Hypoxia-induced miR-92a regulates p53 signaling pathway and apoptosis by targeting calcium-sensing receptor in genetically improved farmed tilapia (Oreochromis niloticus).

miR-92a miRNAs are immune molecules that regulate apoptosis (programmed cell death) during the immune response. Apoptosis helps to maintain the dynamic balance in tissues of fish under hypoxia stress. The aim of this study was to explore the role and potential mechanisms of miR-92a in the liver of tilapia under hypoxia stress. We first confirmed that CaSR (encoding a calcium-sensing receptor) is a target gene of miR-92a in genetically improved farmed tilapia (GIFT) using luciferase reporter gene assays. In GIFT under hypoxia stress, miR-92a was up-regulated and CaSR was down-regulated in a time-dependent manner. Knocked-down CaSR expression led to inhibited expression of p53, TP53INP1, and caspase-3/8, reduced the proportion of apoptotic hepatocytes, and decreased the activity of calcium ions induced by hypoxia in hepatocytes. GIFT injected in the tail vein with an miR-92a agomir showed up-regulation of miR-92a and down-regulation of CaSR, p53, TP53INP1, and caspase-3/8 genes in the liver, resulting in lower serum aspartate aminotransferase and alanine aminotransferase activities under hypoxia stress. These findings suggest that stimulation of miR-92a interferes with hypoxia-induced apoptosis in hepatocytes of GIFT by targeting CaSR, thereby alleviating liver damage. These results provide new insights into the adaptation mechanisms of GIFT to hypoxia stress.

Introduction

miRNAs are a small class of non-coding RNAs that regulate the expression of one or more target genes by binding to their 3′-untranslated region (UTR). In recent years, the potential application of members of the miR-92 family as immune molecules in aquaculture has received extensive attention [1-4]. Previous studies have shown that the miR-92 family is closely related to the immune response of sea cucumber (Apostichopus japonicus) to vibriosis caused by Vibrio splendidus [1]. That study showed that miR-92 can regulate the host–pathogen interaction in sea cucumber by binding to two candidate genes, one encoding polyepidermal growth factor-like domain 6 and the other encoding SMAD-specific E3 ubiquitin protein ligase. In oyster (Crassostrea gigas), miR-92d regulates the expression of tumor necrosis factor (TNF) by targeting the coding region of CgLITAF3, which encodes lipopolysaccharide-induced TNF-a factor 3, thereby triggering an inflammatory response to invading bacteria [2]. In amphioxus (Branchiostoma belcheri), miR-92d regulates the immune response to bacterial infection by binding to the gene encoding complement C3 [3]. In genetically improved farmed tilapia (GIFT), (Oreochromis niloticus), miR-92d-3p is involved in mediating the expression of complement C3, and inhibition of miR-92d-3p promotes complement C3 expression and enhances the inflammatory response to Streptococcus iniae infection [4].

In a previous study, we successfully constructed miRNA expression libraries from uninfected GIFT and those infected with S. iniae. Gene enrichment pathway analyses indicated that one of the differentially expressed miRNAs, miR-92a, may be involved in cell signal transduction or immune regulation processes in GIFT [5]. CaSR, which encodes a calcium-sensing receptor, may be a potential target gene of miR-92a (free energy, −26.5 Kcal/mol) in GIFT. Recent studies have found that activated CaSR is involved in the regulation of cellular inflammatory responses and apoptosis signaling [6-8]. In mammals, CaSR is a sensitive receptor for Ca2+, and its expression is regulated by the concentration of extracellular Ca2+. As a multifunctional regulator, CaSR participates in G protein signal transduction and GPCRs kinase (GPK)-induced desensitization by altering the intracellular Ca2+ concentration, thereby mediating processes such as cell growth, differentiation, and ion channel opening [9]. To expand on our previous studies, we aimed to confirm the relationship between miR-92a and its target gene CaSR, and determine how this relationship regulates cell stress responses in GIFT. We also aimed to clarify its regulatory pathway and activation mechanism. Addressing these questions will shed light on the molecular mechanisms of stress regulation in fish.

Changes in dissolved oxygen (DO) can affect fish survival. Generally, fish grow and develop normally when the DO level is higher than 4.0 mg/L. Low DO levels (<2.0 mg/L) cause a floating phenomenon in farmed fish. If the DO level drops below 1.0 mg/L (hypoxic conditions), most fish will severely float their heads and eventually suffocate to death [10]. There are many causes of fish death under hypoxia stress, including an imbalance of apoptosis [11]. Apoptosis is the orderly death of cells controlled by genes to maintain the stability of the internal environment. Apoptosis and the immune response involve proteins including p53, p53-inducible nuclear protein 2 (TP53INP2), caspase-3, and caspase-8. Among them, the p53 signaling pathway is considered to be a key factor in the regulation of apoptosis, because it directly or indirectly induces multiple regulatory genes to interfere with apoptosis in fish [12]. As a downstream gene of p53, TP53INP encodes a protein that coactivates transcription factors such as p53 in the nucleus to regulate apoptosis and the expression of cell cycle-related genes [13]. Apoptosis-associated caspases are widely expressed in various fish exposed to hypoxia, and their activity leads to impairment of liver metabolism and calcium homeostasis [14, 15]. The expression levels of apoptosis-related genes (caspase-3/8/9) were found to be low in the gill and liver of healthy largemouth bass (Micropterus salmoides) but increased after exposure to hypoxia [16, 17]. Similarly, Atlantic croaker (Micropogonias undulatus) under hypoxia stress showed increased caspase-3/7 activity and elevated expression of the proapoptotic genes Bax and p53 in the ovaries, and the apoptotic pathway was activated [18].

Tilapia is one of the most important freshwater farmed fish in China. High temperatures or deterioration of water quality can cause a sharp drop in DO levels in aquaculture ponds, leading to fish death [19]. Therefore, the main objectives of this study were as follows: 1) to verify that CaSR is a potential target gene of miR-92a; 2) to determine the effect of CaSR knockdown on the Ca2+ concentration and apoptosis pathway in GIFT hepatocytes; and 3) to explore how miR-92a expression is activated in GIFT under hypoxia stress. The results of this study provide new information about stress regulation and adaptation mechanisms in fish.

Materials and methods

Ethics statement

The study protocols and design were approved by the Ethics Committee at the Freshwater Fisheries Research Center of the Chinese Academy of Fishery Sciences (Wuxi, China). The Ethics approval number for this experiment is 2019–028. The GIFT were maintained in well-aerated water and treated with 200 mg/L tricaine methane sulfonate (Sigma, St Louis, MO, USA) for rapid deep anesthesia. The samples were extracted based on the Guide for the Care and Use of Laboratory Animals in China.

