The MicroRNAs-Transcription Factors-mRNA Regulatory Network Plays an Important Role in Resistance to Cold Stress in the Pearl Gentian Grouper

Abstract

Pearl gentian grouper (Epinephelus fuscoguttatus ♀ × E. lanceolatus ♂) is a hybrid fish with high commercial value. It is widely cultured on the Asian coast; however, it is not cold-tolerant. Although we have previously characterized the liver transcriptomic responses of this grouper to cold stress, the roles of miRNAs and transcription factors (TFs) in cold resistance and the underlying regulatory mechanisms are still unclear. In this study, we integrated miRNA and mRNA sequencing data for pearl gentian grouper under cold stress and constructed a miRNA-TF-mRNA regulatory network. Furthermore, we screened seven key miRNAs (i.e., gmo-miR-221-5p, ssa-miR-7132b-5p, ola-let-7c, ssa-miR-25-3-5p, ccr-miR-489, gmo-miR-10545-5p, ccr-miR-122) that regulated target genes (including TF ACSS2, TF PPARD, TF PPP4CB; CYP2J2, EHHADH, RXRs, NR1D2, PPP1CC-A, PPP2R1A, FOXK2, etc.). These miRNAs participated in several important pathways and biological processes by the direct or indirect regulation of target genes, such as antioxidation and membrane fluidity, glucose and lipid metabolism, circadian rhythm, DNA repair, and apoptosis. The key cold-related miRNAs, TFs, and genes and their potential regulatory relationships identified in this study provide a deeper understanding of the complex molecular basis of the response to low-temperature environments in the grouper. In particular, our results provide the first identification for the role of NR1D2 gene in the cold tolerance of fish via the regulation of circadian rhythm. Furthermore, the key miRNAs and genes provide a basis for the molecular breeding of new cold-tolerant varieties of the pearl gentian grouper.

Sea Urchins in Acute High Temperature and Low Oxygen Environments: The Regulatory Role of microRNAs in Response to Environmental Stress

“…We hypothesized that when S. intermedius is damaged by environmental stresses, the expression of genes related to apoptosis and immunity is significant. Our results support the view that miRNA-target gene interactions are complex, and a single miRNA can regulate multiple target genes simultaneously, and a gene can be regulated by multiple miRNAs simultaneously (Agarwal et al 2015;Lan et al, 2016;Lai et al, 2022;Miao et al, 2022). The mechanism of the S. intermedius response to environmental stress is also complex.…”

Transcriptomic Analysis Reveals Circadian Rhythm Homeostasis in Pearl Gentian Grouper under Acute Hypoxia

“…Therefore, the above three miRNAs co-regulated nr1d1, thereby inhibiting the transcriptional expression of bmal1 and facilitating circadian rhythm stability and normal physiological metabolism under hypoxic conditions in pearl gentian grouper. In the previous cold stress transcriptome analysis, we detected increased nr1d2 gene expression via ssa-miR-25-3-5p, and this gene participated in the maintenance of the circadian rhythm balance and lipid homeostasis to enhance cold tolerance [32]. As a homologue of nr1d1, nr1d2 is also considered to be involved in the regulation of the circadian rhythm, energy metabolism, cellular autophagy, and other biological processes [68,69].…”

“…PCR systems for genes and miRNAs were configured using the PerfectStart ® Green qPCR SuperMix Kit (TransGen Biotech, Beijing, China) and the miRNA Fluorescence Quantitative PCR Kit (Sangon Biotech, Shanghai, China), respectively. The selected reference gene for RT-qPCR was 18S rRNA [32]. Triplicate experiments were conducted for each sample, and a negative reaction without a template was set as the control.…”

“…Smadja Storz et al [62] found that the rhythmic expression of per and arylalkylamine N-acetyltransferase 2 (aanat2) in ze-brafish disappears with the inhibition of CK1δ kinase activity, with effects on the behavioral rhythm. Miao et al [32] reported that ccr-miR-489 in the liver of pearl gentian grouper shortens the circadian cycle by regulating the transcriptional expression of ppp1cc-a and participating in the phosphorylation of PER under cold stress. The csnk1db gene regulates the activity and stability of circadian clock elements and determines the length of the circadian cycle by the autophosphorylation of CK1 kinase or in collaboration with other genes [63].…”

“…Therefore, hypoxia would cause circadian rhythm cycle disturbances in fish, thus affecting the rhythm of their behavioral activities and physiological metabolism. In a transcriptomic analysis of the response to cold stress in the pearl gentian grouper, we found that nuclear receptor subfamily 1 group d member 2 (nr1d2) and protein phosphatase 1 catalytic subunit gamma (ppp1cc-a) were up-regulated via ssa-miR-25-3-5p and ccr-miR-489, respectively, and contributed to the regulation of the circadian rhythm core loop to prevent cold shock and enhance cold tolerance [32]. However, the effect of hypoxia on the circadian rhythm regulation of this grouper has not been explored.…”