Anticancer Effection And Mechanism Of Human Original C-type Lectin 2A Intracellular Expression

Nowadays cancer continues to pose a serious threat to human health, has become the world’s largest cause of death.Gene therapy is one of a new therapy, the effect of gene therapy depend on the carrier of carrying gene and the gene. A mature system of virus carrier is oncolytic adenovirus and oncolytic vaccinia virus, at present this system is already very mature. In the past few years our reshearch group made a deep research about the Pinellia pedatisecta agglutinin gene expression in tumor cells, and in-depth study of the PPA in tumor cell cytotoxicity mechanism. The results showed that the effective tumor gene therapy might be present in the lectin family, so in recent years our research group selected plant lectins and marine animal lectins to studied. On the basis of the above, we haven’t choose anthropogenic lectin gene, so this research chose the human CLEC2 A gene. CLEC2 A is expressed on the cell surface, ligands for NK cell surface receptors, Expression of CLEC2 A as gene therapy in tumor cells may activate the body’s own NK cells.CLEC2A gene was cloned into eukaryotic expression vector pEGFP-C1 to construct the recombinant plasmid pEGFP-CLEC2A-C1. Recombinant plasmid pEGFP-CLEC2A-C1 was transfected into human hepatoma cell system 7404,7721 and Hep3 B cells, the gene on tumor cell toxicity were observed. The results showed that the intracellular expression of the fusion protein EGFP-CLEC2 A,which tumor cells will collapse. Suggesting CLEC2 A expression has a certain toxicity on tumor cells. PEGFP-CLEC2A-C1 were co-transfected with recombinant plasmid pCDNA-3.1+-dsRED-PRMT5 respectively. It was found that there was a co-location between CLEC2 A and PRMT5, suggesting that there was an interaction between them. In addition, the packaging of non replicating adenovirus AD-Flag-CLEC2 A is using the Adeasy system, which is carrying the human CLEC2 A expression frame. Identified non wild type virus. Detection of recombinant oncolytic adenovirus of 7421, DU-145, Hep3 B and K562 cell killing effect was determined by MTT assay in vitro. The results show that CLEC2 A gene of the oncolytic adenovirus carrying on cell has certain killing effect. CLEC2 A construction to vector PCB, and recombination with wild vaccinia virus in 293 A cells to obtain recombinant oncolytic vaccinia virus OncoPox-Flag-CLEC2 A. Under the premise of identifying wild virus, Detection of recombinant oncolytic adenovirus of 7421, DU-145, Hep3 B and K562 cell killing effect was determined by MTT assay in vitro.The results show that CLEC2 A gene of oncolytic recombinant vaccinia virus to carry on cell has certain killing effect. And the killing effect of oncolytic adenovirus is obvious. To explore the killing mechanism of AD-Flag-CLEC2 A in prostate cancer cells DU-145. Western blot showed AD-Flag-CLEC2 A enhanced shear Caspase3 and PARP, tips CLEC2 A expression induced apoptosis of tumor cells.In conclusion, express CLEC2 A genes in cells will lead cell to death, CLEC2 A may interact with PRMT5 protein.The cause of the cell death may be due to the down-regulation of Caspase3 and PARP apoptosis inhibitory factor, induced apoptosis. The cause of the cell death may be due to the activation of the caspase pathway, induced apoptosis. Whether to activate the body’s own NK cell system, needs further research on the level of animal experiment.