Analysis of regulatory relationship between miRNA and its target genes

We randomly assigned 180 healthy GIFT (average fish weight: 18.4 g ± 0.8 g) to nine experimental groups (20 fish/group). There were three treatment groups: an miR-92a agomir group; an miR-92a negative agomir group; and a control group treated with phosphate-buffered saline (PBS). Each group had three replicates of 20 fish. The chemically synthesized miRNA agomir (5′-UUGCACUUGUCCCGGCCUGU-3′) and miRNA negative agomir (5′-UU: with a six mismatch mutation in its complementary bases) were designed and synthesized by the Ruibo Biological Technology Co., Ltd. (Guangzhou, China). The miRNA negative agomir was administered by tail vein injection at a dose of 25 mg/kg body weight. The same dose of negative agomir or PBS was administered to fish in the negative agomir or PBS groups, respectively, in the same way. At 0 h, 12 h, 24 h, and 48 h post-injection, three fish were randomly selected from each tank and deeply anesthetized before dissecting liver tissues. The liver samples were snap frozen in liquid nitrogen, and then stored at −80°C until analyses of gene expression and protein levels.

Identification of binding sites of miR-92a-CaSR 3′ UTR

The full-length CaSR 3′ UTR sequence (XM_025910710.1) of GIFT was synthesized and then inserted into the pGL3-control vector, yielding 3′ UTR wild-type. In the seed sequence region where miRNA binds to its target gene, the six complementary bases (AAUUGU) of CaSR 3'-UTR were replaced with GGCAAG to construct the 3'UTR mutant. The procedures for HEK293T cell culture, vector transfection, and luciferase activity determination were as described previously [18]. We used renilla luciferase activity to standardize the luciferase activity of each transfected cell. As the miRNA negative control (NC), we selected the miRNA of Caenorhabditis elegans (5′-UUUGUACUACACAAAAGUACUG-3′), which has minimal homology with all miRNAs in the miRBase database (http://www.mirbase.org/). There were four treatment groups in this experiment: CaSR 3′ UTR-wild type + miRNA mimic; CaSR 3′ UTR-Mutant + miRNA mimic; miRNA NC (Ctrl mimic) + CaSR 3′ UTR-wild type; Ctrl mimic + CaSR 3′ UTR-Mutant. Each treatment had eight replicates. Luciferase activity was detected to determine the binding efficiency of miRNA to its target gene.

Regulatory response of GIFT hepatocytes under hypoxia stress after knocking down expression of CaSR

We constructed an RNA interference (RNAi) expression vector to knock down the expression of CaSR in GIFT. First, according to the GIFT CaSR gene sequence, we designed an RNAi sequence (5′-GAGAGCACAGATGACTGGTGATATT-3′) with no homology to other coding sequences in tilapia. The construction of the positive interference plasmid (CaSR knockdown), the negative control interference plasmid (NC), and the control plasmid (Con) were as described in Qiang et al. (2020). Hepatocytes were cultured, isolated, and purified as described by Chen et al. (2011). This experiment had four experimental groups of cells: three groups were subjected to a hypoxia treatment (CaSR knockdown group, NC group, and Con group), and one group, the normal group (NG), was not subjected to a hypoxia treatment. Each treatment had eight replicates. The primary cultured hepatocytes were grown to about 80%–90%, and then transfected using Lipofectamine 2000. The three hypoxia-treated cell groups were placed in an YQX-II anoxic incubator (JTONE, Hangzhou, China) (27°C, N2 93%, O2 2%, CO2 5%) for 24 h to impose hypoxia stress. The cell lysate was treated for 30 min, and then centrifuged (4°C, 12000 g, 20 min). The supernatant was collected for further analyses. The hepatocytes in the NG were placed in an Herocell 180 incubator (RADOBIO, Shanghai, China) and cultured under normoxic conditions (27°C, O2 20%, CO2 5%) without transfection and hypoxia stress.

miR-92a-mediated stress response in GIFT liver after hypoxia exposure

The experimental design was based on that of Qiang et al. [20]. In total, 360 GIFT juveniles (average size, 26.8 g ± 0.9 g) were randomly assigned to nine 400-L tanks (40 fish/tank). The synthesis, dissolution, and injection of the miR-92a agomir (5′-UUGCACUUGUCCCGGCCUGU-3′) and miRNA NC (5′-UUUGUACUACACAAAAGUACUG-3′) were as described in the section “Analysis of the regulatory relationship between miRNA and its target genes”. The control group was similarly injected with PBS. The experimental GIFT at 12 h post-injection were subjected to low-DO (1.0 mg/L) stress for 96 h. The DO level in water was determined using an LDO101 probe (Hach, Loveland, CO, USA). Nitrogen and air charges were used to regulate and maintain the DO level in water. Three fish were randomly selected from each group at each sampling time point, and blood was drawn and liver tissues were dissected quickly. The liver samples were snap-frozen in liquid nitrogen, and then stored at −80°C until analyses of mRNA levels. Blood samples were left at 4°C for 2 h and then centrifuged (4°C, 3500 g, 10 min) to collect the serum. The serum was kept at −80°C until analysis.

Measurement indices

mRNA expression

Total RNA extraction, reverse transcription (RT), and quantitative real-time PCR (qRT-PCR) were conducted as described elsewhere [21]. The qRT-PCR analyses were conducted using the ABI QuantStudio 5 Real-Time PCR System (Foster City, CA, USA) with gene-specific primers (see Table 1). The endogenous control gene was 18S rRNA.

Table 1

Sequences of primers used for qRT-PCR.

Name	Primer sequence (5'-3')
CaSR	F: 5'-AAAATCTATGATGCTTGTGGCTCC-3'R: 3'-ATTGCCATGCAAGGGGAG-5'
TP53INP2	F: 5’- TACGCGCAGCAATGTTTCAG-3’R: 5’- GGCTCCCTCAGGCAGATTGA-3’
p53	F: 5’-TTTTCTCCTCCCTGTTCGTGG-3’R: 5’- CGGGAACCTCATGCTTCACT-3’
Caspase-3	F: 5’- GAAACGAACAGCAGCAGACC-3’R: 5’- CGAGTGCTCATCCCTGTTGT-3’
Caspase-8	F: 5'- ACAGACAAAGGGCTCGTCAG-3'R: 5'- GAAGACCACGCACAAACCAC-3'
18S rRNA	F: 5'-GGCCGTTCTTAGTTGGTGGA-3'R: 5'-TTGCTCAATCTCGTGTGGCT-3'
miR-92a	5'- AAGCGACCTATTGCACTTGTCC-3'

miRNA expression

The RT reaction and qRT-PCR of the miRNA were as described elsewhere [21]. The primers for miR-92a are shown in Table 1. The reference gene was U6.

