沉默CRAF基因表达对肝癌细胞侵袭和迁移的影响

Effects of silencing expression of CRAF gene on invasion and metastasis of hepatocellular carcinoma cells

目的:探讨肝癌细胞中原癌基因丝氨酸/苏氨酸蛋白激酶(CRAF)高表达对肝细胞癌(HCC)侵袭和转移的影响,并阐明其机制。方法:以CRAF高表达的SMMC-7721细胞作为研究对象,采用shRNA技术下调CRAF表达。将SMMC7721细胞分为对照组和沉默组(shCRAF#1转染组和shCRAF#2转染组)。细胞划痕法观察各组SMMC7721细胞创口愈合指数,Transwell实验检测各组SMMC-7721细胞的穿膜细胞数,Western blotting法检测各组SMMC7721细胞中基质金属蛋白酶2(MMP-2)和基质金属蛋白酶9(MMP-9)的表达水平。结果:细胞划痕实验,与对照组比较,沉默组SMMC7721细胞的创口愈合指数明显降低(P<0.05)。Transwell实验,与对照组比较,沉默组SMMC7721细胞的穿膜细胞数降低(P<0.05)。Western blotting法检测,沉默组细胞中MMP-2和MMP-9表达水平明显低于对照组(P<0.05)。结论:下调CRAF可以通过抑制MMP-2和MMP-9表达抑制肝癌SMMC7721细胞的侵袭和迁移。

Objective：To investigate the effects of high expression of CRAF on the invasion and metastasis of hepatocellular carcinoma cells（HCC）,and to clarify their mechanisms.Methods：The SMMC7721 cells with high expression of CRAF were regarded as the object,and the expressions of CRAF in SMMC7721 cells were downregulated by shRNA.The SMMC7721 cells were divided into control group and silencing groups（shCRAF#1 transfection group and shCRAF#2 transfection group）.The wound closure index of SMMC7721 cells was detected by cell scratch test,the number of transmembrane cells of SMMC7721 cells was detected by Transwell assay,and the expression levels of matrix metalloproteinase-2（MMP-2）and matrix metalloproteinase-9（MMP-9）in SMMC7721 cells were detected by Western blotting method.Results：The scratch test results showed that compared with control group,the wound closure indexes of the SMMC7721 cells in silencing groups were decreased（P〈0.05）.The Transwell assay results show that compared with control group,the number of transmembrane cells in silencing groups was decreased（P〈0.05）.The Western blotting results showed that compared with control group,the expression levels of MMP-2 and MMP-9 in the SMMC7721 cells in silencing group were decreased（P〈0.05）.Conclusion：Silencing CRAF can inhibit the invasion and migration of HCC SMMC7721 cells by inhibiting the expressions of MMP-2 and MMP-9.