-mRNA)网络并加以实验验证,以研究其在晚期膀胱癌发展中的分子调控作用。方法:收集肿瘤基因组图谱(TCGA)和NCBI-GEO数据库431例和492例膀胱癌患者组织样本,Ⅲ、Ⅳ期、高级别、高级别≥病理亚分期2期(pT2期)为晚期膀胱癌,Ⅱ、Ⅰ期、低级别为早期膀胱癌;进行差异表达分析、权重基因共表达网络分析、功能富集分析、生存分析、miRNA靶向预测和Pearson相关分析。利用可视化软件构建网络。统计学方法使用t检验、Fisher精确检验、Pearson相关分析、Log-Rank检验比较组间差异,BH法计算错误发现率校正P值,以FDR<0.05、P<0.05为差异统计学意义。随后,收集22例膀胱癌患者组织样本,高级别肌层浸润性≥T2期、高级别或浸润性TaT1期为晚期膀胱癌,低级别非浸润性TaT1为早期膀胱癌;进行实时荧光定量聚合酶链反应(qPCR)、免疫印迹法和免疫组织化学实验。组间比较采用t检验。结果:获得相似表达模式的8个长链非编码RNA、28个mRNA和8个微小RNA,并以此构建出核心lncRNA-miRNA-mRNA调控网络,该网络与晚期膀胱癌的侵袭、进展、转移及差预后相关。晚期膀胱癌组织Ⅵ型胶原α1(COL6A1)、Ⅱ型钙粘附蛋白(CDH11)、金属蛋白酶和血小板反应蛋白模体Ⅰ型(ADAMTS12)、长链非编码RNA-01705(LINC01705)、长链非编码RNA-01929(LINC01929 RNA)相对内参表达倍数值高于早期膀胱癌组织(-1.213比-4.212,t=4.170,P<0.01;-10.75比-14.64,t=4.602,P<0.01;-7.594比-11.93,t=5.152,P<0.01;-10.18比16.55,t=4.493,P<0.01;-11.62比-15.08,t=2.869,P<0.05),差异均有统计学意义。晚期膀胱癌组织COL6A1、CDH11、ADAMTS12蛋白平均吸光度值高于早期膀胱癌组织(0.2138比0.0909,t=2.302,P<0.05;0.1839比0.0789,t=5.992,P<0.01;0.2791比0.1478,t=4.527,P<0.01),差异均有统计学意义。膀胱癌组织人-微小RNA-6875-5Objective To explore the RNA associated with poor prognosis in the development of advanced bladder cancer(BCa),we constructed and validated an long non-coding RNA-microRNA-mRNA(lncRNA-miRNA-mRNA)network,so as to investigate its molecular regulatory role in the development of BCa.Methods Using transcriptome profiles from The Cancer Genome Atlas and Gene Expression Omnibus databases,431 and 492 cases of BCa tissue samples were collected.Clinical stageⅢandⅣ,high grade,high grade with stage≥pathological stage 2(pT2)were classified as advanced clinical stage BCa(ACS-BCa).Clinical stageⅡandⅠ,low grade were classified as low clinical stage BCa(LCS-BCa).We performed differential expression(DE)analysis,weighted gene co-expression network analysis,functional enrichment analysis,survival analysis,prediction of miRNA targeting,and Pearson correlation analysis.Statistically,t′test,Fisher′s exact test,Pearson correlation analysis,Log-rank test and Flase discovery rate(FDR)calculated with benjamini hochberg(BH)method were used.FDR<0.05 and P<0.05 were used as sifting thresholds.Subsequently,22 cases of BCa tissue samples were collected.High grade and muscle-invasion with stage≥T2,high grade or invasion with stage TaT1 were classified as ACS-BCa,and low grade with stage TaT1 was classified as LCS-BCa.The real time quantitative polymerane chain reaction(qPCR),Western blotting,and immunohistochemistry were carried out.The t′test was used to compare the statistically significant differences between experimental groups.Results Totally,8 lncRNAs-28 mRNAs and 8 miRNAs-28 mRNAs pairs shared similar expression patterns,constituting a core competing endogenous RNA(ceRNA)regulatory network related to the invasion,progression,metastasis and poor prognosis of ACS-BCa.Respectively,the RNA relative expression fold changes of collagen typeⅥα1(COL6A1),cadherin typeⅡ(CDH11),a disintegrin and metalloproteinase with thrombaspondin motif type I(ADAMTS12),long non-coding RNA 01705、01929(LINC01705,LINC01929)in ACS-BCa tissues

关 键 词：长链非编码RNA 微小RNA 膀胱癌 

分 类 号：R737.14[医药卫生—肿瘤]