Western blot analysis

Each liver tissue sample (0.1 g) was ground in liquid nitrogen with a mortar and pestle, then 1 mL ristocetin-induced platelet agglutination buffer (containing 1% v/v 10 mg/ml phenylmethanesulfonyl fluoride) was added and the mixture was homogenized (4°C, 15,000 g, 1 min) using a Polytron (PT2500E, Kinematica, Lucerne, Switzerland). The procedures for SDS-PAGE preparation, protein sample electrophoresis, membrane transfer, blocking, and antibody incubation were as described by Qiang et al. [20]. Color was developed using Immobilon Western HRP substrate (Millipore, Billerica, MA, USA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was the reference protein.

Identification of apoptotic cells and determination of Ca2+ concentration

The culture, collection, and apoptotic cell detection of hepatocytes were conducted as described by Qiang et al. [21]. Hepatocytes were collected and resuspended in pre-cooled PBS, and then the Ca2+-fluorescent probe Fluo-3AM was added to a final concentration of 5 μg/mL. The mixture was incubated in the dark for 40 min at room temperature and then passed through a cell sieve (40 μm mesh size). The Ca2+ concentration was detected using the Incyte module of a FACSCalibur flow cytometer with excitation at 506 nm and emission at 526 nm.

Measurement of activities of aspartate aminotransferase and alanine aminotransferase in serum

The activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in serum were determined using kits obtained from the Jiancheng Bio-Engineering Institute (Nanjing, China), according to the manufacturer’s instructions. Absorbance at the wavelengths specified in the kit manuals was determined using a BioTek Eon Microplate Spectrophotometer (BioTek, Winooski, VT, USA).

Data processing and statistical analysis

The relative expression levels of mRNA and miRNA were calculated using the 2-ΔΔCT method. Data from all experiments were statistically analyzed using SPSS 21.0 software. Shapiro–Wilk’s test was used to test the normal distribution of data (α = 0.1). Levene’s test was used to determine the homogeneity of variance (α = 0.1). The experimental data shown in figures and tables are mean ± standard deviation. When the experimental data met the tests for normality and homogeneity of variance, we performed analysis of variance and selected appropriate statistical methods for comparisons among treatment groups. Independent samples t test was used to compare different treatment times within the same experimental group; Duncan’s multiple comparison was used to compare different treatment groups at the same time. Differences were considered significant at P<0.05. Data that were not normally distributed were log-transformed before statistical analysis.

Results

miR-92a binds to CaSR 3'-UTR to regulate mRNA expression

To analyze the regulatory relationship between miR-92a and its target gene, we injected the miR-92a agomir (negative agomir or PBS (control) into the tail vein of GIFT). The levels of hepatic CaSR mRNA were significantly lower in GIFT treated with the miRNA agomir than in the control group at 12, 24, and 48 h after injection (Fig 1A, P < 0.05). However, at 12, 24, and 48 h after injection, the levels of miR-92a were significantly higher in the miRNA agomir group than in the control group and the negative agomir group (Fig 1B, P < 0.05). The CaSR protein level was significantly lower in the miRNA agomir group (Fig 1C and S1–S3 Figs, P < 0.05) than in the control group at 24 h and 48 h after injection (Fig 1D). There was no significant difference in the levels of CaSR mRNA and miR-92a between the control group and the negative agomir group at each time point (Fig 1A and 1B, P > 0.05). These results showed that up-regulation of miR-92a expression led to down-regulation of CaSR.

Fig 1

Analysis of regulatory relationship between CaSR (A) and miR-92a (B) in vivo. Juvenile GIFT weighing 18.4 g ± 0.8 g were injected in tail vein with miR-92a agomir (dose, 25 mg/kg body weight), miR-92a negative agomir, or PBS (control). (A, C, and D) qRT-PCR (upper panel) and western blot (lower panel) analyses of CaSR expression in GIFT after injection with miR-92a agomir. Bands of interest were cropped from the same membrane. (B) qRT-PCR analysis of miR-92a expression in GIFT after injection with miR-92a agomir. Based on relative levels in control group at 0 h, relative expression levels of mRNA and miRNA in each experimental group were determined by 2-ΔΔCT method. * Indicates significant difference between pre- and post- injection in the same experimental group (Independent samples t test; P < 0.05). Different lowercase letters indicate significant differences among different treatments at each sampling point (Duncan’s multiple range test; P < 0.05).

We generated an miR-92a mimic to target the 3′ end of CaSR, and then tested its efficacy by conducting dual luciferase reporter assays using HEK293T cells. Luciferase activity in the CaSR 3′ UTR wild type + control mimic group (Ctrl group, no mimic) was 1.12 ± 0.03, while that in the UTR wild type + miR-92a mimic group was decreased by 48.47% (Fig 2A) (statistical significance; t = 2.357, P = 0.023). To confirm that binding between the CaSR 3′ UTR and the miR-92a sequence was essential for this activity, we constructed a mutated CaSR 3′ UTR sequence (3′ UTR Mut). The luciferase activity levels in the 3′ UTR Mut + Ctrl mimic group or the miR-92a mimic group were 0.96-times and 0.87-times that in the control group, respectively; these differences were not significant (P > 0.05). These results confirmed that GIFT miR-92a binds to the 3′ UTR of CaSR mRNA. Although the 5′ end of the miR-92a sequence showed only partial complementarity in a 2–8 bp region, a compensation site at the 3′ end enhanced its binding stability to the 3′-UTR of the target CaSR mRNA (Fig 2B).

Fig 2

Verification of binding between miRNA and its potential target gene.

(A) Validation of miRNA binding to 3′ UTR of potential target gene using dual luciferase reporter system. HEK-293T cells in 12-well plates were co-transformed with pGL-CaSR 3′ UTR (3′ UTR-wild type) or pGL- CaSR (3′ UTR-Mutant) and miRNA mimic (miR mimic) or miRNA negative control (Ctrl mimic) using Lipofectamine 2000 transfection reagent. Luciferase activity was determined based on fluorescence. (B) Analysis of binding site between miR-92a and Oreochromis niloticus CaSR (XM_025910710.1): 5′-end of miR-92a can pair with 3048 bp–3056 bp position at the CaSR 3'-UTR. Compensation binding site at 3′-end of miR-92a enhances its binding stability with 3′-UTR of CaSR. Different lowercase letters indicate significant differences among experimental groups (Duncan’s multiple comparison; P < 0.05).

