Molecular characterization and expression analysis of B-cell lymphoma-2 in Trachinotus ovatus and its role in apoptotic process.
Molecular characterization and expression analysis of B-cell lymphoma-2 in Trachinotus ovatus and its role in apoptotic process.
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DOI：
10.3389/fimmu.2023.1129800
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发表时间：
2023
影响因子：
7.3
通讯作者：
中科院分区：
文献类型：
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作者：
B 细胞淋巴瘤-2 (Bcl-2) 是 Bcl-2 家族中第一个被鉴定的成员，在哺乳动物中具有抗凋亡功能。然而，它在硬骨鱼中的作用尚不完全清楚。本研究克隆了卵形鲳鳉Bcl-2（TroBcl2），并探讨了其在细胞凋亡中的作用。本研究通过PCR技术克隆了卵形鲳鳉Bcl-2（TroBcl2）。采用实时定量PCR（qRT-PCR）检测健康状态下和LPS刺激后其mRNA表达水平。将pTroBcl2-N3质粒转染金鲳吻（GPS）细胞进行亚细胞定位，在倒置荧光显微镜DMi8下观察，并通过免疫印迹进一步验证。采用体内过表达和RNAi敲低方法评估TroBcl2在细胞凋亡中的作用。流式细胞术检测TroBcl2的抗凋亡活性。通过带有 JC-1 的增强型线粒体膜电位测定试剂盒测量 TroBcl2 对线粒体膜电位 (MMP) 的影响。采用末端脱氧核苷酸转移酶介导的 dUTP 缺口末端标记 (TUNEL) 方法来评估 TroBcl2 在 DNA 片段化中的作用。使用免疫印迹法验证TroBcl2是否抑制细胞色素c从线粒体释放到细胞质中。 Caspase 3 和 Caspase 9 活性检测试剂盒用于研究 TroBcl2 对 caspase 3 和 caspase 9 活性的影响。通过qRT-PCR和酶联免疫吸附测定（ELISA）评估TroBcl2对体外凋亡相关和核因子-κB（NF-κB）信号通路相关基因表达的影响。荧光素酶报告基因检测用于评估 NF-κB 信号通路的活性。 TroBcl2的全长编码序列含有687bp，编码含有228个氨基酸的蛋白质。在 TroBcl2 中鉴定出四个保守的 Bcl-2 同源 (BH) 结构域和一个位于 BH1 的不变“NWGR”基序。在健康卵形T. ovatus中，TroBcl2广泛分布于11个测试组织中，并且在免疫相关组织中发现较高表达水平，如脾和头肾组织。脂多糖（LPS）刺激后，头肾、脾脏和肝脏中 TroBcl2 的表达显着上调。此外，亚细胞定位分析显示TroBcl2同时定位于细胞质和细胞核。功能实验表明，TroBcl2 可能通过减少线粒体膜电位损失、减少 DNA 碎片、阻止细胞色素 c 释放到细胞质中以及减少 caspase 3 和 caspase 9 激活来抑制细胞凋亡。此外，在 LPS 刺激下，TroBcl2 的过度表达抑制了多种凋亡相关基因的激活，例如 BOK、caspase-9、caspase-7、caspase-3、细胞色素 c 和 p53。此外，TroBcl2 的敲低显着增加了这些凋亡相关基因的表达。此外，TroBcl2过表达或敲低分别诱导或抑制NF-κB的转录，并调节NF-κB信号通路中基因（如NF-κB1和c-Rel）的表达以及下游炎症细胞因子IL-1β的表达。总体而言，我们的研究表明 TroBcl2 通过线粒体途径发挥其保守的抗凋亡功能，并可能作为 T. ovatus 的抗凋亡调节剂。
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. 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- κB (NF-κB) 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-κB signaling pathway. 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-κB and regulated the expression of genes (such as NF-κB1 and c-Rel) in the NF-κB signaling pathway as well as the expression of the downstream inflammatory cytokine IL-1β. 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.
影响因子：
6.4
作者：
Turk, Harmony F.;Kolar, Satya S.;Fan, Yang-Yi;Cozby, Caitlin A.;Lupton, Joanne R.;Chapkin, Robert S.
通讯作者：
Chapkin, Robert S.
影响因子：
3.7
作者：
Iwata A;de Claro RA;Morgan-Stevenson VL;Tupper JC;Schwartz BR;Liu L;Zhu X;Jordan KC;Winn RK;Harlan JM
通讯作者：
Harlan JM