SYK基因甲基化对胃癌预后评估的价值及恶性生物学行为的影响

Associatione of SYK gene methylation with prognosis of patients with gastric cancer and its effect on malignant behavior of gastric cancer cells

目的探讨脾酪氨酸激酶(SYK)基因甲基化对胃癌病情及预后评估的价值及恶性生物学行为的影响。方法回顾性选取2020年1月至2022年1月湖州师范学院附属第一医院诊治的胃癌患者107例为观察组,另择54例同期在本院诊治的胃良性疾病(胃息肉、胃炎等可取得病理组织的疾病)患者为对照组。留取观察组患者肿瘤组织及配对的癌旁组织(距离肿瘤组织边缘>3 cm),对照组患者胃镜活检组织为本研究的组织标本。以购买的MKN-45、SGC-7901、MFC、AGS胃癌细胞系及人胃正常黏膜上皮细胞(GES-1)为细胞标本。采用甲基特异性聚合酶链反应检测各组组织与各组细胞SYK基因甲基化状态。比较不同临床病理特征的观察组肿瘤组织SYK基因甲基化情况。Kaplan-Meier法分析SYK基因甲基化与观察组患者预后的关系。将针对SYK启动子序列设计的MON、UMON、CON1、CON2寡核苷酸序列转染至AGS细胞,分别设为MON组、UMON组、CON1组、CON2组。采用CCK-8法检测细胞增殖活性;Transwell检测细胞迁移及侵袭能力;采用实时荧光定量聚合酶链反应检测SYK及上皮间质转化相关蛋白上皮型钙黏蛋白(E-cadherin)、神经型钙黏蛋白(N-cadherin)和波形蛋白(Vimentin)基因mRNA表达;采用Western blot法检测SYK、E-cadherin、N-cadherin和Vimentin蛋白表达。结果观察组肿瘤组织SYK基因甲基化率(70.1%)高于观察组癌旁组织(14.0%)、对照组胃镜活检组织(14.8%)(均P<0.05),SYK mRNA及蛋白表达量均低于癌旁组织、对照组患者胃镜活检组织(均P<0.05)。MKN-45、SGC-7901、MFC胃癌细胞SYK基因呈甲基化状态,而AGS、GES-1细胞SYK基因呈未甲基化状态,AGS、GES-1细胞SYK mRNA及蛋白表达量均高于MKN-45、SGC-7901、MFC细胞(均P<0.05)。组织学分级3级、肿瘤大小≥5 cm、T_(3)~T_(4)期、区域淋巴结有转移、有远处转移及Ⅲ~Ⅳ期患者肿瘤组织SYK基因甲基化率分别高于1~2级、肿瘤大小<5 cm、T_(1)~T_(2)期、区域淋巴结无转移、无远处转移及Ⅰ~Ⅱ期患者(均P<0.05)。SYK基因甲基化患者中位生存期明显短于未甲基化患者(26.7个月比35.1个月,P<0.05)。与UMON组、CON1组及CON2组比较,MON组AGS细胞SYK mRNA和蛋白表达量均降低(均P<0.05),增殖活性、迁移和侵袭能力均增强(均P<0.05),E-cadherin表达量降低(均P<0.05),N-cadherin和Vimentin表达量均升高(均P<0.05)。结论胃癌组织SYK基因处于高甲基化状态,为患者预后的不利因素。SYK基因高甲基化可促进胃癌细胞增殖、迁移、侵袭及上皮间质转化。

Objective To investigate the association of spleen tyrosine kinase(SYK)gene methylation with disease progression and prognosis of patients with gastric cancer and its impact on its malignant biological behavior of gastric cancer cells.Methods A total of 107 patients with gastric cancer admitted in the First Affiliated Hospital of Huzhou Teachers College from January 2020 to January 2022 were enrolled(GC group)in the study,and 54 patients with benign gastric diseases were selected as the control group.Tumor tissues and paired pericancerous tissues of patients in the GC group,and gastric tissues of patients in the control group taken by endoscopic biopsy were collected.Gastric cancer cell lines MKN 45,SGC 7901,MFC,AGS and human normal gastric mucosal epithelial cells(GES 1)were used for in vitro study.Methyl-specific polymerase chain reaction was used to detect the methylation status of the SYK gene in tissues and cell samples.The Kaplan-Meier method was used to analyze the relationship between SYK gene methylation and the prognosis of patients in the GC group.The MON,UMON,CON1,and CON2 oligonucleotide sequences designed for the SYK promoter sequence were transfected into AGS cells.The cell proliferation activity was detected by CCK 8 method;the cell migration and invasion ability were detected by Transwell test;the mRNA expression of SYK and epithelial-mesenchymal transition(EMT)-related proteins E-cadherin,N-cadherin,and Vimentin genes was detected by real-time fluorescence quantitative PCR;the protein expression of SYK,E-cadherin,N-cadherin,and Vimentin was detected by Western blot.Results The SYK gene methylation rate in the tumor tissue of the GC group(70.1%)was significantly higher than that in the adjacent tissue(14.0%)and the gastric tissue of the control group(14.8%)(all P<0.05),and the SYK mRNA and protein expression levels were lower than those in the adjacent tissue and the gastric tissue of the control group(all P<0.05).The SYK gene in MKN 45,SGC 7901,and MFC gastric cancer cells was methylated,while the SYK gene in AGS and GES 1 cells was unmethylated.The SYK mRNA and protein expression levels in AGS and GES 1 cells were higher than those in MKN 45,SGC-7901,and MFC cells(all P<0.05).The SYK gene methylation rates in tumor tissues of patients with histological grade 3,tumor size≥5 cm,T_(3)-T_(4)stage,regional lymph node metastasis,distant metastasis,andⅢ-Ⅳstage were higher than those of patients with grade 1-2,tumor size<5 cm,T_(1)-T_(2)stage,no regional lymph node metastasis,no distant metastasis,andⅠ-Ⅱstage(all P<0.05).The median survival of patients with SYK gene methylation was significantly shorter than that of patients with unmethylation(26.7 months vs.35.1 months,P<0.05).Compared with the UMON group,CON1 group,and CON2 group of AGS cells,the expression levels of SYK mRNA and protein in the MON group of AGS cells were decreased(all P<0.05),the proliferation activity,migration and invasion abilities were enhanced(all P<0.05),the expression level of E-cadherin was decreased(all P<0.05),and the expression levels of N-cadherin and Vimentin were increased(all P<0.05).Conclusion The SYK gene in gastric cancer tissue is in a hypermethylated state,which is an unfavorable factor for the patient's prognosis.Hypermethylation of the SYK gene can promote the proliferation,migration,invasion and epithelial-mesenchymal transition of gastric cancer cells.