下调肝星状细胞 MACC1对胃癌细胞迁移侵袭能力的影响

摘要

Objective:By in vitro experiment to explore the regulatory effect of MACC1 gene silencing in HSC expressα-SMA,MMP -2,MMP -9 and HGF,and its effects on ability of migration and invasion of co -cultured gastric carcinoma cells.Methods:To construct targeting MACC1 gene eukaryotic expression interference plasmid (MACC1 -shRNA)and non -specific irrelevant interference plasmid (shNC),transient transfect them into HSC with lipofectamine -2000,named low -expression group(shRNA)and negative control group (NC)accordingly,non-transfected HSC treated as a blank control group(Blank -Ctrl).The above three groups of cells were cultured in accordance with routinel,the expression levels of MACC1,α-SMA,HGF,MMP -2 and MMP -9 in human hepatic stellate cells were detected by Western blot and RT -PCR.Three groups of cells were co -cultured with MGC803 gastric carcinoma cells in vitro,the capabilities of migration and invasion were measured by transwell assay.Results:Western -Blot results showed that the expression of MACC1,α-SMA,HGF,MMP -2 and MMP -9 protein in HSC was significantly lower than that in the negative group and blank control group after transfection of MACC1 -shRNA (P <0.05).Real -time PCR results showed that the expression levels of mRNA of MACC1,α-SMA,HGF,MMP -2 and MMP -9 in low -expression group HSC significantly decreased (P <0.01),compared with the others groups. Transwell test indicated that the number of cells through membrane in low -expression groups were significantly less than the negatiave groups and blank control groups in MGC803 cell (P <0.01).There had no statistical significance between blank control group and negative group(P >0.05).Conclusion:Silencing MACC1 in hepatic stellate cells, can inhibit the expression of MMP -2,MMP -9 and HGF,and decrease the migration and invasion of gastric cancer cells in co -culture.%目的：通过体外实验探讨肝星状细胞（hepatic stellate cell，HSC）MACC1基因沉默后调节肝星状细胞激活标志物α－平滑肌肌动蛋白（α－smooth muscle actin，α－SMA）、肝细胞生长因子（hepatocyte growth fac-tor，HGF）、基质金属蛋白酶2（matrix metalloproteinase －2，MMP －2）、基质金属蛋白酶9（matrix metalloprotein-ase －9，MMP －9）的表达，以及对共培养胃癌细胞迁移侵袭能力的影响。方法：利用 RNA 干扰技术，构建针对MACC1基因的真核表达干涉质粒（MACC1－shRNA）、非特异性无关干涉质粒（shNC）。利用 Lipofectamine －2000将其瞬时转染 HSC，分别作为实验组（shRNA 组）、阴性对照组（NC 组），未处理的 HSC 作为空白对照组（Blank －Ctrl 组）。以上三组细胞按照常规培养。应用 RT －PCR 和 Western Blot 技术检测细胞中 MACC1、α－SMA、HGF、MMP －2、MMP －9基因的 mRNA 和蛋白的表达。以上三组细胞分别与胃癌细胞 MGC803体外共培养，Transwell 实验观察三组不同培养条件下对胃癌细胞 MGC803迁移侵袭能力的影响。结果：Western Blot 结果显示，转染 MACC1－shRNA 后，与 NC 组和 Blank －Ctrl 组比较，shRNA 组 HSC 产生的 MACC1、α－SMA、HGF、MMP －2、MMP －9蛋白的表达水平均明显下调，差异有统计学意义（P ＜0．05）。Real －time PCR 检测结果显示，转染 MACC1－shRNA 后，与其他两组比较，shRNA 组 HSC 的 MACC1、α－SMA、HGF、MMP －2、MMP －9基因的 mRNA 的表达水平均显著下调（P ＜0．01）。Transwell 侵袭迁移实验显示，穿过滤膜的 shR-NA 组 MGC803细胞数较 NC 组和 Blank －Ctrl 组明显减少，差异均具有统计学意义（P ＜0．01）。NC 组和Blank －Ctrl 组相比较，差异均无统计学意义（P ＞0．05）。结论：通过下调肝星状细胞 MACC1的表达，可抑制MMP －2、MMP －9、HGF 的表达，并能降低共培养胃癌细胞的迁移和侵袭能力。