摘要
目的探讨长链非编码RNA(lncRNA)在食管鳞状细胞癌(ESCC)患者外周血中的表达及意义。方法收集南京市第一医院2015年6月至2018年12月期间早期(Ⅰ~Ⅱ)ESCC患者(组别1)、中晚期(Ⅲ~Ⅳ)ESCC患者(组别2)、正常成人(对照组)血清标本各5例,采用TRIzol试剂提取总RNA,将转录得到的cRNA为模板反转录合成cDNA,纯化后荧光标记并与lncRNA芯片进行杂交。两两比较筛选出相对表达量比值在2倍以上且差异有统计学意义的lncRNA作为差异表达的lncRNA。应用实时定量反转录聚合酶链反应(RT-qPCR)验证lncTCONS00011689在ESCC样本中的表达情况,并分析其与ESCC患者临床病理特征及预后的关系,随访日期截止2019年12月31日。应用GraphPad Prism 5.0统计学软件分析,采用配对t检验和Log-rank生存分析。结果在组别1比对照组中上调的lncRNA有1 620条,下调的lncRNA有102条。在组别2比对照组中上调的lncRNA有17条,下调的lncRNA有129条。在组别2比组别1中上调的lncRNA有9条,下调的lncRNA有73条。生信数据分析显示这些差异表达基因的功能与细胞间相互作用、代谢、免疫应答、细胞黏附分子等功能均具有显著相关(r>0.99,P<0.05)。不同组别差异表达的lncRNA与差异表达的mRNA共表达分析显示表达结果中关联度最大的前1 000个关系对(r>0.99,P<0.05)。lncTCONS00011689在ESCC患者血浆中表达与正常人比较明显降低,其在ESCC患者血浆中的表达为5.12±0.31,在正常人血浆中的表达为4.03±0.26,差异有统计学意义(t=2.187,P<0.05)。lncTCONS00011689与ESCC临床TNM分期呈负相关(t=2.716,P<0.05),而与年龄、性别、分化程度以及淋巴结转移均无明显相关(P>0.05)。随访结果显示lncTCONS00011689表达低的ESCC患者总生存期相比于高表达的患者更短(F=0.048,P<0.05)。结论建立不同分期的ESCC患者外周血中差异lncRNA表达谱。lncTCONS00011689可作为食管鳞癌诊断、分期及预后监测的血浆分子标志物。
Objective To investigate the expression and significance of long non-coding RNA(lncRNA)in peripheral blood of patients with esophageal squamous cell carcinoma(ESCC).Methods Five serum samples in early-stage(Ⅰ-Ⅱ)ESCC patients(case group 1),mediate-late-stage(Ⅲ-Ⅳ)ESCC patients(case group 2),and normal adult(control group)each from June 2015 to December 2018 in Nanjing First Hospital were collected.Total RNA was extracted using TRIzol reagent and transcribed.The obtained cRNA was used as a template for reverse transcription to synthesize cDNA.After purification,it was fluorescently labeled and hybridized with lncRNA chip.Pairwise comparisons screened out lncRNAs whose relative expression ratio was more than 2 times and the differences were statistically significant as differentially expressed lncRNAs.Real-time fluorescence quantitative PCR(RT-qPCR)was used to verify the expression of lnc_TCONS_00011689 in ESCC samples,and to analyze its relationship with the clinicopathological characteristics and prognosis of ESCC patients.The follow-up date ended December 31,2019.GraphPad Prism 5.0 statistical software was used for analysis and paired t test and Log-rank survival analysis were applied.Results In the case group 1 vs.control group,1620 lncRNAs were up-regulated and 102 lncRNAs were down-regulated.In the case group 2 vs.control group,17 lncRNAs were up-regulated and 129 lncRNAs were down-regulated.In the case group 2 vs.case group 1,9 lncRNAs were up-regulated and 73 lncRNAs were down-regulated.Analysis of bioinformatics data shows that the functions of these differentially expressed genes are related to the functions of cell interaction,metabolism,immune response,and cell adhesion molecules(r>0.99,P<0.05).The co-expression analysis of differentially expressed lncRNAs and differentially expressed mRNAs in different groups showed that the top 1000 relationships were the most relevant in the expression results(r>0.99,P<0.05).The expression of lnc_TCONS_00011689 in plasma of ESCC patients was significantly lower than that in normal people,and it was significantly correlated with clinical stage and prognosis(P<0.05).Conclusion This study established differential lncRNA expression profiles in peripheral blood of ESCC patients with different stages.Lnc_TCONS_00011689 can be used as a molecular marker for the diagnosis,staging and prognosis of ESCC.
作者
胡云
曹秀峰
唐薇薇
Hu Yun;Cao Xiufeng;Tang Weiwei(Department of General Surgery,Jiangsu Cancer Hospital,Jiangsu Institute of Cancer Research,the First Affiliated Cancer Hospital of Nanjing Medical University,Nanjing 210009,China;Department of Oncology,Nanjing First Hospital,Nanjing Hospital,Nanjing Medical University,Nanjing 210006,China)
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2020年第4期745-747,共3页
Chinese Journal of Experimental Surgery
基金
国家自然科学基金项目(81672423)。
关键词
食管鳞状细胞癌
长链非编码RNA
芯片
分子标志物
Esophageal squamous cell carcinoma
Long non-coding RNA
Microarray
Molecular biomarker
