Construction Of Recombinant Adenovirus Co-expressing Apoptin And MEL And Its Inhibitory Effect On Hepatocellular Carcinoma

Hepatocellular carcinoma（HCC）is highly heterogeneous,with high mortality and metastasis,and is mainly characterized by vascular proliferation.Early stage patients have limited clinical symptoms and late stage is difficult to cure,making clinical treatment more difficult.Both human and animal prevalence is increasing year by year,drug resistance is increasing,and targeted gene therapy has become a research hotspot for hepatocellular carcinoma treatment.HIV trans-activated transcription（TAT）protein,derived from the human HIV viral TAT protein,penetrates the cell membrane and promotes the entry of macromolecules into the cytoplasm;Apoptin-induced apoptosis is regulated by Bcl-2 and Apaf-1 apoptotic vesicles,which specifically induce by activating apoptotic vesicles and Bcl-2-regulated death pathways,as well as activating autophagy and mitochondrial apoptosis pathways Tumor homing peptide RGD recognizes integral proteins present on the surface of cancer cells and specifically targets the vascular zone of the tumor;MEL disrupts the cell membrane by binding to the cell membrane and forming transmembrane ring pores,causing leakage of substances from the cell and increasing cell membrane permeability,which eventually leads to cell lysis.The Apoptin and the MEL were selected as the target therapeutic genes for hepatocellular carcinoma,which were efficiently delivered to the target cells with the help of adenovirus as the vector.The fusion expression of the membrane penetrating peptide TAT with Apoptin promotes the secondary internalization of Apoptin protein in lysed hepatocellular carcinoma cells into cancer cells;The targeting peptide RGD targets the integrin receptor in hepatocellular carcinoma blood vessels to fusion expression with MEL to direct MEL to specifically kill tumor cells and mitigate the non-specific damage to normal tissues.By fusion expression of Apoptin and MEL genes,Apoptin was used to kill tumor cells with mitochondrial apoptosis and autophagy specific induction while MEL lysed the cell membrane of cancer cells to destroy the complete structure of cells,achieving the purpose of coordinated dual gene tumor suppression and laying the foundation for gene targeting therapy of Hepatocellular carcinoma.The pMD-TAT-Apoptin+UBI-RGD-MEL gene fragment was amplified with high fidelity specificity and seamlessly cloned into linearized GV314 to construct the adenovirus shuttle vector GV-TAT-Apoptin+UBI-RGD-MEL.The adenovirus shuttle vector was cotransfected with a large backbone plasmid into HEK293A cells and packaged The titer of the obtained recombinant adenovirus Ad-TAT-Apoptin was 10^10.75 pfu/m L,the titer of Ad-RGD-MEL was10^10.38 pfu/m L,the titer of TAT-Apoptin+UBI-RGD-MEL was 10^10.25 pfu/m L,and the titer of Ad-EGFP was 10¹¹ pfu/m L.Each recombinant adenovirus was separately infected with SMMC-7721 cells and successful expression of the target gene in hepatoma cells was detected by indirect immunofluorescence and Western Blotting;Recombinant adenovirus Ad-TAT-Apoptin+UBI-RGD-MEL increased the expression content of reactive oxygen species and contributed to the fragmentation of cell nuclear DNA.The recombinant adenovirus had no significant inhibitory effect on L-02,and the inhibitory effect on SMMC-7721 and Huh 7 cells was time and dose-dependent;the early and late apoptosis rates of Ad-TAT-Apoptin+UBI-RGD-MEL were highly significant compared with the control group after 36 h of infestation of SMMC-7721 cells with 1000 MOI recombinant adenovirus by flow cytometry,and were both highly significant（P<0.01）compared to the control.Recombinant adenovirus Ad-TAT-Apoptin+UBI-RGD-MEL highly significantly inhibited the migration of SMMC-7721 cells and highly significantly inhibited FBS-mediated invasion and chemotaxis of SMMC-7721 cells（P<0.01）.The recombinant adenovirus Ad-TAT-Apoptin+UBI-RGD-MEL was able to inhibit the growth rate of tumors,reduce liver metastasis of subcutaneous tumors,and delay the trend of weight loss in nude mice ectopic tumor model;The Ad-TAT-Apoptin+UBI-RGD-MEL group had strong targeting,less damage to normal tissues and organs,and induced the expression of Csapase3 and P53 protein in tumor and liver,and increased the number of apoptotic cells.In this experiment,recombinant adenovirus Ad-TAT-Apoptin+UBI-RGD-MEL was successfully packaged.The results of in vitro assay confirmed that the recombinant adenovirus Ad-TAT-Apoptin+UBI-RGD-MEL could significantly inhibit the proliferation,migration and invasion and suppression of SMMC-7721.As verified by a subcutaneous ectopic tumor model in nude mice,the recombinant adenovirus Ad-TAT-Apoptin+UBI-RGD-MEL treatment group showed increased expression of P53 and Casepase3 protein in tumors,and delayed the trend of weight loss in nude mice,with no significant killing effect on normal tissues.In summary,this trial demonstrated that recombinant adenovirus Ad-TAT-Apoptin+UBI-RGD-MEL can target and inhibit hepatocellular carcinoma cells,providing a theoretical basis for comparative medicine and tumor-targeted therapy.