Chun-Xiao Huang

The zebrafish miR-462/miR-731 cluster is induced under hypoxic stress via hypoxia-inducible factor 1α and functions in cellular adaptations

Hypoxia, a unique and essential environmental stress, evokes highly coordinated cellular responses, and hypoxia‐inducible factor (HIF) 1 in the hypoxia signaling pathway, an evolutionarily conserved cellular signaling pathway, acts as a master regulator of the transcriptional response to hypoxic stress. MicroRNAs (miRNAs), a major class of posttranscriptional gene expression regulators, also play pivotal roles in orchestrating hypoxia‐mediated cellular adaptations. Here, global miRNA expression profiling and quantitative real‐time PCR indicated that the up‐regulation of the miR‐462/miR‐731 cluster in zebrafish larvae is induced by hypoxia. It was further validated that miR‐462 and miR‐731 are upregulated in a Hif‐1α‐mediated manner under hypoxia and specifically target ddx5 and ppm1da, respectively. Overexpression of miR‐462 and miR‐731 represses cell proliferation through blocking cell cycle progress of DNA replication, and induces apoptosis. In situ detection revealed that the miR‐462/miR‐731 cluster is highly expressed in a consistent and ubiquitous manner throughout the early developmental stages. Additionally, the transcripts become restricted to the notochord, pharyngeal arch, liver, and gut regions from postfertiliztion d 3 to 5. These data highlight a previously unidentified role of the miR‐462/miR‐731 cluster as a crucial signaling mediator for hypoxia‐mediated cellular adaptations and provide some insights into the potential function of the cluster during embryonic development.—Huang, C.‐X., Chen, N., Wu, X.‐J., Huang, C.‐H., He, Y., Tang, R., Wang, W.‐M., Wang, H.‐L. The zebrafish miR‐462/miR‐731 cluster is induced under hypoxic stress via hypoxia‐inducible factor 1α and functions in cellular adaptations. FASEB J. 29, 4901–4913 (2015). www.fasebj.org

Characterization of a novel CC chemokine CCL4 in immune response induced by nitrite and its expression differences among three populations of Megalobrama amblycephala

A novel CC chemokine gene, chemokine CC motif ligand 4 (CCL4), was isolated from Megalobrama amblycephala. The full-length cDNA was 913 bp, encoding 94 amino acid residues. The deduced amino acid sequence possessed the typical arrangement of four cysteines as found in other known CC chemokines. The expression of M. amblycephala CCL4 during the early development showed the mRNA levels before hatching and at 62 h post fertilized (hpf) were significantly higher than other post-hatching stages (P < 0.05). Besides, it was widely expressed in all detected tissues with the highest transcription in liver, followed by intestine, spleen and gill, where a larger number of immune cells including lymphocytes and macrophages are present. Our findings had fully confirmed that CCL4 expression was strongly induced in vitro and quickly up-regulated after nitrite stress, then substantially altered in all tested tissues, supporting a potential pro-inflammatory function. We also indicated that inflammation effect might firstly happen in blood after nitrite stress. Furthermore, the tissue expression differences of CCL4 among three natural populations revealed that CCL4 mRNA in Yuni Lake population was obviously higher than the other two populations, Liangzi Lake population and Poyang Lake population, which will provide valuable insights into breeding strategies for selecting population with better immune property of M. amblycephala.

Study on the immune response to recombinant Hsp70 protein from Megalobrama amblycephala.

The expression of heat shock protein 70 (Hsp70) is induced in response to many factors including high temperature, infection, metal pollutants and toxic chemicals. In this study, Megalobrama amblycephala HSP70 promoter was cloned, and characteristic heat shock elements (HSEs) were identified in the promoter region. The recombinant M. amblycephala Hsp70 protein (rMaHsp70) was expressed and purified from Escherichia coli BL21 (DE3). To evaluate in vivo immune response of rMaHsp70, we administered intraperitoneal (IP) injection, and demonstrated that rMaHsp70 stimulated M. amblycephala immune activity by inducing the expression of HSP70, HIF-1α, HSC70, CXCR4b, TNF-α and IL-1β mRNAs in liver, headkidney, spleen and gill, as well as SOD, glutathione, lysozyme and interferon alpha proteins in serum and liver. The effect of rMaHsp70 as adjuvant against Aeromonas hydrophila was assessed by injecting a mixed vaccine of rMaHsp70 and A. hydrophila (A. hydrophila/Hsp70) into M. amblycephala, and the relative percent survival (RPS) in the A. hydrophila/Hsp70 group was 75% compared to 50% in the A. hydrophila/PBS group. Furthermore, rMaHsp70 also promoted the proliferation and suppressed apoptosis in M. amblycephala fin cells (MAF) in a dose-dependent manner. Taken together, these results suggest that rMaHsp70 can induce organic immune response and improve environmental tolerance.

