m^(6)A甲基化修饰酶YTHDF3对食管鳞癌细胞增殖和迁移的影响及机制

The Effect and Mechanism of m^(6)A Methylation Modification Enzyme YTHDF3 on the Proliferation and Migration of Esophageal Squamous Cell Carcinoma Cells

目的:研究N6–甲基腺嘌呤(m^(6)A)甲基化修饰酶YTH域家族蛋白3(YTHDF3)对人食管癌细胞(Ec109)增殖及迁移的影响及其机制。方法:选择食管癌患者为研究对象,取肿瘤组织及癌旁组织检测YTHDF3基因信使核糖核酸(mRNA)和蛋白表达。设计合成YTHDF3过表达质粒(pcDNA–YTHDF3)、空载质粒(pcDNA)、干扰表达质粒(si–YTHDF3)及对照(si–NC)转染至Ec109细胞。采用实时荧光定量聚合酶链式反应(qRT–PCR)检测YTHDF3、大肿瘤抑制基因1(LAST1)、锚定蛋白重复域蛋白1(ANKRD1)、半胱氨酸丰富血管生成诱导因子61(CYR61)、结缔组织生长因子(CTGF)基因mRNA表达;蛋白质印迹法检测YTHDF3基因蛋白表达;MTT法检测细胞增殖活力;Transwell检测细胞迁移。Kaplan–Meier法比较YTHDF3与生存期的关系。结果:转染si–YTHDF3后Ec109细胞YTHDF3及LAST1基因mRNA和蛋白表达、靶基因ANKRD1、CYR61及CTGF的m RNA表达显著降低,细胞增殖及迁移显著增加,差异有统计学意义(P <0.05)。转染pcDNA–YTHDF3后Ec109细胞YTHDF3及LAST1基因mRNA和蛋白表达、靶基因ANKRD1、CYR61及CTGF的m RNA表达显著升高,细胞增殖及迁移显著降低,差异有统计学意义(P <0.05)。食管鳞癌患者肿瘤组织中YTHDF3基因mRNA及蛋白表达均显著低于癌旁组织,差异有统计学意义(P <0.05)。YTHDF3基因高表达患者中位生存期显著高于YTHDF3基因低表达组患者,差异有统计学意义(P <0.05)。结论:YTHDF3基因可抑制食管鳞癌细胞增殖及迁移,其机制可能与调控LAST1基因表达有关。

Objective To study the effect and mechanism of N6-methyladenine(m^(6)A) methylation modification enzyme YTH domain family protein 3(YTHDF3) on the proliferation and migration of Ec109 esophageal squamous cell carcinoma cells. Methods Patients with esophageal cancer were selected as the research objects, and the expression of YTHDF3 gene mRNA and protein was detected in tumor tissues and adjacent tissues. pcDNA-YTHDF3, pcDNA, si-YTHDF3 and si-NC were transfected into Ec109 cells respectively. The mRNA expressions of YTHDF3, large tumor suppressor gene 1(LAST1), ankyrin repeat domain protein 1(ANKRD1), cysteine-rich angiogenesis-inducible factor 61(CYR61) and connective tissue growth factor(CTGF) were detected by real-time fluorescent quantitative polymerase chain reaction(qRT-PCR);the protein expression of YTHDF3 gene was detected by Western blot;the cell proliferation activity was detected by MTT method. Transwell was used to detect cell migration. The relationship between YTHDF3 and survival was compared by Kaplan-Meier method. Results After transfection with si-YTHDF3, the mRNA and protein expressions of YTHDF3 and LAST1 genes, the mRNA expressions of target genes ANKRD1, CYR61 and CTGF in Ec109 cells were significantly decreased, and the cell proliferation and migration were significantly increased,the differences were statistically significant(P < 0.05). After transfection with pcDNA-YTHDF3, the mRNA and protein expressions of YTHDF3 and LAST1 genes, the mRNA expressions of target genes ANKRD1, CYR61 and CTGF in Ec109 cells were significantly increased, and the cell proliferation and migration were significantly decreased, the differences were statistically significant(P < 0.05). The expression of YTHDF3 gene mRNA and protein in tumor tissues of patients with esophageal squamous cell carcinoma was significantly lower than that in adjacent tissues, the differences were statistically significant(P < 0.05). The median survival time of patients with high expression of YTHDF3 gene was significantly higher than that of patients with low expression of YTHDF3 gene, the differences were statistically significant(P < 0.05). Conclusion YTHDF3 gene can inhibit the proliferation and migration of esophageal squamous cell carcinoma cells, and the mechanism may be related to the regulation of LAST1 gene expression.