CaSR knockdown regulates Ca2+ concentration and expression of genes related to cell signaling pathway in GIFT hepatocytes

Next, we determined the effects of CaSR knockdown on the responses of GIFT hepatocytes under hypoxia stress. This experiment consisted of four groups of GIFT hepatocytes: a normal group (NG, no transfection, no hypoxia); a control + hypoxia stress group (Con + hypoxia); a CaSR-negative control transfection + hypoxia stress group (NC + hypoxia); and a CaSR knockdown + hypoxia stress group (Knockdown + hypoxia).

As shown in Fig 3A, compared with the CaSR transcript levels in the Con + hypoxia group and the NC + hypoxia group, that in the Knockdown + hypoxia group was decreased by 60% (P < 0.05). The transcript levels of TP53INP2 (Fig 3B), p53 (Fig 3C), caspase-3 (Fig 3D), and caspase-8 (Fig 3E) were significantly lower in the Knockdown+ hypoxia group than in the Con + hypoxia and NC + hypoxia groups. The percentage of hepatocytes in the early and late stage of apoptosis was 14.62% in the Con + hypoxia group and 14.28% in the NC + hypoxia group (Q4 +Q2 in Fig 4A), significantly higher than in the NG and Knockdown + hypoxia group (Fig 4A). The Ca2+ concentration was significantly lower in the Knockdown + hypoxia group than in the Con + hypoxia and NC + hypoxia groups (Fig 4B), but not significantly different between the NG and the knockdown + hypoxia group (P>0.05).

Fig 3

Effect of CaSR knockdown on transcript levels of CaSR, p53, p53-inducible nuclear protein 2, caspase-3, and caspase-8 in GIFT hepatocytes under hypoxia stress.

(A) Effect of CaSR knockdown on transcript levels of CaSR in GIFT hepatocytes under hypoxia stress. Hepatocytes were transfected with CaSR knockdown, NC, or Con vector using Lipofectamine 2000 and subjected to 24 h of hypoxia stress in an incubator (knockdown+ hypoxia; NC+ hypoxia, and Con+ hypoxia, respectively). Normal group (NG): hepatocytes under normoxic conditions (no transfection, no hypoxia). (B, C, D, and E) Effect of CaSR knockdown on transcript levels of p53, p53-inducible nuclear protein 2, caspase-3 and caspase-8 in GIFT hepatocytes under hypoxia stress. Based on relative levels of respective mRNAs in NG, relative mRNA levels in each group were determined by 2-ΔΔCT method. Different lowercase letters indicate significant differences among experimental groups (Duncan’s multiple range test, P < 0.05).

Fig 4

Effect of CaSR knockdown on apoptosis and Ca2+ concentration in hepatocytes of GIFT under hypoxia stress.

(A) Proportion of apoptotic cells in NG, knockdown+ hypoxia, NC+ hypoxia and Con+ hypoxia groups. Figure shows percentages of dead cells (Q1), late apoptotic cells (Q2), viable cells (Q3), and early apoptotic cells (Q4). (B) Ca2+ fluorescence intensity in NG, knockdown+ hypoxia, NC + hypoxia, and Con + hypoxia groups. Different lowercase letters indicate significant differences among experimental groups (Duncan’s multiple range test, P < 0.05).

miR-92a regulates p53, TP53INP2, caspase-3/8 and serum AST and ALT activities in GIFT under hypoxia stress

To further analyze the role of miRNA-mediated CaSR expression in the GIFT liver in response to hypoxia stress, juvenile GIFT were injected with miR-92a agomir, miRNA NC, or PBS (control) via the tail vein. At 12 h after miRNA agomir injection, the GIFT were subjected to hypoxia stress. At each time point, the level of miR-92a was significantly higher in the agomir group than in the control and the NC groups (Fig 5A, P<0.05). The expression levels of miR-92a in the control and NC groups were significantly lower at 48 h and 72 h of hypoxia stress than before the stress treatment (P<0.05). There was no significant difference in the expression level of miR-92a between the control group and the NC group at each sampling time (Fig 5A, P>0.05). The transcript level of CaSR was significantly lower in the agomir group than in the control group at 12, 24, 48, 72, and 108 h of hypoxia stress (Fig 5B).

Fig 5

Effect of promoting miR-92a expression on transcript levels of miR-92a (A) and CaSR (B) in GIFT liver. Juveniles weighing 26.8 g ± 0.9 g were injected in tail vein (dose, 25 mg/kg body weight) with miRNA negative control, or miR-92a agomir, or PBS (control) and response was monitored for 108 h. Based on relative expression levels in control group at 0 h, relative expression levels of mRNA and miRNA in each experimental group were determined by 2-ΔΔCT method. * Indicates significant differences between pre- and post- injection in the same experimental group (Independent samples t test; P < 0.05). Different lowercase letters indicate significant differences among different treatments at each sampling point (Duncan’s multiple comparison; P < 0.05).

After 24 h of hypoxia stress, the serum AST (Fig 6A) and ALT (Fig 6B) activities were significantly lower in the agomir group than in the control and the NC groups. In all treatment groups, the serum AST and ALT activities tended to increase under hypoxia stress. Serum ALT activity in the agomir group was significantly higher at 24 and 48 h than at 0 h (pre-stress). The transcript levels of p53, TP53INP2, caspase-3, and caspase-8 in the liver tended to increase under hypoxia stress in the control group and the NC group (Fig 7). The transcript level of TP53INP2 was significantly lower in the agomir group than in the control group at 48 and 96 h of hypoxia stress (Fig 7A). The transcript levels of p53 (Fig 7B) and caspase-8 (Fig 7D) were lower in the agomir group than in the control group at 96 h of hypoxia stress. The transcript levels of caspase-3 were significantly lower in the agomir group than in the control group and the NC group at 24, 48, and 96 h of hypoxia stress (Fig 7C).

Fig 6

Effect of promoting miR-92a expression on serum aspartate aminotransferase (A) and alanine aminotransferase (B) activities in GIFT under hypoxia stress. At 12 h after miR-92a agomir injection or PBS (control), juveniles were subjected to hypoxia stress for 96 h. * Indicates significant differences between of pre- and post- injection in the same experimental group (independent samples t test; P < 0.05). Different lowercase letters indicate significant differences among different treatments at each sampling point (Duncan’s multiple comparison; P < 0.05).

