CTHRC1调控COL1A1过表达促进胃癌细胞增殖的研究

CTHRC1 promotes proliferation of gastric cancer cells by overexpression of COL1A1

目的 探讨胶原三股螺旋重叠蛋白1(CTHRC1)对胃癌细胞的增殖作用机制。方法 收集广州市增城区人民医院2017年6月-2018年6月经病理确诊的8例胃癌患者的癌组织及癌旁组织。采用Western blotting检测胃癌组织、癌旁组织和胃癌细胞系SGC-7901、BGC-823、BGC-803、MKN-45、MKN-28,以及人胃黏膜GES-1细胞中CTHRC1的蛋白表达水平;将si-CTHRC1、si-COL1A1转染至胃癌SGC-7901细胞后,用MTT法检测细胞增殖情况,流式细胞术检测细胞凋亡率;用STRING数据库预测CTHRC1的靶基因;Western blotting和免疫荧光检测si-CTHRC1转染胃癌SGC-7901细胞后CTHRC1、COL1A1蛋白表达情况;Kaplan-Meier分析CTHRC1和COL1A1蛋白在胃癌组织中的表达情况与胃癌患者预后的关系。结果 胃癌组织中CTHRC1蛋白的表达量为0.87±0.13,明显高于癌旁组织(0.31±0.20);胃癌细胞系中CTHRC1蛋白的表达量均高于人胃黏膜GES-1细胞,差异均有统计学意义(P<0.05)。si-CTHRC1转染胃癌SGC-7901细胞后,细胞增殖明显降低,si-CTHRC1与si-COL1A1共转染组胃癌SGC-7901细胞活力(0.40±0.07)明显低于单独si-CTHRC1组(0.87±0.05)和si-COL1A1组(0.83±0.13),差异均有统计学意义(P<0.05);si-CTHRC1转染胃癌SGC-7901细胞后细胞凋亡率为16.77%±1.55%,高于空白对照组的4.80%±1.93%,差异有统计学意义(P<0.05);STRING数据库预测提示COL1A1为CTHRC1作用的靶基因;si-CTHRC1转染胃癌SGC-7901细胞后CTHRC1、COL1A1相对蛋白表达量为0.92±0.16、1.08±0.23,均高于空白对照组的0.55±0.15、0.65±0.12,差异均有统计学意义(P<0.05);免疫荧光检测发现,si-CTHRC1转染胃癌SGC-7901细胞后CTHRC1、COL1A1蛋白表达明显降低;Kaplan-Meier分析结果示胃癌组织中CTHRC1高表达患者的5年生存率(27.4%)明显低于低表达患者(38.4%),胃癌组织中COL1A1高表达患者的5年生存率(20.4%)明显低于低表达患者(49.3%),差异均有统计学意义(P<0.001)。结论 CTHRC1通过过表达COL1A1蛋白促进胃癌细胞的增殖。

Objective To investigate the proliferation and potential mechanism of CTHRC1 on gastric cancer cells.Methods The cancer tissues and adjacent tissues were collected of 8 gastric cancer patients diagnosed by pathology from Jun.2017 to Jun.2018 in Guangzhou Zengcheng People's Hospital.Human gastric mucosal GES-1 cells,and gastric cancer SGC-7901,BGC-823,BGC-803,MKN-45,and MKN-28 cells were purchased from Shanghai Cell Bank of Chinese Academy of Sciences.Western blotting was used to detect the expression of CTHRC1 protein in gastric cancer tissues,paracancerous tissues and gastric cancer cell lines SGC-7901,BGC-823,BGC-803,MKN-45,MKN-28 and human gastric mucosal GES-1 cells.After transfection of gastric cancer SGC-7901 cells with si-CTHRC1 and si-COL1A1,cell proliferation was detected by MTT assay,and the apoptosis rate was detected by flow cytometry.The target gene of CTHRC 1 was predicted by STRING database.Western blotting and immunofluorescence were performed to detect the expression of CTHRC1 and COL1A1 proteins in gastric cancer SGC-7901 cells transfected with si-CTHRC1.The expressions of CTHRC1 and COL1A1 proteins in gastric cancer tissues and the prognosis of gastric cancer patients were analyzed by Kaplan-Meier.Results The expression of CTHRC1 protein was significantly higher in gastric cancer tissues(0.87±0.13)than in adjacent tissues(0.31±0.20,P<0.05).The expression of CTHRC1 protein was higher in gastric cancer cell lines than in human gastric mucosa GES-1 cells with significant difference(P<0.05).The cell proliferation of gastric cancer SGC-7901 cells transfected by si-CTHRC1 decreased significantly.The cell vitality of gastric cancer SGC-7901 cells co-transfected by si-CTHRC1 and si-COL1A1 was lower(0.40±0.07)than those only transfected by si-CTHRC1(0.87±0.05)or by si-COL1A1 groups(0.83±0.13,P<0.05).The apoptosis rate of gastric cancer SGC-7901 cells transfected by si-CTHRC1(6.77%±1.55%)was higher than that in NC group(4.80%±1.93%)with statistical significance(P<0.05).The STRING database predicted that COL1A1 was a target gene for CTHRC1.For gastric cancer SGC-7901 cells transfected by si-CTHRC1,the relative expressions of CTHRC1 and COL1A1 proteins were 0.92±0.16 and 1.08±0.23,which were higher than those in NC group(0.55±0.15 and 0.65±0.12)with significant difference(P<0.05).Immunofluorescence showed that the expressions of CTHRC1 and COL1A1 proteins decreased significantly after transfection of si-CTHRC1 into gastric cancer SGC-7901 cells.Kaplan-Meier analysis showed that the 5-year survival rate of patients with high CTHRC1 expression in gastric cancer tissues was significantly lower(27.4%)than that of patients with low CTHRC1 expression(38.4%);The 5-year survival rate of patients with high COL1A1 expression in gastric cancer tissues(20.4%)was significantly lower than that in low expression patients(49.3%),the difference was statistically significant(P<0.001).Conclusion CTHRC1 promotes the proliferation of gastric cancer cells by overexpressing COL1A1 protein.