RNA干扰沉默CXCR4基因对肝癌细胞增殖和侵袭的影响

Effects of CXCR4 Silence Induced by RNA Interference on Growth and Invasion of Hepatocellular Carcinoma Cell Lines

目的探讨RNA干扰质粒靶向沉默CXCR4基因表达,对人肝癌细胞增殖和侵袭作用的影响及其机制。方法检测不同分化程度的肝癌细胞株CXCR4表达程度。将CXCR4干扰质粒转染肝癌细胞HepG2,经G418筛选出稳定抑制细胞,使用Western blotting和Real time RT-PCR验证干扰质粒对CXCR4基因的靶向沉默效率。MTT法和Transwell小室试验检测CXCR4基因沉默后肝癌细胞的增殖能力和侵袭能力。Western blotting检测基质金属蛋白酶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。结果不同分化程度的肝癌细胞均有CXCR4表达,其中HepG2细胞表达最强。通过RNA干扰技术靶向沉默CXCR4基因,可高效、稳定地抑制CXCR4表达。CXCR4基因靶向沉默后明显抑制肝癌细胞增殖和侵袭。Western blotting提示CXCR4基因靶向沉默导致MMP-2蛋白表达明显下调。结论 CXCR4基因沉默可以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有关,提示CXCR4可能是肝癌治疗的一个有效靶点。

Objective To explore the impact of CXCR4 silence induced by RNAi on the growth and invasion in hepatocellular carcinoma cells.Methods The CXCR4 expressions of hepatocellular carcinoma cell lines were detected by western blotting and Real-time RT-PCR.The shRNA plasmid vector was tranfected into the HepG2 cells,and the stable transfected cells were selected by G418.The inhibitory effect of RNAi on CXCR4 expression was inspected with Western blotting and Real-time RT-PCR.MTT assay and Transwell assay were used to assess the influence of CXCR4 shRNA on cell growth and invasion ability.The expressions of MMP-2 and MMP-9 protein were determined by Western blotting and immunofluorescence.Results HepG2 cells showed highly expression of CXCR4.Stable transfection of CXCR4 shRNA into HepG2 cells caused efficient declined expression of CXCR4 at both mRNA and protein level,and significantly decreased proliferation.Furthermore,the decrease of invasion ability was observed in HepG2 cell with CXCR4 knockdown using Transwell assay,even under the stimulation of CXCR4 ligand,SDF-1.Moreover,Western blotting and immunofluorescence showed that MMP-2 was significant down-regulated due to CXCR4 RNAi on HepG2 cells.Conclusion Inhibition of CXCR4 expression by RNAi reduces the proliferation and invasion in hepatocellular carcinoma cell,suggesting that CXCR4 may serve as an effective target for the treatment of hepatocellular carcinoma.