Fig 7

Effect of promoting miR-92a expression on transcript levels p53-inducible nuclear protein 2 (A), p53 (B), caspase-3 (C), and caspase-8 (D) in GIFT under hypoxia stress. At 12 h after injection with miR-92a agomir or PBS (control), juveniles were subjected to hypoxia stress for 96 h. Based on relative expression level in control group at 0 h, relative expression levels of mRNAs in each experimental group were determined by 2-ΔΔCT method. * Indicates significant differences between pre- and post- injection in the same experimental group (independent samples t test; P < 0.05). Different lowercase letters indicate significant differences among different treatments at each sampling point (Duncan’s multiple comparison; P < 0.05).

Discussion

Calcium ions are second messengers of signal transduction and can trigger a variety of cellular and physiological responses, such as muscle contraction, exocytosis, neurotransmitter release, cell proliferation, and apoptosis [8, 22]. In this study, the Ca2+ concentration in GIFT hepatocytes increased sharply under hypoxia stress, which may explain the increased proportions of cells in the early and late stages of apoptosis. Previous studies have shown that an increase in the Ca2+ concentration induces expression of pro-inflammatory cytokines, thereby activating caspase-9 in the mitochondrial pathway or caspase-8 in the death receptor pathway, via the apoptosis-inducing effects of caspase-3 [23]. However, in hepatocytes of GIFT with knocked-down CaSR expression, the decreased CaSR expression levels may have prevented the accumulation of Ca2+ under hypoxia stress. This would explain the reduced transcript levels of p53, TP53INP2, caspase-3, and caspase-8 in hepatocytes with knocked-down CaSR expression. Therefore, CaSR knockdown helped to maintain the homeostasis of the Ca2+ concentration inside and outside the cell, and relieve hypoxia stress-induced apoptosis.

Hypoxia-induced apoptosis is an anoxic adaptation mechanism that eliminates stressed cells (Shimizu et al., 1996). Under hypoxia stress, activation of p53 can induce cell necrosis, apoptosis, or autophagy, all of which are involved in cell death [12, 24]. Previous studies have shown that hypoxia stress leads to increased expression of the p53 gene and p53 protein in oriental river prawn (Macrobrachium nipponense) and in the hepatopancreas and hemocytes of Pacific white shrimp (Litopenaeus vannamei), leading to apoptosis [24-26]. Similar results were found in this study. The transcript level of p53 in each experimental group gradually increased under hypoxia stress. Therefore, GIFT may initiate the p53 signaling pathway through the central axis of hypoxia-miR-92a-CaSR under hypoxia stress. In the control group, the down-regulation of miR-92a and up-regulation of CaSR at 48 h and 72 h may have increased cell apoptosis and alleviated stress damage caused by hypoxia. However, activated endogenous expression of miR-92a in GIFT by injection of an miRNA agomir inhibited CaSR mRNA expression levels, thereby regulating the apoptosis response. Therefore, decreased CaSR expression in the agomir group may have helped to reduce the increase in p53 expression and alleviate apoptosis induced by hypoxia stress. As a key regulatory gene in the p53 regulatory network, TP53INP2 plays an important role in the apoptosis and invasion of tumor cells. In previous studies, up-regulation of this gene was shown to induce cell apoptosis of glioma cells, breast cancer cells, and osteosarcoma cells [27, 28]. Up-regulation of p53 may result in a significant up-regulation of TP53INP2, leading to increased hepatocyte apoptosis and liver damage. However, in our study, p53 was significantly down-regulated in the liver of GIFT during prolonged exposure to hypoxia stress. Liu et al. [29] suggested that p53 is a universal receptor for environmental stress. Therefore, down-regulation of p53 and TP53INP2 may be a self-adaptive mechanism under hypoxia stress in aquatic animals. In another study, after 48 h of hypoxia stress, Pacific white shrimp with silenced p53 showed enhanced expression of cyclin-dependent kinase 2 (which is involved in cell cycle progression) and decreased caspase-3 expression, and these changes in expression regulated hepatocyte apoptosis and the cell cycle [30].

In our study, the transcript levels of caspase-3 and caspase-8 increased significantly in the liver tissues of GIFT under hypoxia stress, suggesting that the caspase signaling pathway may play a role in stress adaptation. Caspase signaling under various stress conditions has been observed in other fish species. For example, cadmium stress was found to induce apoptosis of purse red common carp (Cyprinus carpio) hepatocytes, and caspase-3A activity was significantly increased in liver tissues [31]. However, copper stress did not lead to up-regulation of caspase-3 in Nile tilapia, nor did it increase the proportion of apoptotic cells, suggesting that caspase-3-dependent or caspase-independent apoptotic pathways may not exist in this fish [32]. In our study, up-regulation of miR-92a in the agomir group inhibited the expression of the target gene CaSR and reduced the increase in caspase-3 and caspase-8 transcript levels under hypoxia stress. This may have prevented apoptosis of GIFT liver cells under hypoxia stress. In another study, down-regulation of miR-532-5p expression in H9c2 cells exposed to hypoxia led to up-regulation of its target gene encoding programmed cell death protein 4, and increased the expression of caspase-3, thereby promoting hypoxia-induced apoptosis of H9c2 cells [33]. Inhibition of miR-9 up-regulated the gene encoding Yes-associated protein 1, promoted cell proliferation, and inhibited apoptosis and caspase-3/7 activity in hypoxic H9c2 cells [34].

Under normal conditions, AST and ALT in fish are mainly located in the liver, and only small amounts are released into the blood. Thus, serum AST and ALT activities are important indicators of normal liver function [35]. In other studies, increased serum AST and ALT activities in GIFT have been detected under high temperature [36] and crowding [37]. In our study, the GIFT liver may have been damaged by hypoxia stress. An increase in the membrane permeability of liver cells would lead to the release of AST and ALT, resulting in increased serum AST and ALT activities. Inhibition of CaSR in the agomir group may have helped to alleviate hepatocyte damage, thereby reducing serum AST and ALT activities.

Conclusions

This study confirmed the central hypothetic axis of the hypoxia-miR-92a-CaSR-apoptotic phenotype. Up-regulation of miR-92a led to down-regulation of its target gene CaSR in GIFT. Stimulation of miR-92a interfered with hypoxia-induced apoptosis in GIFT hepatocytes by targeting CaSR, and alleviated liver damage. Our study provides novel insights into the adaptation mechanism of GIFT to hypoxia, and suggests that miR-92a might be a target for relieving stress in farmed fish.

Marker map of CaSR protein expression in GIFT.

