Investigation On Gene Therapy For Hepatocellular Carcinoma By HrDNA Targeting Vector-mediated Melanoma Differentiation-associated Gene-7/Interleukin-24

The melanoma differentiation associated gene-7 (mda-7 gene) wasidentified by subtraction hybridization from a human melanoma cell line,which is a specific targeting tumor cell gene. Structural and sequencehomology indicates that this gene belongs to the interleukin (IL)-10family of cytokines and has therefore been redesignated IL-24 in 2001.IL-24 has immunity functions. As a cytokine, it can stimulate productionof inflammatory factors and participate in immunity activation. However,what interests people greatly is its specific anti-tumor potential. Up tonow, many independent groups have demonstrated that MDA-7/IL-24protein has obvious anti-tumor function, which selectively inducesgrowth suppression and programmed cell death (apoptosis) in a broadspectrum of human cancer and terminal differentiation in humanmelanoma cells, but spares the human normal cells. Ad.mda-7 iscurrently undergoing phaseâ…¡clinical trails in American.The rDNA regions targeted non-viral vector——pHrneo is a novelnon-viral human system derived from human chromosomes, which wasdeveloped in our laboratory. It shows a great advantage in study on genetherapy for single gene inheritance diseases such as hemophilia andDuchenne Muscular Dystrophy(DMD). To further investigate thepotential of pHrneo in tumor gene therapy, we cloned the mda-7 gene byPCR. Then mda-7 gene was ligated with a green fluorescence proteingene to form a fusion gene, which was further inserted into the ribosometargeting vector phrneo to construct the pHr-CMG. The expression offusion gene was controlled by the CMV enhancer/promoter. The tumorsuppressor vector pHr-CMG was transferred to human hepatocellularcarcinoma cell line Bel-7402 by lipofectamine 2000. Transfectionefficiency was determined using flow cytometer. The mda-7/GFP fusiongene expression was confirmed by RT-PCR and Western blotting and afluorescent microscope. The effect of growth suppression and apoptosisinduced by pHr-CMG was confirmed by Hoechst 33258 staining,cell-cycle and hypodiploidy analyses and MTT assay. The results showed: (1) The transfection efficiency could attain 37%-44%. (2) The mRNAand protein expression of MDA-7/GFP was detected by RT-PCR andWestern blotting. Furthermore, the location of green fluorescence signalin cell cytoplasmatic of Bel-7402 transfected with pHr-CMG wasdetected by a fluorescent microscope.(3)The effect of apoptosis inducedby pHr-CMG was confirmed by Hoechst33258 staining. (4) pHr-CMGmarkedly induced a G₂/M accumulation in Bel-7402 cell lines bycell-cycle and hypodiploidy analyses. (5)The relative cell viability ofpHr-CMG treated cells was 78%, 22%, 13% for 24h, 48h, 72hrespectively by MTT assay. These results confirmed induction ofapoptosis and growth suppression by the mda-7 gene with hrDNAtargeting vector in human hepatocellular carcinoma cell line Bel-7402and suggested that pHr-CMG might contribute to the cancer genetherapy.