Effects of Acute Hypoxia and Reoxygenation on Physiological and Immune Responses and Redox Balance of Wuchang Bream (Megalobrama amblycephala Yih, 1955)

To study Megalobrama amblycephala adaption to water hypoxia, the changes in physiological levels, innate immune responses, redox balance of M.amblycephala during hypoxia were investigated in the present study. When M. amblycephala were exposed to different dissolved oxygen (DO) including control (DO: 5.5 mg/L) and acute hypoxia (DO: 3.5 and 1.0 mg/L, respectively), hemoglobin (Hb), methemoglobin (MetHb), glucose, Na+, succinatedehydrogenase (SDH), lactate, interferon alpha (IFNα), and lysozyme (LYZ), except hepatic glycogen and albumin gradually increased with the decrease of DO level. When M. amblycephala were exposed to different hypoxia time including 0.5 and 6 h (DO: 3.5 mg/L), and then reoxygenation for 24 h after 6 h hypoxia, Hb, MetHb, glucose, lactate, and IFNα, except Na+, SDH, hepatic glycogen, albumin, and LYZ increased with the extension of hypoxia time, while the above investigated indexes (except albumin, IFNα, and LYZ) decreased after reoxygenation. On the other hand, the liver SOD, CAT, hydrogen peroxide (H2O2), and total ROS were all remained at lower levels under hypoxia stress. Finally, Hif-1α protein in the liver, spleen, and gill were increased with the decrease of oxygen concentration and prolongation of hypoxia time. Interestingly, one Hsp70 isoforms mediated by internal ribozyme entry site (IRES) named junior Hsp70 was only detected in liver, spleen and gill. Taken together, these results suggest that hypoxia affects M. amblycephala physiology and reduces liver oxidative stress. Hypoxia-reoxygenation stimulates M. amblycephala immune parameter expressions, while Hsp70 response to hypoxia is tissue-specific.

The zebrafish miR-125c is induced under hypoxic stress via hypoxia-inducible factor 1α and functions in cellular adaptations and embryogenesis

Hypoxia is a unique environmental stress. Hypoxia inducible factor-lα (HIF-lα) is a major transcriptional regulator of cellular adaptations to hypoxic stress. MicroRNAs (miRNAs) as posttranscriptional gene expression regulators occupy a crucial role in cell survival under low-oxygen environment. Previous evidences suggested that miR-125c is involved in hypoxia adaptation, but its precise biological roles and the regulatory mechanism underlying hypoxic responses remain unknown. The present study showed that zebrafish miR-125c is upregulated by hypoxia in a Hif-lα-mediated manner in vitro and in vivo. Dual-luciferase assay revealed that cdc25a is a novel target of miR-125c. An inverse correlation between miR-125c and cdc25a was further confirmed in vivo, suggesting miR-125c as a crucial physiological inhibitor of cdc25a which responds to cellular hypoxia. Overexpression of miR-125c suppressed cell proliferation, led to cell cycle arrest at the G1 phase in ZF4 cells and induced apoptotic responses during embryo development. More importantly, miR-125c overexpression resulted in severe malformation and reduction of motility during zebrafish embryonic development. Taken together, we conclude that miR-125c plays a pivotal role in cellular adaptations to hypoxic stress at least in part through the Hif-1α/miR-125c/cdc25a signaling and has great impact on zebrafish early embryonic development.Hypoxia is a unique environmental stress. Hypoxia inducible factor-lα (HIF-lα) is a major transcriptional regulator of cellular adaptations to hypoxic stress. MicroRNAs (miRNAs) as posttranscriptional gene expression regulators occupy a crucial role in cell survival under low-oxygen environment. Previous evidences suggested that miR-125c is involved in hypoxia adaptation, but its precise biological roles and the regulatory mechanism underlying hypoxic responses remain unknown. The present study showed that zebrafish miR-125c is upregulated by hypoxia in a Hif-lα-mediated manner in vitro and in vivo. Dual-luciferase assay revealed that cdc25a is a novel target of miR-125c. An inverse correlation between miR-125c and cdc25a was further confirmed in vivo, suggesting miR-125c as a crucial physiological inhibitor of cdc25a which responds to cellular hypoxia. Overexpression of miR-125c suppressed cell proliferation, led to cell cycle arrest at the G1 phase in ZF4 cells and induced apoptotic responses during embryo development. More importantly, miR-125c overexpression resulted in severe malformation and reduction of motility during zebrafish embryonic development. Taken together, we conclude that miR-125c plays a pivotal role in cellular adaptations to hypoxic stress at least in part through the Hif-1α/miR-125c/cdc25a signaling and has great impact on zebrafish early embryonic development.