CaSR protein (66kD) was detected in GIFT liver sample. The procedures for SDS-PAGE preparation, protein sample electrophoresis, membrane transfer, blocking, and antibody incubation were as described by Qiang et al. [20]. Color was developed using Immobilon Western HRP substrate (Millipore, Billerica, MA, USA).

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Click here for additional data file.

Western blot of CaSR expression in GIFT both miR-92a agomir group and PBS group.

CaSR protein (66kD) in miR-92a agomir group (A-D) was gradually weaken after 24 hours but was absent by 48 hours. CaSR protein (66kD) in PBS group (E-H) was used as a control and was present in liver samples. The procedures for SDS-PAGE preparation, protein sample electrophoresis, membrane transfer, blocking, and antibody incubation were as described by Qiang et al. [20]. Color was developed using Immobilon Western HRP substrate (Millipore, Billerica, MA, USA).

(PDF)

Click here for additional data file.

Western blot of GAPDH expression in GIFT both miR-92a agomir group and PBS group.

GAPDH (38 kD) was used as a loading control and was present in GIFT liver samples (A-H). The procedures for SDS-PAGE preparation, protein sample electrophoresis, membrane transfer, blocking, and antibody incubation were as described by Qiang et al. [20]. Color was developed using Immobilon Western HRP substrate (Millipore, Billerica, MA, USA).

(PDF)

Click here for additional data file.

11 Sep 2020

PONE-D-20-25810

Hypoxia-induced miR-92a regulates p53 signalling pathway and apoptosis by targeting calcium-sensing receptor in Genetically Improved Farmed Tilapia (Oreochromis niloticus)

PLOS ONE

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Comments to the Author

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The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: No

**********

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Reviewer #1: Yes

Reviewer #2: No

**********

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Reviewer #1: No

Reviewer #2: No

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5. Review Comments to the Author

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Reviewer #1: General comments.

The author studied hypoxia introduces the liver damage by though which the miR-92a and CaSR-apoptosis axis. Then, the author identifies the usefulness of miR-92a to preventing liver dysfunction in fish. The study is exciting and scientifically sound. The author investigated using a cell culture model following the in vivo assay, which is very good. However, there are several missing sentences or insufficient explanations in the text. Usually, this type of careless miss is not suitable for the judgment of the review process, as such, I may not be able to correctly justify the results and may point out with my miss-understand. Please carefully consider and correct the manuscript.

Major comments,

L68-77: Introduce miR-92a in a tumor, but this study did not focus on the oncogenesis. Do you think this part is necessary? I may assume that the author may want to conduct tumor biology and the apoptosis phenomenon, but the author clearly introduces this issue later in the introduction.

L147: Although the author describes that the Liver sample was frozen in liquid nitrogen within 1 h, I think 1 hour is relatively long periods. I am sorry, but I am not familiar with sample collection from fish, is this a typical method for fish? To my knowledge, especially, the mRNA profile in the sample may change during 1 hour. I assume that there is some time lag for sample correcting and frozen in the first sample and last sample. So this means that the author’s experimental condition may not be suitable for downstream analysis. Is this really 1h? If so, the author needs to explain the situation for sample collecting. In addition, the author also collects the liver sample to describe L172; the description is a little different. Did the author use different procedures in each experiment?

In figure 1, the results show protein expression as western blot band signals. However, there is no description of miR-92a, and there is no indication “Figure 2C, 2D” in the text.

L 247: There is (Figure 3B), is this correct? All of Figure 3 shows similar results, isn’t it?

In Figure 5 and L 255-260: The author did not indicate the change of miR-92a by the time; I mean, the author should explain and describe the decreasing of miR-92a by hypoxic stimulation. I think that this is most important for this study hypothesis. So the central hypothetic axis is hypoxia-miR-92a-CaSR-Apsoptotic phenotype, isn’t it? If I miss understanding, I am sorry. The author also needs to clearly discuss this phenomenon in the discussion as well.

Minor comments.

For reference citation, there are many “Qiang et al., 2017”, so I can not clearly evaluate which is which? Besides, the reference citation style is not correct for PLoS One.

Please add the number of the Ethics approval.

L131: It is better to add the sequence of C. elegans in the text for reproducibility by other researchers if necessary.

L139-140: I think that the author needs to show the sequence of miR-92a agomir and negative agomir in text. The author describes the sequence for RNAi experiments.

L158: “normal” means normoxia condition? The author also needs to show how much O2 tension in the control group? I know 20% in most of the cell culture, but still better to describe.

L168: What is section 2.2?

L184: I think I can not find U6 sequence in Qiang 2020, Front. Physiol. It will be better to add.

L212-214: Please describe the exact methods for comparative statistics. Also, if the data did not meet the normal distribution, the author used what statistics methods?

L257-260: Figure A and B may be better to change order.

L261-265: I think it is better to coordinate the order AST and ALT, the author mixture both in text and figure.

Reviewer #2: miR-92a RNAs are important immune molecules involved in regulating cell apoptosis. The research found that miR-92a mediate the immune mechanism of reducing liver cell damage in genetically improved farmed tilapia by targeting CaSR.

My specific comments are as follows:

Question 1:

Line 51-52: “sea cucumber Apostichopus japonicas”, revise to “sea cucumber (Apostichopus japonicas)”; Line 60: “In genetically improved farmed tilapia (GIFT, Oreochromis niloticus)”, revise to “In genetically improved farmed tilapia (GIFT), (Oreochromis niloticus)”, and unify the format about same question in manuscript.

Question 2:

Line 71: “Bcl-2 interacting mediator of cell death (Bim)”, revise to “ Bcl-2 interacting mediator (Bim) of cell death ”.

Question 3:

Line 85: If there is any research on apoptosis mediated by CaSR, please add in the introduction.

Question 4:

Line 103: Please introduce the research on the effects of DO supplementation on the expression of fish apoptosis and the immune response involve proteins.

Question 5:

Line 105: “Photobacterium damselae ssp. (Reis et al., 2007; 2010). ” , revise to: “ Photobacterium damselae ssp. piscicida (Reis et al., 2007; 2010). ”

And the same question,

Line 497-499: “ Reis, M.I. Costa-Ramos, C., do Vale, Ana., dos Santos, N.M.S., 2010. Molecular cloning of sea bass (Dicentrarchus labrax L.) caspase-8 gene and its involvement in Photobacterium damselae ssp. piscicida triggered apoptosis.”, revise to “… involvement in Photobacterium damselae ssp. piscicida …”.

Question 7:

Line 130: “We used Renilla luciferase activity… ”, revise to “We used renilla luciferase activity”.

Question 8:

Line 132: “in the miRBase database”, please add the URL of web site.

