Role Of Megalobrama Amblycephala Bcl2l13 In Cellapoptosis

Megalobrama amblycephala is an important economic fish in China,however,compared with other Cyprinids,its hypoxia tolerance is poor.Bcl-2-like protein 13（Bcl2l13）is a member of the Bcl-2 protein family and has pro-apoptotic or anti-apoptotic activities under different conditions.Unlike other Bcl-2 family proteins,Bcl2l13 contains four BH domains,one BHNo domain,and one TM domain.Our previous study found that hypoxia can lead to the up-regulation of bcl2l13 expression in M.amblycephala.But the functions of M.amblycephala Bcl2l13 and its different domains in fish cell apoptosis are still unclear.Therefore,in order to study the role of Bcl2l13 in the cell apoptosis of fish,in this study,the role of this gene and its different domains in cell proliferation and apoptosis were analyzed based on overexpression and knockdown experiments.The main results are as follows:1.The effects of Bcl2l13 and its domains on cell proliferation and apoptosis were detected by CCK-8,RT-qPCR and hochest 33258 staining.The results showed that after overexpression of Bcl2l13,the cell proliferation was significantly reduced,and the apoptosis rate increased.After the TM domain or BHNo domain of Bcl2l13 was deleted,the ability of Bcl2l13 to inhibit cell proliferation and apoptosis decreased to varying degrees,and its function was further weakened after the deletion of both these two domains.It was suggested that Bcl2l13 can inhibit cell proliferation and promote cell apoptosis,and its TM and BHNo domains,especially TM domain,are important for the regulation of cell proliferation and apoptosis.2.Based on RT-qPCR and Calcein AM-Co Cl₂staining,the pathways of Bcl2l13mediated apoptosis were analyzed.The results showed that the expression of caspase 3,cyt-c,fas and other genes was significantly down-regulated when the expression of bcl2l13 was inhibited,indicating that Bcl2l13 may mediate apoptosis through the endogenous mitochondrial pathway and death receptor pathway.Further study found that m PTP was increased after overexpression of Bcl2l13 in FHM cells and EPC cells,whereas it was relatively decreased after deletion of the TM domain.Therefore,Bcl2l13apoptosis may be regulated by the m PTP mediated pathway.3.RT-qPCR and Annexin V FITC/PI method were used to detect the apoptosis after overexpression of Bcl2l13 and TM domain deletion in different cells.The results showed that the overexpression of Bcl2l13 had a tendency to promote apoptosis in FHM cells and EPC cells,and after the deletion of the TM domain,the change of apoptosis was significant in EPC but not in FHM.Bcl2l13 has a significant anti-apoptotic function in He La,and this function was significantly weakened after the deletion of the TM domain.The alignment of the amino acid sequences of M.amblycephala and Homo sapiens Bcl2l13 indicated that low homology of the BHNo domain maybe lead to different function of Bcl2l13 in apoptosis in different species.4.The study found that overexpression of bcl2l13 could significantly down-regulate the expression of ndufa2,cox17 and sdha,and negatively regulate the oxidative phosphorylation pathway.In addition,overexpression of bcl2l13 will lead to upregulation of rps21,and subcellular colocalization showed that they had some colocalization region in mitochondrion.It was speculated that bcl2l13 may directly regulate the expression of rps21.