miR-375 upregulates lipid metabolism and inhibits cell proliferation involved in chicken fatty liver formation and inheritance via targeting recombination signal binding protein for immunoglobulin kappa J region (RBPJ)

Abstract

Poultry is susceptible to fatty liver which lead to decrease egg production and increase mortality. But the potential molecular mechanisms remain largely unclear. In the current study, in combination with transcriptome sequencing and miRNA sequencing data analysis from F1 generation of the normal liver and fatty liver tissues, the differentially expressed miR-375 and its target gene RBPJ were screened and verified. The expression levels of miR-375 and RBPJ gene in the liver between control and fatty liver groups of F0-F3 generation for Jingxing-Huang (JXH) chicken are different significantly (P < 0.05 or P < 0.01). And downregulated RBPJ expression can promote TG content and lipid droplets in primary hepatocytes cultured in vitro (P < 0.01). Cell proliferation-related genes, including PMP22, IGF-1, IGF-2, and IGFBP-5, increased or decreased significantly after overexpression or knock-down RBPJ (P < 0.05 or P < 0.01), respectively. This study uniquely revealed that miR-375 induced lipid synthesis and inhibited cell proliferation may partly due to regulation of RBPJ expression, thereby involving in fatty liver formation and inheritance in chicken. The results could be useful in identifying candidate genes and revealing the pathogenesis of fatty liver that may be used for disease-resistance selective breeding in chicken.

Keywords: RBPJ; chicken; fatty liver; lipid metabolism; miR-375.

Figure 1

The histogram of candidate target genes involved KEGG pathway. Histogram of candidate target genes involved KEGG pathway, where the vertical axis represents pathway name, the horizontal axis represents the number of candidate target genes in this pathway.

Figure 2

The histogram of GO enrichment of candidate target genes. The histogram of GO enrichment of candidate target genes. The horizontal axis is GO term of the next level in the three major categories of GO, and the vertical axis is the number of candidate target genes annotated to this term (including sub-terms of this term) and the proportion of the number of candidate target genes in the total number of annotated candidate target genes. The three different classifications represent the three basic classifications of GO term (from left to right, cell components, biological processes, and molecular functions).

Figure 3

Targeting validation of miR-375 and RBPJ. Targeting validation of miR-375 and RBPJ. (A) miR-375 and RBPJ targeted binding sites predicted by software TargetScan (http://www.targetscan.org/vert_72/). (B) Dual luciferase reporter assay results for gga-miR-375+NC, the gga-miR-375+wild-type RBPJ and gga-miR-375+ mutant RBPJ groups. ^⁎⁎ indicates P ≤ 0.01. Abbreviation: RBPJ, recombination signal binding protein for immunoglobulin kappa J region.

Figure 4

Expression level of genes in different groups of chicken primary hepatocytes. Expression level of genes in different groups of chicken primary hepatocytes. In (A and B), qRT-PCR was used to indicate the expression of miR-375 and RBPJ in miR-375 mimic NC, miR-375 mimic, miR-375 inhibitor NC and miR-375 expression levels in the inhibitor group. * Indicates P ≤ 0.05, ^⁎⁎ indicates P ≤ 0.01 and ^⁎⁎⁎ indicates P ≤ 0.001. Abbreviation: RBPJ, recombination signal binding protein for immunoglobulin kappa J region.

Figure 5

Expression level of miR-375 and RBPJ in normal liver and fatty liver from F0 to F3 generation. Expression level of miR-375 and RBPJ in normal liver and fatty liver groups from F0 to F3 generation. (A and B) represent expression levels of miR-375 and RBPJ, respectively, in control groups and fatty liver groups from F0 to F3 generation. * Indicates P ≤ 0.05, ^⁎⁎ indicates P ≤ 0.01 and ^⁎⁎⁎ indicates P ≤ 0.001. Abbreviation: RBPJ, recombination signal binding protein for immunoglobulin kappa J region.

Figure 6

Gene expression levels, TG content and Oil red O staining in different groups. Gene expression levels, TG content and Oil red O staining in different groups. (A and B) represent RBPJ expression levels and TG content in siRBPJ NC, siRBPJ, pcDNA3.1 (+) -GFP and pcDNA3.1 (+) -RBPJ groups. (C) represents TG content in miR-375 mimic NC, miR-375 mimic, miR-375 inhibitor NC, miR-375 inhibitor group. (D) shows lipid droplets in different transfected groups, a represents siRBPJ NC group, b represents siRBPJ group, c represents pcDNA3.1 (+) -GFP group, d represents pcDNA3.1 (+) -RBPJ group, e represents miR-375 mimic NC group, f represents miR-375 mimic group, g represents miR-375 inhibitor NC group, h represents miR-375 inhibitor group. * Indicates P ≤ 0.05 and ^⁎⁎ indicates P ≤ 0.01.

Figure 7

Cell proliferation related genes detection in primary hepatocytes cultured in vitro. Relative expression analysis results of cell proliferation related genes in different groups. (A–D) represent relative expression analysis results of PMP22, IGF-1, IGF-2, and IGFBP-5 in siRBPJ NC, siRBPJ, pcDNA3.1 (+) -GFP and pcDNA3.1 (+) -RBPJ groups, respectively. * Indicates P ≤ 0.05 and ^⁎⁎ indicates P ≤ 0.01. Abbreviations: PMP22, peripheral myelin protein 22; IGF-1, insulin-like growth factor-1; IGF-2, insulin-like growth factor-2; IGFBP-5, insulin-like growth factor binding protein-5.