靶向沉默CXCR4基因抑制肝细胞癌侵袭体外研究

Inhibition effect of silencing CXCR4 on the invasion of hepatocellular carcinoma cell lines in vitro

目的:采用RNAi技术构建CXCR4shRNA干扰载体,探讨靶向沉默CXCR4基因表达后对人肝细胞癌增殖和侵袭作用的影响及可能机制。方法:通过蛋白质印迹法和RT-PCR,检测不同分化程度的肝癌细胞株CXCR4表达程度。通过RNAi技术,将CXCR4shRNA干扰载体转染CXCR4高表达的肝癌细胞HepG2,并采用G418筛选出稳定表达株,蛋白质印迹法和RT-PCR验证shRNA对CXCR4基因的靶向沉默效率。以转染空载体细胞为阴性对照组,MTT法检测CXCR4基因沉默后肝癌细胞的增殖能力,Transwell小室侵袭试验检测CXCR4基因沉默后肝癌细胞的侵袭能力,蛋白质印迹法检测MMP-2和MMP-9蛋白表达水平,免疫荧光检测MMP-2在细胞内表达。结果:CXCR4靶向沉默的细胞CXCR4表达受到显著抑制。HepG2、HepG2-Vector和HepG2-CXCR4-细胞CXCR4蛋白相对表达量分别为0.56±0.07、0.54±0.04和0.14±0.05,F=57.42,P<0.001。正常HepG2、HepG2-Vector和HepG2-CXCR4-细胞的CXCR4mRNA相对表达量分别为1.04±0.05、1.05±0.11和0.19±0.03,P<0.001。MTT结果显示,CXCR4基因沉默能明显抑制HepG细胞的增殖。72h时正常HepG2、HepG2-Vector和HepG2-CXCR4-细胞的相对增殖速度分别为1.34±0.05,1.32±0.03和1.14±0.03。Transwell试验显示,10%FBS趋化作用下,HepG2、HepG2-Vector和HepG2-CXCR4-穿膜细胞数分别为85±13、89±17和23±6,差异有统计学意义,F=63.91,P<0.001;SDF-1趋化作用下,HepG2、HepG2-Vector和HepG2-CXCR4-穿膜细胞数分别为168±20、171±24和30±9。蛋白质印迹法显示,相对于正常HepG2细胞(0.83±0.04),HepG2-CXCR4-细胞MMP-2相对表达量(0.31±0.06)明显下降,P<0.001;而HepG2-Vector细胞MMP-2相对表达量(0.85±0.07)改变不明显,P=0.75。HepG2、HepG2-Vector和HepG2-CXCR4-细胞MMP-9相对表达量分别为0.35±0.04、0.33±0.07和0.32±0.06,差异无统计学意义,F=0.23,P=0.79。结论:通过RNAi技术成功靶向干扰CXCR4基因表达,可抑制肝癌细胞增殖和侵袭,可能与其抑制侵袭相关分子MMP-2表达有关。

OBJECTIVE：To investigate the effect of CXCR4 knockdown by RNA interference on the proliferation and invasion in hepatocellular carcinoma cells. METHODS： The CXCR4 expression level of different hepatocellular carcinoma cell lines was detected by western blotting at protein level and Real time RT-PCR at mRNA level. The shRNA vector tar geting to CXCR4 was transfected into the HepG2 cells, and the stable transfected cells were selected by G418. Western blotting and Real time RT-PCR were performed to observe the inhibitory effect of RNAi on CXCR4 expression. MTT as say was used to assess the influence of CXCR4 shRNA on cell proliferation. Transwell assay was applied to observe the cell invasion ability. The expressions of MMP-2 and MMP-9 protein were determined by Western blotting and immunoflu orescenee. RESULTS：Stable transfeetion of CXCR4 shRNA into HeDG2 cells resulted in efficiently declined exrression ofCXCR4. CXCR4 protein expression in HepG2, HepG2 Vector and HepG2 CXCR± ceils were 0.56 ±0.07,0. 54 ± 0.04 and 0.14±0.05 respectively （F= 57.42, P〈0. 001）. CXCR4 mRNA expression in HepG2, HepG2-Vector and HepG2 CXCR± cells were 1.04±0.05,1. 05±0.11 and 0.19±0.03 respectively （P〈0. 001）. MTT experiments showed that si- lencing CXCR4 significantly decreased proliferation of HepG2 cells. In 72h, the proliferation of HepG2, HepG2 Vector and HepG2 CXCR cells were 1. 34±0.05,1.32 ± 0.03 and 1. 14± 0.03. The number of migrated ceils which attracted by FBS were 85±13,89± 17 and 23± 6 in HepG2, HepG2 Vector and HepG2-CXCRgroup respectively （F= 63.91, P〈 0. 001 ）. Furthermore, the number of migrated cells which attracted by SDF-1 were 168 20,171 ± 24 and 30 ± 9. Using Western blot,we found that the relative expression of MMP-2 was obviously declined in HepG2-CXCR± cells （0. 31 ± 0.06） comparing with normal HepG2 cells （0. 83±0.04）（F= 78.34, P（0. 001）, and the relative expression of MMP-9 were no significant difference in HepG2 （0. 35±0.04） ,HepG2-Vector （0. 33±0.07） and HepG2-CXCR± cells （0. 32± 0.06） （F = 0. 23, P = 0. 79）. CONCLUSION： CXCR4 knockdown is successfully performed by CXCR4 shRNA transfec tion. CXCR4 knockdown inhibits the proliferation and invasion in hepatocellular carcinoma cell.