The Inhibitory Effect Of Antisense Matrix Metalloproteinase-2 Mediated By Adenovirus On Hepatocellular Carcinoma Growth And Invasion In Vitro And In Vivo

Objective This study was conducted (1) to construct a recombinant adenoviral vector carrying antisense matrix metalloproteinase-2(MMP2) gene (Ad-MMP2^AS); (2) to investigate the fact that the Ad-MMP2^AS inhibit the invasiveness of hepatocellular carcinoma(HCC) cells in vitro and decide the role of Ad-MMP2^AS in inhibiting the invasiveness of HCC;. (3) to detect the change of the human vascular endothelial cell (VEC) infected with Ad-MMP2^AS and elucidate the molecular mechanism of Ad-MMP2^AS in inhibiting the angiogenesis of HGC; (4) to observe the growth and angiogenesis of HCC in vivo models, and demonstrat the effect of Ad-MMP2^AS in treatment of HCC. Methods: Total RNA was extracted from HCC, and then A 500-bp fragment at the 5' end of human MMP2 cDNA was synthesized by reverse-transcription polymerase chain reaction, (RT-PCR) and was reversely inserted into the multiclone site (MCS) of the shuttle plasmid pAdTrack-CMV,with the resultant plasmid and the backbone plasmid pAdEasy-l,the homologous recombination took place in the E.coli BJ5183 and the recombinant adenoviral plasmid carrying the antisense MMP2 gene was constructed was generated. The adenoviruses(Ad-MMP2^AS) were packaged and amplified in the HEK 293 cells.Then the viral titer was checked by GFP. Ad-MMP2^AS infected the human HCC cell line (Bel-7402). Then the invasiveness of the Bel-7402 cells was assayed in Matrigel, and the production of MMP2 in the Bel-7402 cells was detected with Western Blot Analysis and Gelatin zymography. Following the Ad-MMP2^AS-infected cells was subcutaneously inoculated in nude mice, and injected intratumorally into pre-existing tumors. The tumors were removed, sectioned, stained with H&E and stained with an anti-CD31 monoclonal antibody. In addition, Ad-MMP2^AS infected the human vascular endothelial cell (VEC) . The VEC was detected with Flow-cytometry (FCM) and Electron-microscope Analysis. Results: The recombinant adenovirus vector carrying antisense MMP2 was constructed successfully, the strong green fluorescence was observed in HEK 293 cells under a fluorescence microscopy, The viral titer was 1×10⁸. Compared with PBS or Ad-CMV-infected cells, infection of Bel-7402 cells with Ad-MMP2^AS significantly reduced MMP2 enzyme activity, invasiveness resulted in 52% reduction in Matrigel assays, and the tumor volume resulted in 4.3-fold reduction in nude mice. In addition,direct intratumoral injection of Ad-MMP2AS into pre-existing tumors significantly impaired the further expansion of the tumor mass and resulted in a 63% reduction in tumor cell growth. Ad-MMP2AS therapy resulted in a 4.3-fold reduction in tumor vessel density, and the tumor cells show atrophy and necrobiosis. Beside these results, the growth rate significantly reduced and the apoptosis rate increased in VEC infected with Ad-MMP2AS. Conclusion: (1) MMP2 plays a pivot role in growth and invasiveness of HCC; (2)The recombinant adenovirus with antisense MMP2(Ad-MMP2AS) can effectively inhibit the invasiveness of Bel-7402 cells via the pathway of attenuating the expressing of MMP2 cDNA in HCC cells and reducing the excreting MMP2 of HCC cells; (3)Ad-MMP2AS can effectively promote the apoptosis and inhibit the growth of VEC; (4) Ad-MMP2AS can effectively attenuate the growth and angiogenesis of HCC in vivo models, and demonstrat a therapeutic potential for HCC.