新加良附方抑制胃癌AGS细胞增殖迁移及其初步生物信息学分析

Modified Liangfu Granule inhibits proliferation and migration of gastric cancer AGS cells and its preliminary bioinformatics analysis

目的 探讨新加良附方对胃癌AGS细胞的抑制作用及初步机制。方法 体外培养胃癌AGS细胞及正常人胃黏膜上皮细胞GES-1,以不同质量浓度新加良附方(4、5、6、7、8 g/L)进行干预。CCK-8法检测新加良附方对胃癌AGS细胞增殖的抑制作用,划痕实验检测新加良附方对胃癌AGS细胞迁移作用的影响,实时荧光PCR检测胃癌AGS细胞中N6-甲基腺嘌呤(m6A)甲基转移酶甲基转移酶样蛋白3(METTL3)基因表达水平。通过生存期分析网站(http://kmplot.com/analysis/)分析METTL3表达对胃癌患者生存期的影响,采用Illuminal平台测序,通过基因本体论(GO)富集分析及京都基因与基因组数据库(KEGG)信号通路富集分析探究新加良附方干预后差异表达基因在分子功能、生物学过程、细胞组成及信号通路方面的作用。结果 CCK-8法结果显示新加良附方在各质量浓度、各时间点均可抑制胃癌AGS细胞增殖(P<0.01)。划痕实验显示新加良附方组胃癌AGS细胞迁移能力降低,各质量浓度新加良附方在24、48 h均可抑制胃癌AGS细胞迁移(P<0.05,P<0.01)。实时荧光PCR结果显示胃癌AGS细胞METTL3表达水平高于正常胃黏膜上皮细胞GES-1中METTL3表达(P<0.01)。胃癌患者生存期分析显示,METTL3低表达患者生存期长于METTL3高表达患者(P<0.05)。新加良附方干预后共筛选出1 245个表达上调基因及1 156个表达下调基因,其中METTL3表达水平下调(P<0.05)。新加良附方作用后所致差异表达基因参与细胞对趋化因子的反应、mRNA分解代谢过程的负调控及酶、蛋白质及核苷酸等的结合过程;信号通路方面主要涉及细胞凋亡、自噬调控、p53信号通路、细胞周期及哺乳动物雷帕霉素靶蛋白、丝裂原激活蛋白激酶、肿瘤坏死因子等经典肿瘤相关信号通路。结论 新加良附方可抑制胃癌细胞增殖迁移,其作用可能与调控m6A甲基转移酶METTL3水平相关。

Objective To investigate the inhibitory effect and preliminary mechanism of Modified Liangfu Granule(MLFG) on gastric cancer AGS cells.Methods Gastric cancer AGS cells and normal human gastric epithelial cells GES-1 were cultured in vitro, and treated with different mass concentrations of MLFG(4, 5, 6, 7 and 8 g/L). The CCK-8 method was used to detect the inhibitory effect of MLFG on gastric cancer AGS cell proliferation, the scratch test was used to detect the effect of MLFG on the migration of gastric cancer AGS cells, and the expression level of m6 A transmethylase METTL3 gene in AGS cells was observed by real-time PCR method. The survival analysis website(http://kmplot.com/analysis/) was used to analyze the effect of METTL3 expression on the survival time of patients with gastric cancer. The Illuminal platform was used for sequencing, and GO enrichment analysis and KEGG signaling pathway were used to analyze the role of differentially expressed genes in molecular function, biological process, cell composition and signaling pathway after the intervention of MLFG.Results The result of CCK-8 showed that MLFG could inhibit the proliferation of AGS cells in each mass concentration and each time(P<0.01). Scratch test showed that the migration ability of AGS cells in MLFG group was significantly reduced, and each mass concentration of MLFG could inhibit the migration of AGS cells at 24 h and 48 h(P<0.05, P<0.01). The result of real-time PCR showed that the expression level of METTL3 in AGS cells was significantly higher than that in GES-1 cells(P<0.01). Survival analysis of gastric cancer patients showed that the survival time of patients with low METTL3 expression was longer than that of patients with high METTL3 expression(P<0.05). After MLFG intervention, a total of 1 245 up-regulated genes and 1 156 down-regulated genes were screened, among which the expression level of METTL3 was down-regulated(P<0.05). The differentially expressed genes induced by MLFG were involved in the response of cells to chemokines, the negative regulation of mRNA catabolism and the binding process of enzymes, proteins and nucleotides;while the signal pathways were mainly related to cell apoptosis, autophagy regulation, p53 signaling pathway, cellular cycle and classic tumor-related signaling pathways, such as mTOR, MAPK and TNF.Conclusion MLFG can inhibit the proliferation and migration of gastric cancer cells, and its effect may be related to regulating the level of m6 A methyltransferase METTL3.