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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 construct the CXCR4 shRNA interference vector using RNAi technique and to explore the effect of targeting CXCR4 gene silencing on the proliferation and invasion of human hepatocellular carcinoma and its possible mechanism. Methods: The expression of CXCR4 was detected by Western blotting and RT-PCR. The CXCR4 shRNA interference vector was transfected into HepG2 cells with high expression of CXCR4 by RNAi technique. Stable expression strains were screened by G418. The efficiency of targeted silencing of CXCR4 gene by shRNA was verified by Western blotting and RT-PCR. Transfected empty vector cells as negative control group, MTT assay CXCR4 gene silencing the proliferation of hepatocellular carcinoma cells, Transwell chamber invasion assay CXCR4 gene silencing invasion ability of hepatocellular carcinoma cells, Western blotting detection of MMP-2 and MMP-9 The expression of MMP-2 in the cells was detected by immunofluorescence. Results: CXCR4 targeting silencing cells CXCR4 expression was significantly inhibited. The relative expression levels of CXCR4 in HepG2, HepG2-Vector and HepG2-CXCR4-cells were 0.56 ± 0.07, 0.54 ± 0.04 and 0.14 ± 0.05 respectively, F = 57.42, P <0.001. The relative expression levels of CXCR4 mRNA in normal HepG2, HepG2-Vector and HepG2-CXCR4- cells were 1.04 ± 0.05, 1.05 ± 0.11 and 0.19 ± 0.03 respectively, P <0.001. The results of MTT showed that CXCR4 silencing could significantly inhibit the proliferation of HepG cells. The relative proliferation rates of normal HepG2, HepG2-Vector and HepG2-CXCR4- cells at 72h were 1.34 ± 0.05, 1.32 ± 0.03 and 1.14 ± 0.03, respectively. Transwell assay showed that the numbers of transmembrane cells of HepG2, HepG2-Vector and HepG2-CXCR4-transfected cells were 85 ± 13, 89 ± 17 and 23 ± 6 respectively with 10% FBS chemotaxis, the difference was statistically significant (F = 63.91, P <0.001. Under the chemotactic effect of SDF-1, the numbers of transmembrane cells of HepG2, HepG2-Vector and HepG2-CXCR4- were 168 ± 20, 171 ± 24 and 30 ± 9, respectively. Western blotting showed that the relative expression level of MMP-2 in HepG2-CXCR4- cells (0.31 ± 0.06) was significantly lower than that in normal HepG2 cells (0.83 ± 0.04, P <0.001), while the relative expression of MMP-2 in HepG2-Vector (0.85 ± 0.07) change is not obvious, P = 0.75. The relative expression of MMP-9 in HepG2, HepG2-Vector and HepG2-CXCR4-cells was 0.35 ± 0.04, 0.33 ± 0.07 and 0.32 ± 0.06, respectively. There was no significant difference between the two groups (F = 0.23, P = 0.79). Conclusion: The successful targeting of CXCR4 by RNAi technology can inhibit the proliferation and invasion of hepatocellular carcinoma cells, which may be related to the inhibition of the expression of MMP-2.