miR-34a Regulates the Activity of HIF-1a and P53 Signaling Pathways by Promoting GLUT1 in Genetically Improved Farmed Tilapia (GIFT, Oreochromis niloticus) Under Hypoxia Stress

Abstract

In fish under hypoxia stress, homeostasis can become imbalanced, leading to tissue and organ damage and decreased survival. Therefore, it is useful to explore the molecular and physiological regulation mechanisms that function in fish under hypoxia stress. The microRNA miR-34a is involved in fat and glycogen metabolism, and in apoptosis. In this study, we first verified that GLUT1, the gene encoding glucose transporter 1, is a potential target gene of miR-34a in genetically improved farmed tilapia (GIFT, Oreochromis niloticus) by dual luciferase reporter assays. Then, we clarified the regulatory relationship between miR-34a and GLUT1 by qRT-PCR analyses. We analyzed the regulatory effects of knockdown or promotion of GLUT1 expression in vitro and in vivo in GIFT under hypoxia stress. The results confirm that GLUT1 is a target gene of miR-34a in GIFT. Down-regulation of miR-34a significantly promoted GLUT1 expression. Knockdown of GLUT1 reduced the glycogen content in GIFT liver cells, inhibited HIF-1a gene expression, up-regulated the expression of genes involved in P53 signaling pathways (P53 and CASPASE-3 genes), and accelerated hepatocyte apoptosis under hypoxia stress. Compared with the control group, the group injected in the tail vein with miR-34a antagomir showed up-regulated expression of GLUT1 in the liver, increased liver glycogen content at 96 h of hypoxia stress, down-regulated expression of P53 and CASPASE-3, and decreased serum aspartate aminotransferase and alanine aminotransferase enzyme activities. Our results provide information about the molecular regulation mechanism of miRNAs and their target genes in fish during the response to hypoxia stress.

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

“…First, according to the GIFT CaSR gene sequence, we designed an RNAi sequence (5 0 -GAGAGCACAGATGACTGGTGATATT-3 0 ) 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).…”

“…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).…”

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

“…First, according to the GIFT CaSR gene sequence, we designed an RNAi (5′-GAGAGCACAGATGACTGGTGATATT-3′) sequence with no homology to other coding sequences of 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).…”

“…Total RNA extraction, reverse transcription (RT), and quantitative real-time PCR (qRT-PCR) were as described elsewhere (Qiang et al, 2020). qRT-PCR (see Table 1 for primers) analyses were conducted using the ABI QuantStudio 5 Real-Time PCR System (Foster City, CA, USA), with 18S rRNA as the endogenous control (reference gene).…”

“…The culture, collection, and apoptotic cell detection of hepatocytes were as described by Qiang et al (2020). Hepatocytes were collected and resuspended in pre-cooled PBS, and then the Ca 2+fluorescent probe Fluo-3AM was added to a final concentration of 5 μg/mL.…”