TMEM100调控Notch信号对胃癌细胞增殖、迁移、侵袭与凋亡影响的实验研究

Experimental study on the effect of Notch signaling regulated by TMEM100 on proliferation,migration,invasion and apoptosis of gastric cancer cells

目的探讨TMEM100对胃癌细胞增殖、迁移、侵袭与凋亡的影响及其可能分子机制。方法通过GEPIA数据库分析胃癌组织中TMEM100表达,实时荧光定量PCR(qPCR)检测胃癌细胞株中TMEM100表达。将胃癌SGC-7901细胞分为对照组(空载体组)、TMEM100过表达(转染过表达载体pcDNA3.0-HA-TMEM100)组、TMEM100过表达+Notch1激动剂(VPA-d4)组。MTT、划痕实验、Transwell小室和流式细胞术分别检测细胞增殖、迁移、侵袭和凋亡情况。结果TCGA数据显示,与正常组织比较,胃癌组织中的TMEM100表达显著降低,差异具有统计学意义(P<0.05);GES-1细胞中TMEM100表达量为1.000±0.103,均高于胃癌AGS细胞(0.693±0.091)、MKN-45细胞(0.567±0.079)、MGC80-3细胞(0.491±0.094)和SGC-7901细胞(0.415±0.0.84),差异有统计学意义(P<0.05)。与空载体组(1.000±0.184)比较,TMEM100过表达组细胞TMEM100蛋白(2.648±0.249)和mRNA(3.462±0.328)表达均升高(P<0.05);TMEM100过表达组细胞Notch1蛋白(0.417±0.103)和mRNA(0.424±0.091)表达均降低(P<0.05)。与空载体组比较,TMEM100过表达组细胞增殖活性显著降低(P<0.05);与TMEM100过表达组比较,TMEM100过表达+VPA-d4组细胞增殖活性明显升高(P<0.05)。划痕实验显示,空载体组细胞划痕愈合率为(80.3±12.3)%,高于TMEM100过表达组的(35.1±7.38)%;而TMEM100过表达+VPA-d4组细胞划痕愈合率为(79.2±12.6)%,低于TMEM100过表达组(P<0.05)。Transwell小室实验结果显示,空载体组穿膜细胞数为233±30个,高于TMEM100过表达组的72±19个,差异有统计学意义(P<0.05);与TMEM100过表达组比较,TMEM100过表达+VPA-d4组穿膜细胞数增加至257±35个,差异有统计学意义(P<0.05)。空载体组细胞凋亡率为(3.6±0.3)%,TMEM100过表达组细胞凋亡率为(19.6±1.6)%,两组差异有统计学意义(P<0.05);与TMEM100过表达组比较,TMEM100过表达+VPA-d4组细胞凋亡率降至(4.0±0.4)%,差异有统计学意义(P<0.05)。结论TMEM100在胃癌组织和细胞中表达降低,且可能通过抑制Notch1表达在胃癌中发挥抑癌作用。

Objective To investigate the effects of TMEM100 on proliferation,migration,invasion and apoptosis of gastric cancer cells and its possible molecular mechanism.Methods The expression of TMEM100 in gastric cancer tissues was analyzed by GEPIA database,and the expression of TMEM100 in gastric cancer cell lines was detected by real-time fluorescence quantitative PCR(qPCR).Gastric cancer SGC-7901 cells were divided into control group(empty carrier group),TMEM100 overexpressed group(transfected with overexpressed vector pcDNA3.0-HA-TMEM100),TMEM100 overexpressed+Notch1 agonist group(VPA-d4).MTT,scratch assay,Transwell chamber and flow cytometry were used to detect cell proliferation,migration,invasion and apoptosis,respectively.Results The TCGA data showed that TMEM100 in gastric cancer tissues was significantly decreased compared with normal gastric tissues,the difference was statistically significant(P<0.05).The expression of TMEM100 in GES-1 cells was 1.000±0.103.They were higher than AGS cells(0.693±0.091),MKN-45 cells(0.567±0.079),MGC80-3 cells(0.491±0.094)and SGC-7901 cells(0.415±0.0.84),and the difference was statistically significant(P<0.05).Compared with empty carrier group(1.000±0.184),the expression of TMEM100 protein(2.648±0.249)and mRNA(3.462±0.328)were increased in TMEM100 overexpressed group(P<0.05).The expression of Notch1 protein(0.417±0.103)and mRNA(0.424±0.091)were decreased in TMEM100 overexpressed group(P<0.05).Compared with empty carrier group,the proliferation activity of TMEM100 overexpressed group was significantly decreased(P<0.05).Compared with TMEM100 overexpressed group,the cell proliferation activity in TMEM100 overexpressed+VPA-d4 group was significantly increased(P<0.05).Scratch test showed that the scratch healing rate of empty carrier group was(80.3±12.3)%,higher than that of TMEM100 overexpressed group(35.1±7.38)%.The scratch healing rate of TMEM100 overexpressed+VPA-d4 group was(79.2±12.6)%,which was lower than that of TMEM100 overexpressed group(P<0.05).The results of Transwell assay showed that the number of transmembrane cells in empty carrier group was 233±30,which was higher than that in TMEM100 overexpressed group(72±19),and the difference was statistically significant(P<0.05).Compared with TMEM100 overexpressed group,the number of transmembrane cells in TMEM100 overexpressed+VPA-d4 group increased to 257±35,and the difference was statistically significant(P<0.05).The apoptosis rate was(3.6±0.3)%in the empty carrier group and(19.6±1.6)%in the TMEM100 overexpressed group,the difference was statistically significant(P<0.05).Compared with TMEM100 overexpressed group,the apoptosis rate of TMEM100 overexpressed+VPA-d4 group decreased to(4.0±0.4)%,and the difference was statistically significant(P<0.05).Conclusion The expression of TMEM100 is decreased in gastric cancer tissues and cells,and it may play a role in tumor inhibition in gastric cancer by inhibiting Notch1 expression.