Investigation Of APOBEC3Family Genes For Their Function And Mediating Regulation

Tumorigenesis is a stepwise process concerning many genes mutatioa Given the genome instability, LINE-1(L1) retrotransposon, a genetic endogenous virus, is focused owing that it is consisted of17%of human genomes. Competent L1can transcribe in the cells like viruses when it activates, which will cause instability of our genome. In addition, L1retrotransposons can be detected in almost all carcinoma cells and the activation of them is closely related to carcinogenesis. To constraint L1retrotransposition, the measure of inhibitory mechanism in cells is taken during long-term evolution, such as A3s mediated regulation. A3s gene is a sort of stress response gene and usually can lead to cytidine deamination of target DNA sequence. When retroviruses replicate to get a single strand DNA, A3s will cause cytidine deamination to become thymine at TCW site. Due to the mutations of DNA, the replications of DNA and L1retrotransposons will be repressed. In a similar way, APOBECs also inhibit other retroviruses. Given that mutation is a main reason for carcinogenesis, it is worth studying the effects of expression of A3s genes on cellar biological behavior and on tumor regulatory factors.Purpose:To investigate the effect of A3s gene on cellular biological characters and the function mediated by A3s in carcinogenesis.Methods:Extracting RNA from Hela cells or A549cells, we prepared the A3s genes(A3B, A3C, A3F, A3G and A3H) by RT-PCR. We connected the genes to expression vector, pcDNA3.1(+) after DNA sequencing. Then we transfected human embryonic kidney293cells(HEK293) to investigate the biological functions of A3s. We used western blot to identify the expression of those genes. The effects of expression of A3s on proliferation, apoptosis and cell cycle of HEC293in vitro were observed by cell wound healing, CCK-8and AnnexxinV-FITC staining and flow cytometry. In study of carcinogenesis, the interactionof A3s and L1-ORF1p was detected by co-IP. TP53and K-RAS genes with the feature of most mutation in many cancers were exampled as A3s mediating mutation, with which a3D-PCR approach was employed.Results:With transfected HEK293cells, the cell proliferation was detected by an approach of CCK-8and showed that over expression of A3B, A3C, A3F and A3G increases cell growth upon19.5%,19.9%,18.9%and15.7%respectively (p<0.01). Also, the apoptosis of transfected HEK.293cells with different time-points was evaluated by flow cytometry using AnnexinV-FITC-PI staining and found that over expressions of A3B, A3C, A3F and A3G suppress apoptosis10.53%,19.02%,12.54%and13.95%respectively, which the control is21.31%. Cell cycle analysis showed that over expressions of expression of A3B, A3C, A3F and A3G shorten phases of GO and G1, but extend phases of S, G2and M, which may account for the results of facilitating cell proliferation.Regarding A3s mediated regulation, it indicated that (1) A3s(A3C, A3F and A3G) can interact with L1ORF-lp by a RNA dependent manner.(2) over-expression of A3s enhances the mutations of TP53body genes as well as its promoter region, whereas there is no significant effect on K-RAS genes.(3) The mutation of TP53promoter contributes to alteration of its activity.Conclusion:Over expression of APOBEC3genes in human embryonic kidney293cells facilitates the cell proliferation by affecting cell cycles and inhibits apoptosis.. The interaction of APOBEC3proteins with LINE1ORF-lp with a RNA-dependent manner accounts for a new mecansism for APOBEC3mediated regulation in tumor. The mutation of TP53and its promoter regions, but not K-RAS, elicited by A3s may reinforce the function of A3s in tumorigenesis.