PMID- 37006242 OWN - NLM STAT- MEDLINE DCOM- 20230404 LR - 20230417 IS - 1664-3224 (Electronic) IS - 1664-3224 (Linking) VI - 14 DP - 2023 TI - Molecular characterization and expression analysis of B-cell lymphoma-2 in Trachinotus ovatus and its role in apoptotic process. PG - 1129800 LID - 10.3389/fimmu.2023.1129800 [doi] LID - 1129800 AB - INTRODUCTION: B-cell lymphoma-2 (Bcl-2) is the first identified member of the Bcl-2 family that performs an anti-apoptotic function in mammals. However, its role in teleosts is not fully understood. In this study, Bcl-2 of Trachinotus ovatus (TroBcl2) was cloned, and its role in apoptosis was investigated. METHODS: In this study, Bcl-2 of Trachinotus ovatus (TroBcl2) was cloned by PCR. Quantitative real-time PCR (qRT-PCR) was used to detect its mRNA expression level in healthy condition and after LPS stimulation. Subcellular localization was performed by transfecting the pTroBcl2-N3 plasmid into golden pompano snout (GPS) cells and observed under an inverted fluorescence microscope DMi8 and further verified by immunoblotting. In vivo overexpression and RNAi knockdown method were performed to evaluate the role of TroBcl2 in apoptosis. The anti-apoptotic activity of TroBcl2 was detected by flow cytometry. The effect of TroBcl2 on the mitochondrial membrane potential (MMP) was measured by an enhanced mitochondrial membrane potential assay kit with JC-1. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method was performed to evaluate the role of TroBcl2 in the DNA fragmentation. Immunoblotting was used to verify whether TroBcl2 inhibits the release of cytochrome c from mitochondria into the cytoplasm. The Caspase 3 and Caspase 9 Activity Assay Kits were used to investigate the effect of TroBcl2 on caspase 3 and caspase 9 activities. The effects of TroBcl2 on the expression of apoptosis-related and nuclear factor- kappaB (NF-kappaB) signaling pathway-related genes in vitro were evaluated by qRT-PCR and Enzyme linked immunosorbent assay (ELISA). Luciferase reporter assay was used to evaluate the activity in NF-kappaB signaling pathway. RESULTS AND DISCUSSION: The full-length coding sequence of TroBcl2 contains 687 bp and encodes a protein containing 228 amino acids. Four conserved Bcl-2 homology (BH) domains and one invariant "NWGR" motif located in BH1 were identified in TroBcl2. In healthy T. ovatus, TroBcl2 was widely distributed in the eleven tested tissues, and higher expression levels were found in immune-related tissues, such as spleen and head kidney tissues. After stimulation with lipopolysaccharide (LPS), the expression of TroBcl2 in the head kidney, spleen, and liver was significantly upregulated. In addition, subcellular localization analysis revealed that TroBcl2 was localized in both the cytoplasm and nucleus. Functional experiments showed that TroBcl2 inhibited apoptosis, possibly by reducing mitochondrial membrane potential loss, decreasing DNA fragmentation, preventing cytochrome c release into cytoplasm, and reducing the caspase 3 and caspase 9 activations. Moreover, upon LPS stimulation, overexpression of TroBcl2 suppressed the activation of several apoptosis-related genes, such as BOK, caspase-9, caspase-7, caspase-3, cytochrome c, and p53. Furthermore, knockdown of TroBcl2 significantly increased the expression of those apoptosis-related genes. In addition, TroBcl2 overexpression or knockdown induced or inhibited, respectively, the transcription of NF-kappaB and regulated the expression of genes (such as NF-kappaB1 and c-Rel) in the NF-kappaB signaling pathway as well as the expression of the downstream inflammatory cytokine IL-1beta. Overall, our study suggested that TroBcl2 performs its conserved anti-apoptotic function via the mitochondrial pathway and may serve as an anti-apoptotic regulator in T. ovatus. CI - Copyright (c) 2023 Cao, Yang, Li, Liu, Li, Zhou and Sun. FAU - Cao, Zhenjie AU - Cao Z AD - State Key Laboratory of Marine Resource Utilization in South China Sea, Hainan University, Haikou, China. AD - Collaborative Innovation Center of Marine Science and Technology, Hainan University, Haikou, China. FAU - Yang, Xin AU - Yang X AD - State Key Laboratory of Marine Resource Utilization in South China Sea, Hainan University, Haikou, China. AD - Collaborative Innovation Center of Marine Science and Technology, Hainan University, Haikou, China. FAU - Li, Tao AU - Li T AD - Collaborative Innovation Center of Marine Science and Technology, Hainan University, Haikou, China. FAU - Liu, Zhiru AU - Liu Z AD - Collaborative Innovation Center of Marine Science and Technology, Hainan University, Haikou, China. FAU - Li, Pengfei AU - Li P AD - Guangxi Key Laboratory of Marine Natural Products and Combinatorial Biosynthesis Chemistry, Guangxi Beibu Gulf Marine Research Center, Guangxi Academy of Sciences, Nanning, Nanning, China. FAU - Zhou, Yongcan AU - Zhou Y AD - State Key Laboratory of Marine Resource Utilization in South China Sea, Hainan University, Haikou, China. AD - Collaborative Innovation Center of Marine Science and Technology, Hainan University, Haikou, China. FAU - Sun, Yun AU - Sun Y AD - State Key Laboratory of Marine Resource Utilization in South China Sea, Hainan University, Haikou, China. AD - Collaborative Innovation Center of Marine Science and Technology, Hainan University, Haikou, China. LA - eng PT - Journal Article PT - Research Support, Non-U.S. Gov't DEP - 20230316 PL - Switzerland TA - Front Immunol JT - Frontiers in immunology JID - 101560960 RN - 0 (NF-kappa B) RN - EC 3.4.22.- (Caspase 3) RN - EC 3.4.22.- (Caspase 9) RN - 9007-43-6 (Cytochromes c) RN - 0 (Lipopolysaccharides) RN - 0 (Proto-Oncogene Proteins c-bcl-2) SB - IM MH - Animals MH - *NF-kappa B/metabolism MH - Caspase 3/metabolism MH - Caspase 9/genetics/metabolism MH - *Cytochromes c/metabolism MH - Lipopolysaccharides/pharmacology MH - Apoptosis MH - Fishes/metabolism MH - Proto-Oncogene Proteins c-bcl-2/genetics/metabolism MH - Mammals/metabolism PMC - PMC10063160 OTO - NOTNLM OT - Bcl-2 OT - NF-kappaB OT - Trachinotus ovatus OT - apoptosis OT - mitochondria COIS- The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. EDAT- 2023/04/04 06:00 MHDA- 2023/04/04 06:42 PMCR- 2023/01/01 CRDT- 2023/04/03 03:44 PHST- 2022/12/24 00:00 [received] PHST- 2023/03/07 00:00 [accepted] PHST- 2023/04/04 06:42 [medline] PHST- 2023/04/03 03:44 [entrez] PHST- 2023/04/04 06:00 [pubmed] PHST- 2023/01/01 00:00 [pmc-release] AID - 10.3389/fimmu.2023.1129800 [doi] PST - epublish SO - Front Immunol. 2023 Mar 16;14:1129800. doi: 10.3389/fimmu.2023.1129800. eCollection 2023.