食管鳞状细胞癌中 MGP 基因的表达和启动子区甲基化状态及其临床意义

MGP gene expression and promoter methylation status in esophageal squamous cell carcinoma and its clinical significance

目的研究基质Gla蛋白(MGP)在食管鳞状细胞癌(ESCC)中的表达和启动子区甲基化状态,探讨MGP对ESCC细胞侵袭的影响。方法收集2014年5月至2019年5月于中国人民解放军空军第九八六医院就诊的253例ESCC患者的ESCC组织标本和癌旁非癌组织标本,分别设为ESCC组和癌旁非癌组。采用实时荧光定量PCR(Real-time qPCR)法检测MGP mRNA的相对表达量,采用蛋白质印迹法检测MGP的蛋白表达水平,采用免疫组织化学染色法评估MGP蛋白在组织中的定位和分布,采用甲基化酶消化法、DNA亚硫酸氢盐修饰法及Real-time qPCR法分析MGP基因启动子区的甲基化状态。构建重组MGP过表达质粒和DNA甲基化转移酶1的沉默质粒(si-DNMT1)。将人ESCC细胞系KYSE30分为对照组、si-DNMT1组、阴性对照(si-NC)组、MGP过表达组和空载体(EV)组,采用Transwell小室法检测各组细胞的侵袭能力。结果与癌旁非癌组织相比,ESCC组织中MGP mRNA和蛋白表达水平均显著升高,差异均有统计学意义(P均<0.05)。免疫组织化学染色结果表明,ESCC组织中MGP的阳性率高于癌旁非癌组织。与癌旁非癌组相比,ESCC组中MGP基因启动子区CpG岛甲基化水平降低,两组差异有统计学意义(P<0.05)。与对照组相比,MGP过表达组KYSE30细胞的侵袭能力较强;与si-NC组相比,si-DNMT1组KYSE30细胞的侵袭能力较强,组间比较差异均有统计学意义(P均<0.05)。结论ESCC组织中MGP表达上调或MGP基因启动子区甲基化下调可以介导ESCC细胞的侵袭。

Objective To investigate the expression and promoter methylation of matrix Gla protein(MGP)in esophageal squamous cell carcinoma(ESCC),and to investigate the effect of MGP on invasion of ESCC cells.Methods From May 2014 to May 2019,two hundred and fifty-three patients with ESCC were enrolled in the 986th Hospital of PLA Air Force for ESCC and paracancer non-cancer tissue samples,which were divided into the ESCC group and the paracancer non-cancer group respectively.The relative expression of MGP mRNA was detected by real-time quantitative PCR(Real-time qPCR).Western blotting was used to detect the expression of MGP protein,and immunohistochemistry was used to evaluate the location and distribution of MGP protein in tissues.Methylation status of the promoter region of MGP gene was analyzed by utilizing the methylase digestion and DNA bisulfite modification method and Real-time qPCR method.Recombinant MGP overexpression plasmid and methyltransferase DNMT1 silencing plasmid(si-DNMT1)were constructed.The human ESCC cell line KYSE30 was assigned to the control group,the si-DNMT1 group,the si-NC group,the MGP overexpression group,and the empty vector(EV)group.The transwell cell method was used to detect the invasion ability of each group of cells.Results Compared with non-cancerous tissues adjacent to cancer,the levels of MGP mRNA and protein in ESCC tissues are significantly higher,with statistically significant differences(P<0.05).The results of immunohistochemical staining show that the positive rate of MGP in ESCC tissues is higher than that in non-cancerous tissues adjacent to cancer.Compared with the adjacent non-cancerous group,the methylation level of CpG island in the MGP gene promoter region in the ESCC group is reduced,with a statistically significant difference(P<0.05).Compared with the control group,the invasion ability of KYSE30 cells in the MGP overexpression group is stronger;compared with the si-NC group,the invasion ability of KYSE30 cells in the si-DNMT1 group is stronger,with statistically significant differences(P<0.05).Conclusion Up-regulation of MGP expression or down-regulation of MGP gene promoter region methylation in ESCC tissues can mediate ESCC cell invasion.