Question 9:

Line 162: “and then centrifuged (12000 g, 20 min, 4 °C)”, revise to “ and then centrifuged (4 °C, 12000 g, 20 min) ”; also,

Line 188: “……was added and the mixture was homogenized (15,000 g, 1 min, 4 °C)”, revise to “……was added and the mixture was homogenized (4 °C, 15,000 g, 1 min)”.

Question 10:

Line 167: “……were as described in section 2.2.”, revise to “……were as described in section “Identification of binding sites of miR-92a-CaSR 3’UTR”.”.

Question 11:

Line 216: Please unify the header format in the results.

Question 12:

Line 229: “To analyse the regulatory relationship between miR-92a and its target gene, we injected the miR-92a agomir. negative control (NC) or PBS (control) into the tail vein of GIFT.”, revise to “To analyse the regulatory relationship between miR-92a and its target gene, we injected the miR-92a agomir (negative control (NC) or PBS (control) into the tail vein of GIFT).”

Question 13:

Line 291: “and white shrimp”, revise to “and Pacific white shrimp”.

Question 14:

Line 374 & Line 417: “western blot”, revise to “ Western blot”.

Question 15:

Figures: Please note the significant difference in the figure and who has the difference compared with whom, unify the format of each figure.

Question 16:

According to the journal request, unify the format of reference in the text.

**********

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Reviewer #1: No

Reviewer #2: Yes: Qinghua Zhang

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

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20 Oct 2020

Detailed responses to comments from the editors and reviewers are provided below. Changes are shown in the TrackedCopy manuscript.

Editors:

Comment 1: statistical analysis and language may need a checkup by specialists Response: We have again revised the language and added details of the statistical analyses.

Comment 2: all figures (i.e. 2C,D) should be referred to in the main text

Response: We have ensured that all figures are referred to in the text.

Comment 3: Please ensure that your manuscript meets PLOS ONE's style requirements Response: We have revised the manuscript to ensure that it meets the style requirements of PLoS ONE.

Comment 4: We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form

Response: We have revised funding information from the paper.

Comment 5: PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files

Response: We have supplied the original result in the Supporting Information file, and revised the format and file name. Please see S1_raw_images.

Reviewer 1:

Comment 1: L68-77: Introduce miR-92a in a tumor, but this study did not focus on the oncogenesis. Do you think this part is necessary? I may assume that the author may want to conduct tumor biology and the apoptosis phenomenon, but the author clearly introduces this issue later in the introduction.

Response: We agree with this point, we have rearranged the sentences in the introduction and removed unnecessary information. (Line 68)

Comment 2: L147: Although the author describes that the Liver sample was frozen in liquid nitrogen within 1 h, I think 1 hour is relatively long periods. I am sorry, but I am not familiar with sample collection from fish, is this a typical method for fish? To my knowledge, especially, the mRNA profile in the sample may change during 1 hour. I assume that there is some time lag for sample correcting and frozen in the first sample and last sample. So this means that the author’s experimental condition may not be suitable for downstream analysis. Is this really 1h? If so, the author needs to explain the situation for sample collecting. In addition, the author also collects the liver sample to describe L172; the description is a little different. Did the author use different procedures in each experiment?

Response: We have revised the incorrect information in our manuscript. During the sampling process, liver was rapidly excised, snap frozen in liquid nitrogen, and then stored at −80 °C until later use [Lines 124-125]. For the whole experiment, sampling was completed within 1 h. After extracting RNA, we further analyzed its integrity (see figure 1 below) and the OD value (OD 260/280 nm), the OD value was 1.9–2.1. We used a Nanodrop 2000 instrument to measure the RNA concentration (800–1000 ng/μL), and the quality of the RNA was confirmed to be sufficient for subsequent analysis. The liver sample collection process described in L172 is the same as that in L147. In our original manuscript, we deleted some of our descriptions to avoid repeating information. We have revised the descriptions of our methods to be more accurate in the revised manuscript [Lines 168-169].

Figure 1. Analysis of completeness of RNA extraction from some samples.

Comment 3: In figure 1, the results show protein expression as western blot band signals. However, there is no description of miR-92a, and there is no indication “Figure 2C, 2D” in the text.

Response: We have added a description of miR-92a in the text of the Results section [Lines 223-225; 227-228]. We have replaced the order of Fig 1 and Fig 2 according to the recommendations of reviewer. Figure 1C and 1D were shown in Line 225-227.

Comment 4: L 247: There is (Figure 3B), is this correct? All of Figure 3 shows similar results, isn’t it?

Response: We have carefully checked the results and figures and confirmed that the results are correct. In this experiment, the normal group (NG, no transfection, no hypoxia) served as the control; that is, the expression level of each gene was set to 1 in the NG samples. The figure shows that hypoxia stress significantly upregulated CaSR (Figure 3A), TP53INP2 (Figure 3B), p53 (Figure 3C), caspase-3 (Figure 3D), and caspase-8 (Figure 3E) in GIFT hepatocytes. However, these genes were not upregulated under hypoxia stress in the CaSR-knockdown line. The experimental results indicated that CaSR may regulate the p53 signaling pathway to interfere with apoptosis in GIFT under hypoxia stress. We also analyzed other genes in related pathways and apoptosis genes in our experiments. Some showed no significant differences or differences that could not be explained. The genes discussed in this paper showed significant differences in their transcript levels among the different treatment groups and/or between hypoxia stress and normal conditions. Thus, these genes can better explain the regulatory mechanism of the hypoxia-miR-92a-CaSR-apoptotic phenotype.

Comment 5: In Figure 5 and L 255-260: The author did not indicate the change of miR-92a by the time; I mean, the author should explain and describe the decreasing of miR-92a by hypoxic stimulation. I think that this is most important for this study hypothesis. So the central hypothetic axis is hypoxia-miR-92a-CaSR-Apoptotic phenotype, isn’t it? If I miss understanding, I am sorry. The author also needs to clearly discuss this phenomenon in the discussion as well.

Response: The reviewer is correct, the central hypothetic axis of this study is the hypoxia-miR-92a-CaSR-apoptotic phenotype. We have added a detailed description in the results [Lines 305-309] and discussion sections [Lines 368-372; 394-397].

Comment 6: For reference citation, there are many “Qiang et al., 2017”, so I can not clearly evaluate which is which? Besides, the reference citation style is not correct for PLoS One.

Response: We have revised the format of citations and references in the paper according to the PLoS ONE style.

Comment 7: Please add the number of the Ethics approval.

