下调肝星状细胞MACC1对胃癌细胞迁移侵袭能力的影响

Effect of down-regulating MACC1 by shRNA on invasion and migration of gastric carcinoma cells

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 factor,HGF)、基质金属蛋白酶2(matrix metalloproteinase-2,MMP-2)、基质金属蛋白酶9(matrix metalloproteinase-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(sh NC)。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shRNA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。

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（ sh NC）,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.