Research On The Molecular Mechanism Of GADD45? Regulating The Proliferation And DNA Repair Of Rat Liver Cells BRL-3A

Studies have shown that GADD45?(growth arrest and DNA damage-inducible 45 alpha)is a stress-induced gene that expressed constitutively in low abundance but is transcriptionally activated by a variety of stress stimuli,including ultraviolet and ionizing radiation,and interact with many intracellular signaling molecules involved in cell cycle regulation,apoptosis,maintenance,genomic stability,DNA repair and immune response.In this study,GADD45? was found to be significantly enhanced in hepatocytes of rat regenerated liver.However,the roles of GADD45? in liver regeneration and regulatory mechanisms have not been reported.To this end,this study intends to delve into the GADD45? regulatory rat normal liver cells BRL-3A and injury-induced BRL-3A molecular mechanism.In order to understand the effect of GADD45? on rat liver regeneration and its mechanism,GADD45?was found on the basis of the gene expression profiling of regenerated liver based on the laboratory in the stage of liver regeneration(0-2 h)and progression stage(6-72 h),suggesting that it may be involved in DNA damage repair during rapid cell proliferation after hepatectomy.Quantitative RT-PCR was used to verify the reliability of the results.The signal pathways of GADD45? were constructed by analyzing IPA,GenMAPP,KEGG,BIOCARTA,QIAGEN and Biocompare databases,and the possible mechanism of GADD45? was analyzed.The results showed that GADD45? might be detected by P38 MAPK,JNK,CDC2/CCNB1,AKT and MTOR signaling pathway regulates normal BRL-3A cells and FZD/UVCinduced proliferation,apoptosis,cell cycle and DNA damage repair of BRL-3A cells.In this study,GADD45? overexpression or down-regulated in BRL-3A cells were obtained by gene addition and gene interference.MTT,Ed U,flow cytometry,qRT-PCR and western blot were used toinvestigate the effects of GADD45? on the viability,proliferation,apoptosis,DNA damage and repair of normal BRL-3A cells and FZD/UVC-treated BRL-3A cells.The results showed that overexpression of GADD45? significantly inhibited the cell viability,proliferation,the number of cells in G1 and S phases,and of FZD/UVC induced apoptosis of BRL-3A cells and decreased the inhibition of FZD/UVC on the viability,proliferation of BRL-3A cells,while increased the number of cells in G2/M phase of BRL-3A cells and FZD/UVC induced S phase arrest.Downregulated GADD45? induced the viability,proliferation,the number of cells in S and G2/M phases and the inhibition of FZD/UVC on the viability,proliferation of BRL-3A cells increased,while decreased apoptosis,the number of cells in G1 phases of BRL-3A cells and FZD/UVC induced S phase arrest.q RT-PCR and western blot were employed to detect the genes/proteins expression profiles of GADD45? signaling pathway in BRL-3A cells after up/down-regulating GADD45?,and the results showed that genes/proteins related to P38 MAPK,JNK,CDC2/CCNB1,AKT and MTOR signaling pathways were significantly changed in normal BRL-3A cells.The expression profiles of cell cycle,proliferation and apoptosis related genes/proteins in FZD/UVC treated BRL-3A cells were also detected by qRT-PCR and western blot,and the results indicated that the expression of Myc,Bcl-2,Ccnd1,PCNA,P21,Ccnb1,Caspase3,Caspase8,Caspase9 and Bax have significantly changes.Above all,GADD45? regulate cell proliferation and apoptosis of BRL-3A via P38 MAPK,JNK,CDC2/CCNB1,AKT and MTOR signaling pathways,and regulate the repair of FZD/UVC induced DNA damage of BRL-3A cell through Myc,Bcl-2,Ccnd1,Ccnb1,PCNA,P21,Caspase3,Caspase8,Caspase9 and Bax.