Recombinant adenovirus vectors carrying antisense MMP2 inhibit invasion of HCC cells in vitro / 中华肝脏病杂志

<p><b>OBJECTIVES</b>To construct a recombinant adenoviral vector carrying antisense matrix metalloproteinase-2 (MMP2) and to study its inhibitory effects on the invasiveness and migratory capacity of hepatocellular carcinoma (HCC) cell line HepG2 in vitro.</p><p><b>METHODS</b>Total RNA was extracted from HCC. Then a 500 bp fragment at the 5' end of the human MMP2 cDNA sequence was synthesized by polymerase chain reaction (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-1, the homologous recombination took place in the E.coli BJ5183 and the recombinant adenoviral plasmid carrying the antisense MMP2 gene was constructed. The adenovirus (Ad-MMP2AS) was packaged and amplified in the HEK 293 cells and the viral titer was checked by GFP. Using the Boyden chamber model, the influence of Ad-MMP2AS on the invasion ability of HepG2 cells was determined in vitro.</p><p><b>RESULTS</b>The recombinant adenovirus vector carrying antisense MMP2 was constructed successfully and a strong green fluorescence was observed in HepG2 cells under a fluorescence microscope. The viral titer was 1 x 10(8); Ad-MMP2AS can effectively inhibit the penetrating capacity of HepG2 cells through Matrigel in vitro.</p><p><b>CONCLUSION</b>The recombinant adenovirus with antisense MMP2 can effectively inhibit the invasiveness and migratory capacity of HepG2 in vitro and may have potential in treating HCC.</p>

Inhibition of adenovirus-mediated gene transfer of antisense matrix metalloproteinase-2 on hepatocellular carcinoma growth in vivo / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate if a recombinant adenoviral vector carrying antisense matrix metalloproteinase-2 (MMP2) gene would inhibit the growth of hepatocellular carcinoma (HCC) in vivo.</p><p><b>METHODS</b>Using the recombinant adenoviral vector carrying antisense MMP2 gene (Ad-MMP2AS) which was constructed by us previously, to infect 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 blotting analysis and Gelatin zymography. Then the Ad-MMP2AS-infected cells were subcutaneously inoculated in nude mice. After tumors developed, Ad-MMP2AS was injected intratumorally into pre-existing tumors. The tumors were removed, sectioned, and stained with H E.</p><p><b>RESULTS</b>Compared with PBS or Ad-CMV-infected cells, the infected Bel-7402 cells with Ad-MMP2AS injections significantly reduced their MMP2 enzyme activity and invasiveness about 52.05% in Matrigel assays, and the tumor volumes in nude mice resulted in a 3.3-fold reduction. In addition, direct intratumoral injection of Ad-MMP2AS into pre-existing tumors significantly prevented further expansion of the tumor masses and resulted in a 63.06% reduction in tumor cell growth.</p><p><b>CONCLUSION</b>The recombinant adenovirus with antisense MMP2 can effectively inhibit the invasiveness and growth of Bel-7402 cells in vitro and in vivo, and it has a therapeutic potential for HCC.</p>