郝利娜 王觅柱 陈玉

[摘要] 目的 檢测胃肠道间质瘤（GIST）组织及瘤旁组织中DNA甲基转移酶1（DNMT1）、DNA甲基转移酶3a（DNMT3a）和DNA甲基转移酶3b（DNMT3b）的表达，并研究整体DNA甲基化。 方法 选取包头医学院第二附属医院2017年1月至2020年1月内镜下切除的GIST组织30例及瘤旁组织30例，应用免疫组化和qRT-PCR方法检测GIST组织和瘤旁组织中DNMT1、DNMT3a和DNMT3b蛋白和mRNA的表达情况;应用亚硫酸氢盐限制性酶切分析技术（COBRA）分析重复序列LINE-1的甲基化水平。 结果 GIST中DNMT1 mRNA的表达与瘤旁组织比较，差异无统计学意义（P>0.05）;GIST中的DNMT3a mRNA表达高于瘤旁组织，差异有统计学意义（P<0.001）;GIST中的DNMT3b mRNA表达高于瘤旁组织，差异有统计学意义（P<0.001）。DNMT1蛋白在GIST及瘤旁组织中的阳性率比较，差异无统计学意义（P=1.000）;DNMT3a蛋白在GIST中的阳性率高于瘤旁组织，差异有统计学意义（P=0.045）;DNMT3b蛋白在GIST中的阳性率高于瘤旁组织，差异有统计学意义（P=0.017）。GIST中LINE-1序列的甲基化水平低于瘤旁组织，差异有统计学意义（P<0.001）;GIST中LINE-1序列的非甲基化水平高于瘤旁组织，差异有统计学意义（P=0.001）。 结论 DNMT1、DNMT3a和DNMT3b的异常表达与GIST的发生发展有密切关系，具体机制可能与降低整体甲基化水平有关。

[关键词] 胃肠道间质瘤;DNA甲基转移酶;基因表达;免疫组化;甲基化

[中图分类号] R573          [文献标识码] A          [文章编号] 1673-9701（2021）08-0039-04

Expression and DNA methylation of DNMT1， DNMT3a and DNMT3b in gastrointestinal stromal tumors

HAO Li′na1   WANG Mizhu2   CHEN Yu3

1.Clinical Medicine， Graduate School， Baotou Medical College， Inner Mongolia University of Science and Technology， Baotou   014040， China; 2.Department of Gastroenterology， the Second Affiliated Hospital of Baotou Medical College， Baotou   014030， China; 3.General Practice， Inner Mongolia Baogang Hospital， Baotou   014010， China

[Abstract] Objective To detect the expression of DNA methyltransferase 1（DNMT1）， DNA methyltransferase 3a （DNMT3a） and DNA methyltransferase 3b （DNMT3b） in gastrointestinal stromal tumor （GIST） tissues and adjacent tissues， and to study the overall DNA methylation. Methods A total of 30 cases of GIST and 30 cases of adjacent tissues were collected， all of which were resected under endoscopy in the Second Affiliated Hospital of Baotou Medical College from January 2017 to January 2020. Immunohistochemistry and qRT-PCR methods were used to detect the expression of DNMT1， DNMT3a and DNMT3b proteins and mRNA in GIST and adjacent tissues; combined bisulfite restrition analysis （COBRA） was used to analyze the methylation level of repetitive sequence LINE-1. Results The expression of DNMT1 mRNA in GIST was not different from that of adjacent tissues， and the difference was not statistically significant（P>0.05）. The expression of DNMT3a mRNA in GIST was higher than that in adjacent tissues， and the difference was statistically significant（P<0.001）; the expression of DNMT3b mRNA in GIST was higher than that of adjacent tissues， and the difference was statistically significant （P<0.001）. There was little difference in the positive rate of DNMT1 protein in GIST and adjacent tissues， and the difference was not statistically significant（P=1.000）; the positive rate of DNMT3a protein in GIST was higher than that in adjacent tissues， and the difference was statistically significant（P=0.045）; the positive rate of DNMT3b protein in GIST was higher than that in adjacent tissues， and the difference was statistically significant （P=0.017）. The methylation level of LINE-1 in GIST was lower than that in the adjacent tissue， and the difference was statistically significant （P<0.001）; the non-methylation level of LINE-1 in GIST was higher than that in adjacent tissues， and the difference was statistically significant （P=0.001）. Conclusion The abnormal expression of DNMT1， DNMT3a and DNMT3b is closely related to the occurrence and development of GIST， and the specific mechanism may be related to the reduction of overall methylation level.

[Key words] Gastrointestinal stromal tumor; DNA methyltransferase; Gene expression; Immunohistochemistry; Methylation

胃肠道间质瘤（Gastrointestinal stromal tumors，GIST）在胃肠道肿瘤中最常见，可能来自于Cajal间质细胞（Interstitial cells of cajals，ICCs）或干细胞前体细胞。在GIST中，存在异常甲基化的报道[1]，从消化道上皮增生到形成浸润病灶的过程中，DNA甲基化扮演了重要的角色。DNA甲基转移酶催化并维持DNA的基本结构和功能[2]，到目前为止，有3种DNA甲基转移酶（DNA methyltransferase，DNMT）被发现[3]，即DNA甲基转移酶1（DNA methyltransferase 1，DNMT1）、DNA甲基转移酶3a（DNA methyltransferase 3a，DNMT3a）和DNA甲基转移酶3b（DNA methyltransferase 3b，DNMT3b），此三者同时调节DNA的甲基化。本研究旨在通过检测DNA甲基转移酶在GIST和瘤旁组织中的表达变化，以及整体DNA的甲基化水平，观察DNA甲基转移酶在GIST发生发展中的作用，以期寻找新的消化道肿瘤生物标志物。

1 资料与方法

1.1 一般资料

随机选取包头医学院第二附属医院2017年1月至2020年1月内镜下切除的GIST组织30例及瘤旁组织30例，全部病例均经病理确诊。纳入标准：患者未经任何抗肿瘤药物治疗或未接受放化疗治疗;瘤旁组织距离肿瘤切缘>5 cm，且标本未经任何生物化学处理。排除标……