| ?BACKGROUND/OBJECTIVE?Fibrinogen-like protein 2, also known as fibroleukin,encompassed a membrane and a soluble form. Mfgl2 expressed on the surface of tumor, macrophages, endothelial cells and epithelial cells. It can directly catalyze prothrombinase to thrombinase, thus catalyzing fibrinogen to fibrin formation, which facilitates tumor growth. According to associated research results, we successfully constructed a micro RNA against hfgl2 carried by adenovirus(Ad-hfgl2-mi RNA). This study was to investigate the effect of adenovirus on human tumor cells' proliferation, and further studyed the interference effect on tumor growth in xenograft mice model of HCC. ?METHODS?1.we detected the expression of fgl2, and coxsackie and adenovirus receptor(CAR) on human cell lines including LO2? LM3?Hela?CNE?NCIH460 with flowcytometry.2.Western-Bloting analysis was performed to measure hfgl2 expression.3.Infected LM3?Hela?CNE and NCIH460 cells with different concentrations of Ad-hfgl2-mi RNA to confirm the optimal multiplicity of infection.4.Used CCK-8 arrays to detect the proliferation effects of Ad-hfgl2-mi RNA on LM3?Hela?CNE and NCIH460 cells.5.We established the tumor-bearing mice model of HCCLM3 in nude mice firstly, later we isolated tumor tissues from mice and passaged it twice in vivo, then we obtained a reliable model which better displayed the biology of HCCLM3. When the tumor volume reached about 100mm3, nude mice were randomly assigned to four groups, each received intratumoral injection of high?medium?low doses(3.3*1010IU?1.0*1010IU?3.3*109IU) of Ad-hfgl2-mi RNA or non-related control, respectively. The intervention was once a week with a total of three times. And the measurement of tumor size, relative tumor volume and relative tumor proliferation rate were calculated. ?RESULTS?1.The expression of fgl2 on LO2?LM3?Hela?CNE?NCIH460 were 0%?15%?12%?19.1%?15.9%, respectively, and CAR expression on four human tumor cell lines mentioned above were 6%?13.2%?13%?32%, respectively;2.WB results showed that Ad-hfgl2-mi RNA inhibited the expression of target protein fgl2 obviously.3.The values of MOI of Ad-hfgl2-mi RNA on LM3?Hela?CNE and NCIH460 cell were 200?50?100?100, respectively;4.CCK-8 assay showed that infection of different MOIs of Ad-hfgl2-mi RNA resulted in inhibition of cell proliferation in dose-dependent manners in LM3?Hela?CNE and NCIH460 cells. Compared with negative control group, the experimental group had a stronger inhibitory effect, the difference was statistically significant(p<0.05). These data indicated that the knock down of hfgl2 expression was effective in inhibiting LM3?Hela?CNE and NCIH460 cells proliferation.5.We have been successfully established human hepatocellular carcinoma subcutaneous model in nude mice. The mean tumor volume in intervention group(3.3*1010IU?1.0*1010IU?3.3*109IU) were much smaller than that in the control group,that were 523.0 ± 44.19 mm3 VS 1042 ± 138.8 mm3,P<0.05;546.5 ± 22.41 mm3 VS 1042 ± 138.8 mm3,P<0.05; 622.4 ± 82.93 mm3 VS 1042 ± 138.8 mm3,P<0.05; moreover, at the treatment of day 19, compared with the control group, tumor volume in the intervention group were significantly reduced(307.2 ± 22.30 mm3 VS2351 ± 593.7 mm3,P<0.01;704.4 ± 60.18 mm3 VS 2351 ± 593.7 mm3,P<0.01;1042 ± 50.07mm3 VS 2351 ± 593.7mm3,P<0.05), these results suggested that micro RNA targeting hfgl2 carried by adenovirus obviously inhibited tumor growth, and this anti-tumor effects is related with dose. ?CONCLUSIONS?1. The Ad-hfgl2-mi RNA adenovirus particles has inhibited LM3?Hela?CNE and NCIH460 cells proliferation significantly;2.Ad-hfgl2-mi RNA down-regulated fgl2 expression and inhibited subcutaneous tumor growth. |
PDF Full Text Request