COL3A1对胃癌细胞增殖及侵袭影响的实验研究

Experimental study on the effect of COL3A1 on proliferation and invasion of gastric cancer cells

目的 研究COL3A1在胃癌细胞增殖、迁移及侵袭过程中的作用。方法 以靶向COL3A1的干扰慢病毒(3个靶点)及阴性对照慢病毒转染胃癌细胞株MGC803,嘌呤霉素筛选稳转细胞株。qRT-PCR验证转染后MGC803细胞中COL3A1的mRNA表达,筛选出2个转染效率最高的细胞株,再以Western blot法进一步检测转染后胃癌细胞中COL3A1的蛋白表达量。采用MTT、Transwell法检测COL3A1干扰组、对照组胃癌细胞增殖、侵袭、迁移表型,观察COL3A1敲减前后胃癌细胞生物学特性的变化。结果 (1)COL3A1的mRNA表达水平在胃癌细胞转染shRNA-COL3A1慢病毒后出现显著下降,其中抑制效果最好的是shRNA 1组(P<0.01)、shRNA 3组(P<0.001);重新分组后行Western blot法,发现与NC组相比,shRNA 1组、shRNA 2组COL3A1蛋白表达均下降,其中shRNA 2组抑制效果更显著(P<0.01);(2)MTT结果显示,shRNA-COL3A1慢病毒转染后48h胃癌细胞的生长被明显抑制,在各时间点吸收值均低于NC组(P<0.05),其中shRNA 2组最明显(P<0.0001);(3)通过Transwell小室对shRNACOL3A1胃癌细胞迁移能力进行检测,发现shRNA-COL3A1转染后,shRNA 1组(P=0.0029)及shRNA 2组(P<0.0001)胃癌细胞的迁移能力显著降低,其中以shRNA 2组最为显著(P<0.0001);(4)通过Transwell小室检测shRNA-COL3A1胃癌细胞侵袭能力,发现shRNA 1组(P=0.0046)及shRNA 2组(P=0.0002)胃癌细胞的侵袭能力均显著下降,其中shRNA 2组最为显著(P<0.001)。结论 敲减COL3Al基因可有效抑制胃癌细胞的增殖、侵袭及迁移,COL3Al在胃癌发生发展中发挥了重要作用。

Objective To investigate the role of COL3A1 in the proliferation,migration and invasion of gastric cancer cells.Methods The gastric cancer cell line MGC803 was transfected with the interference lentivirus targeting COL3A1(3 targets)and the negative control lentivirus,and the stable cell line was screened for purinomycin resistance.The mRNA expression of COL3A1 in MGC803 cells after transfection was verified by qRT-PCR,and two cell lines with the highest transfection efficiency were screened out.The protein expression of COL3A1 in gastric cancer cells after transfection was further detected by Western blot.MTT and Transwell assay were used to detect the proliferation,invasion and migration phenotypes of gastric cancer cells in the COL3A1 interference group and the control group,and the changes in the biological characteristics of gastric cancer cells before and after COL3A1 knockdown were observed.Results (1)The mRNA expression level of COL3A1 was significantly decreased after gastric cancer cells were transfected with shRNA-COL3A1 lentivirus,and the inhibitory effect was best in shRNA 1 group(P<0.01)and shRNA 3 group(P<0.001).Western blot analysis was performed after regrouping.Compared with NC group,the expression of COL3A1 protein in shRNA group 1 and shRNA group 2 was decreased,and the inhibitory effect of shRNA group 2 was more significant(P<0.01).(2)MTT results showed that 48 hafter shRNA-COL3A1 lentivirus transfection,the growth of gastric cancer cells was significantly inhibited,and the absorption values at all time points were lower than those in NC group(P<0.05),and shRNA 2 group was the most obvious(P<0.0001).(3)Transwell chamber was used to detect the migration ability of shRNA-COL3A1 gastric cancer cells,and it was found that after shRNA-COL3A1 transfection,the migration ability of gastric cancer cells in shRNA 1 group(P=0.0029)and shRNA 2 group(P<0.0001)was significantly decreased.The shRNA group 2 was the most significant(P<0.0001).(4)The invasion ability of shRNA-COL3A1 gastric cancer cells was detected by Transwell chamber,and it was found that the invasion ability of gastric cancer cells was significantly decreased in shRNA 1 group(P=0.0046)and shRNA 2 group(P=0.0002),and the invasion ability of shRNA 2 group was the most significant(P<0.001).Conclusion Knockdown of COL3 Al gene can effectively inhibit the proliferation,invasion and migration of gastric cancer cells,and COL3 Al played an important role in the development of gastric cancer.