miR-6867及其宿主基因RAPGEFL1在食管鳞状细胞癌组织和细胞系中的表达及甲基化状态

目的:检测miR-6867及其宿主基因Rap型鸟苷酸交换因子1(rap guanine nucleotide exchange factor like l,RAPGEFL1)在食管癌细胞系及食管鳞状细胞癌(esophageal squamous cell carcinoma,ESCC)组织中的表达水平及甲基化状态,并探讨其在ESCC发生发展中的作用.方法:选取河北医科大学第四医院2014年1月至2016年1月收治的ESCC手术患者组织标本87例.应用实时荧光定量PCR(qRT-PCR)检测miR-6867及RAPGEFL1在食管癌细胞系和ESCC组织及其相应癌旁组织中的表达水平,分析miR-6867和RAPGEFL1基因表达之间的相关性.应用甲基化特异性PCR法(MSP)检测RAPGEFL1在食管癌细胞系和ESCC组织及其相应癌旁组织中的甲基化状态.结果:miR-6867在ESCC组织中的相对表达量显著低于其相应癌旁组织(P＜0.05),并与淋巴结转移及TNM分期有关(P＜0.05);RAPGEFL1基因在ESCC组织中的表达显著低于其相应癌旁组织(P＜0.05),并与淋巴结转移、组织分化程度及TNM分期相关(P＜0.05);miR-6867与RAPGEFL1在ESCC组织中的表达呈明显正相关(P＜0.05).5-氮杂-2'-脱氧胞苷(5-Aza-2’-deoxycytidine,5-Aza-dC)处理后,4种食管癌细胞系中miR-6867和RAPGEFL1基因的表达均增高,并且其甲基化程度明显降低.RAPGEFL1基因在ESCC组织中的甲基化率显著高于其相应癌旁组织(P＜0.05),并与淋巴结转移、组织分化程度及TNM分期有关(P＜0.05).结论:ESCC的发生发展可能与miR-6867和RAPGEFL1的异常低表达及RAPGEFL1高甲基化状态有关,miR-6867与RAPGEFL1表达具有一致性,且RAPGEFL1基因启动子区甲基化可能是导致miR-6867与RAPGEFL1表达沉默的机制之一.

Expression and methylation status of miR-6867 and its host gene RAPGEFL1 in human esophageal squamous cell carcinoma tissues and cell lines

Objective:To detect the expression and methylation status of miR-6867 and its host gene RAPGEFL1 (rap guanine nucleotide exchange factor like 1) in human esophageal carcinoma cell lines and esophageal squamous cell carcinoma (ESCC) tissues,and to explore the role of miR-6867 and RAPGEFL1 in occurrence and development of ESCC.Methods:Tissue specimens were obtained from 87 ESCC patients treated in the Fourth Affiliated Hospital of Hebei Medical University between January 2014 and January 2016.The expression ofmiR-6867 and RAPGEFL1 gene in esophageal cancer cell lines and ESCC tissues as well as the corresponding noncancerous tissues were detected by real-time fluorescent quantitative PCR.The relationship of miR-6867 and RAPGEFL1 gene expression were analyzed.The methylation status of RAPGEFL1 in esophageal cancer cell lines and ESCC tissues and the corresponding noncancerous tissues was detected by methylation specific PCR methods.Results:The relative expression of miR-6867 in ESCC tissues was significantly lower than that in corresponding normal tissues (P＜0.05),and its expression was closely correlated with lymph node metastasis and TNM staging (P＜0.05).The relative expression of RAPGEFL1 in ESCC tissues was significantly lower than that in corresponding normal tissues (P＜0.05),and its expression was closely correlated with lymph node metastasis,pathological differentiation and TNM staging (P＜ 0.05).The expression of miR-6867 and RAPGEFL1 was positively correlated in ESCC tissues (P＜0.05).After 5-Aza-dC treatment,the relative expression of miR-6867 and RAPGEFL1 in four cell lines was increased,and the methylation status of RAPGEFL1 was significantly decreased.The methylation rate of RAPGEFL1 in ESCC tissues was significantly higher than that in corresponding normal tissues (P＜0.05),which was closely correlated with lymph node metastasis,pathological differentiation and TNM staging (P＜0.05).Conclusions:The occurrence and development of ESCC may be related to the abnormally decreased expression of miR-6867 and RAPGEFL1 and the high methylation status of RAPGEFL1;the expression of miR-6867 was consistent with RAPGEFL1 expression.Moreover,the methylation of RAPGEFL1 gene promoter might be one of the mechanisms underlying the silence of miR-6867 and RAPGEFL1.