Response: The registration number of this experiment is 2019-028. We have added this information to the revised manuscript. [Line 109]

Comment 8: L131: It is better to add the sequence of C. elegans in the text for reproducibility by other researchers if necessary.

Response: We added the sequence. [Lines 133-134]

Comment 9: L139-140: I think that the author needs to show the sequence of miR-92a agomir and negative agomir in text. The author describes the sequence for RNAi experiments.

Response: We have added the sequences of the miR-92a agomir and negative agomir. [Lines 117–119]

Comment 10: L158: “normal” means normoxia condition? The author also needs to show how much O2 tension in the control group? I know 20% in most of the cell culture, but still better to describe.

Response: Yes, normal conditions were normoxic conditions. We have added this description to the text. In the experiment, our normally cultured hepatocytes were placed in an Herocell 180 incubator (RADOBIO, Shanghai, China) under normoxic conditions (27 °C, O2 20%, CO2 5%).

Comment 11: L168: What is section 2.2?

Response: We have revised the incorrect description [Lines 162-163].

Comment 12: L184: I think I can not find U6 sequence in Qiang 2020, Front. Physiol. It will be better to add.

Response: The U6 gene that we used was from the Takara miRNA SYBR Green qRT-PCR kit and the sequence information is confidential to the company.

Comment 13: L212-214: Please describe the exact methods for comparative statistics. Also, if the data did not meet the normal distribution, the author used what statistics methods?

Response: We have described the statistical analyses in more detail. [Lines 213–216]

Comment 14: L257-260: Figure A and B may be better to change order.

Response: We have revised the order of Figures A and B, and modified the description in the results section accordingly.

Comment 15: L261-265: I think it is better to coordinate the order AST and ALT, the author mixture both in text and figure.

Response: We now describe AST and ALT in the same order in the text as in the figure.

Reviewer 2

Comment 1: Line 51-52: “sea cucumber Apostichopus japonicas”, revise to “sea cucumber (Apostichopus japonicas)”; Line 60: “In genetically improved farmed tilapia (GIFT, Oreochromis niloticus)”, revise to “In genetically improved farmed tilapia (GIFT), (Oreochromis niloticus)”, and unify the format about same question in manuscript.

Response: We have standardized the style of common and species names in the revised manuscript [Lines 54; 62].

Comment 2: Line 71: “Bcl-2 interacting mediator of cell death (Bim)”, revise to “ Bcl-2 interacting mediator (Bim) of cell death ”.

Response: We deleted this part during the revision of our manuscript.

Comment 3: Line 85: If there is any research on apoptosis mediated by CaSR, please add in the introduction.

Response: We have added information about how CaSR regulates cellular inflammatory responses and apoptosis signaling, as well as supporting references. [Lines 69 -71]

Comment 4: Line 103: Please introduce the research on the effects of DO supplementation on the expression of fish apoptosis and the immune response involve proteins.

Response: We have added more information about the effect of hypoxia stress on fish apoptosis and the immune response, supported by references 12–18. [Lines 88-96]

Comment 5: Line 105: “Photobacterium damselae ssp. (Reis et al., 2007; 2010). ” , revise to: “ Photobacterium damselae ssp. piscicida (Reis et al., 2007; 2010). ”

And the same question,

Line 497-499: “ Reis, M.I. Costa-Ramos, C., do Vale, Ana., dos Santos, N.M.S., 2010. Molecular cloning of sea bass (Dicentrarchus labrax L.) caspase-8 gene and its involvement in Photobacterium damselae ssp. piscicida triggered apoptosis.”, revise to “… involvement in Photobacterium damselae ssp. piscicida …”.

Response: We removed these references during the revision of the manuscript. In the revised manuscript, we have focused on the impact of hypoxia stress on apoptosis in fish.

Comment 6: Line 130: “We used Renilla luciferase activity… ”, revise to “We used renilla luciferase activity”.

Response: Corrected [Line 132].

Comment 7: Line 132: “in the miRBase database”, please add the URL of web site.

Response: We have added the URL of the website [Line 135].

Comment 8: Line 162: “and then centrifuged (12000 g, 20 min, 4 °C)”, revise to “ and then centrifuged (4 °C, 12000 g, 20 min) ”; also,

Line 188: “……was added and the mixture was homogenized (15,000 g, 1 min, 4 °C)”, revise to “……was added and the mixture was homogenized (4 °C, 15,000 g, 1 min)”.

Response: We revised the description as you suggested [Lines 154; 186-187].

Comment 9: Line 167: “……were as described in section 2.2.”, revise to “……were as described in section “Identification of binding sites of miR-92a-CaSR 3’UTR”.”

Response: We have revised the text as you suggested [Lines 162-163].

Comment 10: Please unify the header format in the results.

Response: We have used a standard header format in the revised manuscript.

Comment 11: Line 229: “To analyse the regulatory relationship between miR-92a and its target gene, we injected the miR-92a agomir. negative control (NC) or PBS (control) into the tail vein of GIFT.”, revise to “To analyse the regulatory relationship between miR-92a and its target gene, we injected the miR-92a agomir (negative control (NC) or PBS (control) into the tail vein of GIFT).”

Response: We have revised the text as you suggested [Lines 220-221].

Comment 12: Line 291: “and white shrimp”, revise to “and Pacific white shrimp”.

Response: Corrected [Line 266].

Comment 13: Line 374 & Line 417: “western blot”, revise to “ Western blot”.

Response: The correct spelling of ‘western blot’ is with a lowercase letter. (Only Southern blot is spelled with an uppercase letter, because it is named after a person.)

Comment 14: Figures: Please note the significant difference in the figure and who has the difference compared with whom, unify the format of each figure.

Response: In the figures, * indicates significant difference between pre- and post- injection in the same experimental group (Independent samples t test; P < 0.05), and different lowercase letters indicate significant differences among different treatments at each sampling point (Duncan’s multiple range test; P < 0.05).

This information is provided in each figure legend.

Comment 15: According to the journal request, unify the format of reference in the text.

Response: We have revised the references in the manuscript.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

28 Oct 2020

Hypoxia-induced miR-92a regulates p53 signalling pathway and apoptosis by targeting calcium-sensing receptor in Genetically Improved Farmed Tilapia (Oreochromis niloticus)

PONE-D-20-25810R1

Dear Dr. Qiang,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Klaus Roemer

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

4 Nov 2020

PONE-D-20-25810R1

Hypoxia-induced miR-92a regulates p53 signaling pathway and apoptosis by targeting calcium-sensing receptor in Genetically Improved Farmed Tilapia (Oreochromis niloticus)

Dear Dr. Qiang:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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