The Role Of Apoptosis Regulatory Protein BIM In The Gill Remodeling Of Goldfish

Proapoptotic Bcl-2 homology 3-only protein Bim plays an important role in apoptosis.Bim appears to act as a ’death ligand’ which can neutralize certain members of the pro-survival Bcl-2 sub-family such as Mcl-1,Bcl-2 and Bcl-xL.The ability to induce apoptosis of Bim is determined by a ratio of binding to pro-survival Bcl-2 sub-family members.In addition,Bim can also up-regulate the level of pro-apoptotic Bax sub-family members.The three major Bim isoforms(Bim EL,Bim L and Bim S)are generated by alternative splicing.In general,BimEL is the most abundant and BimS is almost undetectable.All 3 isoforms induce apoptosis,the shortest being the most potent.Goldfish,which belongs to cyprinid genus Carassius,were kept as ornamental fish that date back to Southern Song Dynasty.Goldfish is one of the most potent hypoxic-tolerant freshwater fish with some unique hypoxic adaptation mechanisms such as gill remodeling.The size of interlamellar cell mass(ILCM)is reduced in hypoxia,warm water,and during exercise,whereby the respiratory surface area and the capacity for oxygen uptake are greatly increased.The ability of some fishes to reversibly remodel their gill morphology has become a focus of research after the discovery of extreme morphological gill plasticity in crucian carp and goldfish.HIF-la is generally considered as the main switch in many hypoxia-inducible responses,which is stable and up-regulated in hypoxia.A variety of miRNAs have the effect of inhibiting apoptosis,such as miR-24,which is induced by HIF-1.Bim is a target gene of miR-24.The molecular mechanism of apoptosis of goldfish ILCM is not clear.It is significance to study the effect of Bim on the mechanism of ILCM regresses in goldfish which adaptation to hypoxia and high temperature.In addition,the studies on the reversibly remodel of goldfish gill morphology provide a good idea for breeding new aquaculture resistant varieties through genetic engineering.To explore the role of Bim gene in the apoptosis of ILCM in goldfish gill,the main contents of this study are shown as follows through the DNA recombination technology,real-time quantitative PCR,Western Blot and RNA pull-down experiments:1.Goldfish Bim gene cloning and antibody productionThe Bim CDS sequences were cloned by PCR.Two sequences were 546bp and 213bp,encoding 182 and 71 amino acids,respectively,as determined from DNA sequence analysis.The two Bim isoforms may be generated by alternative splicing of Bim precursor mRNA.Through the comparison of Bim amino acid sequences of goldfish,zebrafish and human,we used "VARELRRIGDEFNRLYC" as the antigenic to Bim antibody of goldfish.The Bim 3’-UTR sequence of 2952bp was amplified by 3’RACE and the pGEM-T-Bim 3’-UTR vector was constructed.Sequence analysis showed that Bim mRNA 3’UTR contained nine AU-rich elements(AUUU-UA,AU-rich elements,AREs).2.Analysis of miR-24 and Bim expression of goldfish in hypoxia adaptationHealthy goldfish were randomly divided into three groups(n=6)for 8 hours:normoxia group(dissolved oxygen of 8.5～9.5ppm),hypoxia group(dissolved oxygen of 1.8ppm～2.1ppm),hypoxia and injected ERK inhibitor group.The gill tissue of goldfish were fixated with glutaraldehyde,dehydrated and dried.Using a digital microscope,we found that goldfish gill ILCM regressed significantly compared with normoxic conditions,while 12.5 mg/kg ERK inhibitor treatment partially inhibited the reduction of ILCM in the gills of goldfish in hypoxic conditions.Real-time PCR analysis showed that hypoxia could significantly increase the expression level of miR-24 in goldfish,while the level of Bim mRNA showed no significant difference.Western Blot analysis showed that expression level of Bim protein reduced in hypoxic conditions,while injected ERK inhibitors leads to reverse this inhibitory effect.It was speculated that overexpression of miR-24 and induction of ERK1/2 kinase in hypoxia decreased the protein level of Bim,thereby promoting the repression of ILCM in goldfish.ERK inhibitor treatment inhibited hypoxia-induced decrease of Bim protein level and ILCM in goldfish gill tissue.3.Bim expression and RNA Pull-down Analysis in goldfish during temperature stressHealthy goldfish were randomly divided into three temperature groups(n=6)10℃,17℃ and 25℃for 24 hours.Real-time PCR analysis showed that the relative expression levels of Bim mRNA and Bim protein levels decreased at elevated temperature.To further explore the effect of temperatures on Bim mRNA level,we use RNA pull-down assay to study the binding of HSC70 and HSP40 to Bim mRNA at different temperatures.We cloned the HSC70 and HSP40 genes of goldfish and constructed the prokaryotic expression vectors pET32a-HSC70 and pET32a-HSP40.The recombinant protein Trx-HSC70 and Trx-HSP40 are expressed by IPTG-induction.pGEM-T-Bim 3’-UTR is cutted into linear DNA after Sal I digestion.We generated biotin-Bim-3’-UTR RNA by in vitro transcription.After the Biotin-Bim 3’-UTR RNA was enriched with the avidin resin,Trx-HSC70 and/or Trx-HSP40 protein were added to the avidin resin and mixed for 3 hours at 10℃,17℃ and 25℃.The binding capacities of HSC70 with Biotin-Bim3’-UTR RNA were analyzed by Western blot in different temperatures.High temperature repressed the protein levels of HSC70 protein binding to Bim mRNA 3’-UTR by the assistance of HSP40.The results suggested that high temperature decreased the binding capacity between HSC70 protein and Bim mRNA 3’-UTR region and thus play an important role for destabilization of Bim mRNA,which was consistent with the decrease of Bim protein level at 25 ℃.This study showed that hypoxia and hyperthermia inhibited Bim expression through ERK/miR-24 and HSC70/HSP40,respectively,and induced decreased ILCM in gill tissue of goldfish.Bim has the dual regulatory function of promoting apoptosis and promoting cell survival,suggesting that Bim mainly plays a role in promoting cell survival in goldfish ILCM cell.Further research is needed for the regulatory mechanism of Bim dual function in a particular cell type.