METTL14在卵巢上皮性癌组织中的表达及对A2780、SKOV3细胞增殖、侵袭和迁移的影响

目的研究甲基转移酶样蛋白14(METTL14)在卵巢上皮性癌(卵巢癌)组织中的表达及其临床意义, 探讨上调和下调METTL14表达对卵巢癌细胞系A2780、SKOV3细胞增殖、侵袭和迁移的影响。方法 (1)组织标本检测:选取2019年12月—2020年11月在广西医科大学附属肿瘤医院行手术治疗的20例卵巢癌患者的新鲜癌组织标本, 收集同期因子宫肌瘤行子宫及双侧附件切除术的15例患者的新鲜正常卵巢组织标本作为对照, 免疫组化法检测卵巢癌与正常卵巢组织中METTL14蛋白表达的差异。另收集2014年1月—2019年10月在广西医科大学附属肿瘤医院行手术治疗的121例卵巢癌患者的癌组织蜡块, 免疫组化法检测卵巢癌组织中METTL14蛋白的表达, 并分析METTL14蛋白表达与卵巢癌患者临床病理特征及预后的关系。(2)细胞实验:分别使用慢病毒载体、小分子干扰RNA(siRNA)技术, 构建上调、下调METTL14表达的卵巢癌A2780、SKOV3细胞系, 并采用逆转录(RT)-实时荧光定量PCR(qPCR)技术和蛋白印迹(western blot)法分别验证其METTL14 mRNA和蛋白的表...

Expression of METTL14 in epithelial ovarian cancer and the effect on cell proliferation,invasion and migration of A2780 and SKOV3 cells

Objective To study the expression of methyltransferase-like protein 14(METTL14)in epithelial ovarian cancer and its clinical significance,and to explore the effect of METTL14 expression on the proliferation,invasion and migration of ovarian cancer cells.Methods Immunohistochemistry(IHC)was used to detect METTL14 expression in tumor tissue samples,and analyze the relationships among METTL14 expression,clinicopathological factors,and prognosis in ovarian cancer.Lentiviral vectors and small interfering RNA(siRNA)were used to up-regulate and down-regulate the METTL14 expression in ovarian cancer cell lines A2780 and SKOV3,respectively.Liquid chromatography-tandem mass spectrometry(LC-MS/MS)method was used to detect the N6-methyladenosine(m6A)content in ovarian cancer cells.Cell counting kit-8(CCK-8),wound healing assay,and transwell assay were used to examine the function of METTL14 expression in the cells.Results(1)The IHC score of METTL14 protein was 6.2±3.7 in 20 samples of ovarian cancer tissues and 3.3±2.5 in 15 samples of normal ovarian tissues,and the difference was statistically significant(t=-2.64,P=0.012).Among the patients who suffered from ovarian cancer,there were 69 cases with high expression of METTL14 protein(IHC score≥6),accounting for 57.0%(69/121),and the cases with low expression of METTL14 protein(IHC score<6)accounting for 43.0%(52/121).Compared with the patients with low expression of METTL14,the patients with high expression of METTL14 had later stages,higher rates of lymph node metastasis,abdominal metastasis,and more ascite amount.The differences were statistically significant(all P<0.05).The overall survival rate was significantly lower in patients with high METTL14 expression than the low expression(P=0.009).(2)LC-MS/MS data showed that the relative expression of m6A in A2780 and SKOV3 cells in the lentivirus(LV)-METTL14 group were 0.213±0.024 and 0.181±0.018,which were significantly higher than those in the LV-normal control(NC)group(0.109±0.022 and 0.128±0.020;all P<0.05).While the relative expression of m6A in A2780 and SKOV3 cells in the si-METTL14 group were 0.063±0.012 and 0.069±0.015,which were significantly lower than the expression in si-NC group of 0.108±0.014 and 0.121±0.014(all P<0.05).CCK-8 assay showed that the absorbance values were significantly lower in the si-METTL14 group compared with the si-NC group at 36,48,60 hours(all P<0.05);while were significantly increased in the LV-METTL14 group compared with the LV-NC group at 48,60 hours(all P<0.01).Scratch wound assays showed that the migration rate of the si-METTL14 group was lower than those of the si-NC group,while the LV-METTL14 group were higher than the LV-NC group by 24 hours,the differences were statistically significant(all P<0.01).Cell migration and invasion were detected by transwell migration and invasion assays.After cultivated for 24 hours,the invasion cell number and the migration cell number in the si-METTL14 group were less than those in the si-NC group.While the invasion cell number and the migration cell number in the LV-METTL14 group were more than those in the LV-NC group,respectively.The differences were statistically significant(all P<0.01).Conclusion Patients with high METTL14 expression have a worse prognosis in ovarian cancer,which may increase the m6A modification of ovarian cancer cells and promote cells proliferation,invasion and migration.