TFF3在胃癌中的表达及对胃癌细胞增殖的影响

Expression of TFF3 in gastric cancer and its effect on proliferation of gastric cancer cells

目的探讨三叶因子3(trefoil factor 3,TFF3)在胃癌中的表达及对胃癌细胞增殖的调控机制。方法采用RT-PCR及免疫组化法检测66例胃癌患者癌组织及癌旁组织中TFF3表达水平的差异。稳定培养SGC-7901细胞,通过感染GV248慢病毒载体沉默细胞中TFF3基因的表达(SGC-7901/KD组),并设置对照组(SGC-7901/NC),采用CCK-8法、流式细胞检测、Transwell等手段检测细胞活性的变化。在20只BALB/C裸鼠右侧腋下分别注射SGC-7901/KD或SGC-7901/NC细胞,利用免疫组化及Western blot法检测小鼠肿瘤中VEGF表达的变化。结果TFF3 mRNA在胃癌癌组织中的表达水平(15.31±2.18)明显高于癌旁组织(4.58±1.69)(P<0.05)。感染重组慢病毒后,SGC-7901/KD细胞中TFF3基因表达水平为0.08±0.006,明显低于其他两组;培养5 d后,SGC-7901/KD组细胞的OD值1.28±0.05,明显低于SGC-7901/NC组的1.78±0.08,细胞集落形成数量也明显减少,集落更小。与对照组SGC-7901/NC组细胞相比,SGC-7901/KD细胞处于G0/G1期的细胞比例及细胞凋亡率明显更高,而处于G2/M期的细胞比例明显较低。划痕愈合试验结果显示,SGC-7901/KD细胞的伤口边缘之间的距离明显短于SGC-7901/NC细胞。Western blot检测发现,SGC-7901/KD细胞中VEGF蛋白相对表达水平(0.98±0.05),明显低于SGC-7901/NC细胞(2.87±0.17)(P<0.05)。与SGC7901/NC组相比,注射SGC-7901/KD细胞2周后,小鼠的肿瘤体积为(184.4±50.2)mm 3,显著小于SGC7901/NC组(326.8±76.9)mm 3(P<0.05),且SGC7901/KD组移植瘤标本中VEGF蛋白的表达明显低于对照组(P<0.05)。结论在胃癌细胞中,TFF3基因沉默可通过抑制VEGF的表达对胃癌细胞生长发挥抑制效应,有望成为一个新的分子靶点。

ObjectiveTo investigate the expression of trefoil factor 3(TFF3)in gastric cancer and its regulation mechanism on the gastric cancer cells proliferation.MethodsThe differences in the expression levels of TFF3 in 66 cases of gastric cancer were detected by RT-PCR and immunohistochemical method.SGC-7901 cells were cultured stably.Through the expression of TFF3 gene in silent cells infected with gv248 lentivirus vector(SGC-7901/KD group)and the setting up of control group(SGC-7901/NC group),CCK-8 method,flow cytometry and Transwell method were used to detect the changes of cell activity.SGC-7901/KD or SGC-7901/NC cells were injected into the right armpit of 20 BALB/C nude mice respectively.The expression of VEGF in tumor was detected by immunohistochemical method and Western blot method.ResultsThe expression level of TFF3 mRNA in gastric cancer tissues(15.31±2.18)was significantly higher than that in adjacent tissues(4.58±1.69)(P<0.05).After infection with recombinant lectin virus,the expression level of TFF3 gene in SGC-7901/KD cells was 0.08±0.006,significantly lower than that in the other two groups.After 5 days of culture,the OD value of cells in SGC-7901/KD group was 1.28±0.05,significantly lower than that in SGC-7901/NC group(1.78±0.08).The number of cell colonies formed was also significantly reduced,and the colonies were smaller.Compared with the control group,the proportion and apoptosis rate of SGC-7901/KD cells in G0/G1 phase were significantly higher,while the proportion of cells in G2/M phase was significantly lower.The results of scratch healing test showed that the distance between wound edges of SGC-7901/KD cells was significantly shorter than that of SGC-7901/NC cells.Western blot analysis showed that the relative expression level of VEGF protein in SGC-7901/KD cells(0.98±0.05)was significantly lower than that in SGC-7901/NC cells(2.87±0.17)(P<0.05).Compared with SGC7901/NC group,after 2 weeks of injection of SGC-7901/KD cells,the tumor volume of mice was(184.4±50.2)mm 3,significantly smaller than that of SGC7901/NC group(326.8±76.9)mm 3(P<0.05).The expression of VEGF protein in SGC7901/KD group was significantly lower than that in control group(P<0.05).ConclusionIn gastric cancer cells,TFF3 gene silencing can inhibit the growth of gastric cancer cells by inhibiting the expression of VEGF,which is expected to become a new molecular target.