小干扰RNA沉默结肠癌转移相关基因1对肝癌细胞增殖、迁移及侵袭的影响

Effects of small interfering RNA silencing metastasis - associated in colon cancer - 1 gene on the proliferation, migration and invasion of liver cancer cells

目的 观察小干扰RNA（siRNA）沉默结肠癌转移相关基因1（MACC1）对肝癌细胞增殖、迁移、侵袭力的影响及其机制。方法培养人肝癌HepG2细胞，根据转染物不同，将细胞分为siRNA-MACC1组、siRNA-NC组和空白对照组，噻唑蓝（MTT）法检测各转染组细胞增殖能力，流式细胞术检测各转染组细胞凋亡情况，Transwell法检测各转染组细胞迁移和侵袭能力，实时荧光定量聚合酶链反应（FQ-PCR）检测各转染组细胞中MACC1、肝细胞生长因子（HGF）和c-Met基因表达，Western blot法检测各组细胞中MACC1、HGF、c-Met、丝裂原细胞外激酶（MEK）、磷酸化MEK（p-MEK）、细胞外信号调节激酶（ERK）和磷酸化ERK（p-ERK）蛋白表达。结果（1）MTT检测结果显示，siRNA-MACC1组细胞24、48、72、96 h时细胞吸光度（A）值（0.27±0.05、0.36±0.06、0.46±0.05、0.58±0.07）均显著低于siRNA-NC组（0.39±0.06、0.47±0.05、0.64±0.07、0.75±0.08）和空白对照组（0.41±0.05、0.49±0.07、0.62±0.06、0.77±0.09），差异均有统计学意义（F＝25.945、32.286、40.264、38.165，P＝0.000、0.000、0.000、0.000）。（2）siRNA-MACC1组细胞凋亡率[（13.52±2.61）%]，均高于siRNA-NC组和空白对照组[（4.94±1.84）%、（5.10±2.13）%，F＝73.918，P＝0.000]。（3）siRNA-MACC1组迁移细胞数和侵袭细胞数[（73.62±12.71）个、（59.23±11.53）个]均低于siRNA-NC组和空白对照组[（98.53±14.22）、（85.44±13.12）个和（102.51±15.13）、（87.62±14.24）个，F＝11.149、9.500，P＝0.000、0.000]。（4）与siRNA-NC组和空白对照组比较，siRNA-MACC1组细胞中MACC1、HGF和c-Met mRNA相对表达量均降低（F＝51.235、137.507、139.708，P＝0.000、0.000、0.000）。（5）与siRNA-NC组和空白对照组比较，siRNA-MACC1组细胞中MACC1、HGF、c-Met、p-MEK、p-ERK蛋白相对表达量均降低（F＝49.803、33.564、24.391、228.400、137.410，P＝0.000、0.000、0.000、0.000、0.000），而MEK、ERK蛋白相对表达量均升高（F＝12.751、23.829，P＝0.000、0.000）。结论特异性抑制肝癌HepG2细胞中MACC1基因表达可有效抑制细胞增殖，减弱细胞迁移及侵袭能力，促使细胞凋亡发生，其机制可能与HGF/Met和MEK/ERK信号通路有关。

Objective To investigate the effects of small interfering RNA （siRNA） silencing metastasis-associated in colon cancer-1 （MACC1） gene on the proliferation, migration and invasion of liver cancer cells, and the possible mechanism.Methods HepG2 cells were cultured. According to the different transfectants, the cells were divided into siRNA-MACC1 group, siRNA-NC group and blank control group. The cell proliferations in the different transfected groups were measured by methyl thiazol tetrazolium （MTT） assay. The cell apoptosis in the different transfected groups was examined by flow cytometry. The cell migration and invasion abilities in the different transfected groups were tested by Transwell methods. The expression levels of MACC1, hepatocyte growth factor （HGF） and c-Met genes in the different transfected groups were detected by real-time fluorescent quantitative polymerase chain reaction（FQ-PCR）. The expression levels of MACC1, HGF, c-Met, MEK, phosphorylated MEK （p-MEK）, extracellular signal-regulated kinase （ERK） and phosphorylated ERK （p-ERK） proteins in the different transfected groups were detected by Western blotting. Results （1） MTT results showed that absorptivity （A） values in the siRNA-MACC1 group at 24, 48, 72 and 96 h （0.27±0.05, 0.36±0.06, 0.46±0.05, 0.58±0.07） were lower than the siRNA-NC group （0.39±0.06, 0.47±0.05, 0.64±0.07, 0.75±0.08） and blank control group （0.41±0.05, 0.49±0.07, 0.62±0.06, 0.77±0.09）, the differences were statistically significant （F=25.945, 32.286, 40.264, 38.165, P=0.000, 0.000, 0.000, 0.000）. （2） Flow cytometry results showed that the apoptosis rate in the siRNA-MACC1 group was （13.52±2.61）%, which was significantly higher than the siRNA-NC group and blank control group [（4.94±1.84）%, （5.10±2.13）%, F=73.918, P=0.000]. （3） The number of migration cells and the number of invasive cells in the siRNA-MACC1 group （73.62±12.71, 59.23±11.53） were significantly lower than those in the siRNA-NC group and blank control group （98.53±14.22, 85.44±13.12 and 102.51±15.13, 87.62±14.24, F=11.149, 9.500, P=0.000, 0.000）. （4） Compared with siRNA-NC group and blank control group, the relative expression levels of MACC1, HGF and c-Met mRNA in the siRNA-MACC1 group were significantly lower than the siRNA-NC group and blank control group （F=51.235, 137.507, 139.708, P=0.000, 0.000, 0.000）. （5） Compared with the siRNA-NC group and blank control group, the relative expression levels of MACC1, HGF, c-Met, p-MEK and p-ERK proteins in the siRNA-MACC1 group were decreased （F=49.803, 33.564, 24.391, 228.400, 137.410, P=0.000, 0.000, 0.000, 0.000, 0.000）, while the the relative expression levels of MEK and ERK proteins were increased （F=12.751, 23.829, P=0.000, 0.000）.Conclusion Specific inhibition of MACC1 gene expression in HepG2 cells could effectively inhibit cell proliferation, decrease cell migration and invasion, and induce cell apoptosis. The mechanism might be related to HGF/Met and MEK/ERK signaling pathways.