The molecular characterization, expression pattern and alternative initiation of Megalobrama amblycephala Hif prolyl hydroxylase Phd1

HIF prolyl hydroxylase 1 (PHD1) functions in prolyl hydroxylation on mammal hypoxia-inducible factors (HIF), important transcription factors involved in hypoxia, however the roles of Phd1 in fish remain unclear. In this study, the full-length cDNA and promoter sequences of blunt snout bream (Megalobrama amblycephala) phd1 gene were isolated by a modified RACE strategy. The phd1 cDNA was 2672 bp for encoding 481 amino acid residues. In silico assays indicated that phd1 had 5 exons, and a 348 bp CpG island in the exon1, and several transcription factor binding sites (CAAT box, HRE, ARNT, FOX, etc) were also found on the promoter. The quantitative real-time PCR results suggested that phd1 mRNA was constitutively expressed in all detected tissues, and higher in the blood, brain and heart in normoxia, but significantly decreased after hypoxia in all detected tissues except for gill. Western blot assays indicated that two Phd1 isoforms were generated by alternative translation initiation. Moreover, these two isoforms were both localized in the nucleus, therein only the senior isoform promoted cell proliferation. Taken together, the present study firstly describes the functions of M. amblycephala two Phd1 isoforms in hypoxia and cell proliferation.

Zebrafish let-7b acts downstream of hypoxia-inducible factor-1α to assist in hypoxia-mediated cell proliferation and cell cycle regulation

Aims: Hypoxia‐inducible factor‐1&agr; (HIF‐1&agr;) is a transcriptional regulator of cellular responses to hypoxic stress. MicroRNAs (miRNAs) play an essential role in hypoxia‐mediated cellular responses. Previous studies have identified some let‐7 family members as hypoxia‐regulated miRNAs (HRMs). In the present study, we aimed to investigate whether zebrafish let‐7b/7f contribute cellular hypoxic response in a Hif‐1&agr;‐dependent manner. Main methods: Stable suppression of zebrafish hif‐1&agr; was achieved by microinjection of an optimized short‐hairpin RNA (shRNA) expression vector. Next‐generation sequencing was conducted to characterize miRNA and mRNA expression profiles. MiRNA promoter analysis and target detection was performed by dual‐luciferase assay. Quantitative real‐time PCR (qRT‐PCR) and western blot were used to determine the expression of let‐7b/7f, Hif‐1&agr; and Foxh1. Proliferation of ZF4 cells was examined using Cell Counting Kit‐8 (CCK‐8) and cell cycle progression was analyzed by flow cytometry assay. Key findings: Correlation between 7 miRNAs and 76 putative targets was identified based on integrated analysis of miRNA‐mRNA profiles. Let‐7b and let‐7f were further considered as potential HRMs, with let‐7b further validated as Hif‐1&agr; up‐regulated. In addition, Forkhead‐box H1 (Foxh1) was confirmed as a bona fide downstream target of let‐7b. Furthermore, overexpression of both let‐7b and let‐7f repressed cell proliferation through blocking cell cycle progression of the G1‐S transition. Significance: Our findings for the first time suggest zebrafish let‐7b acts downstream of Hif‐1&agr; to assist in hypoxia‐mediated cell proliferation and cell cycle regulation at least in part through the downregulation of foxh1. We also identified 4 novel potential HIF‐1&agr;‐regulated miRNAs in zebrafish.

Effect of low temperature on globin expression, respiratory metabolic enzyme activities, and gill structure of Litopenaeus vannamei

Low temperature frequently influences growth, development, and even survival of aquatic animals. In the present study, physiological and molecular responses to low temperature in Litopenaeus vannamei were investigated. The cDNA sequences of two oxygen-carrying proteins, cytoglobin (Cygb) and neuroglobin (Ngb), were isolated. Protein structure analysis revealed that both proteins share a globin superfamily domain. Real-time PCR analysis indicated that Cygb and Ngb mRNA levels gradually increased during decrease in temperatures from 25 to 15°C and then decreased at 10°C in muscle, brain, stomach, and heart, except for a continuing increase in gills, whereas they showed a different expression trend in the hepatopancreas. Hemocyanin concentration gradually reduced as the temperature decreased. Moreover, the activities of respiratory metabolic enzymes including lactate dehydrogenase (LDH) and succinate dehydrogenase (SDH) were measured, and it was found that LDH activity gradually increased while SDH activity decreased after low-temperature treatment. Finally, damage to gill structure at low temperature was also observed, and this intensified with further decrease in temperature. Taken together, these results show that low temperature has an adverse influence in L. vannamei, which contributes to systematic understanding of the adaptation mechanisms of shrimp at low temperature.