miR-19b-3p regulated by estrogen controls lipid synthesis through targeting MSMO1 and ELOVL5 in LMH cells.

miR-19b-3p is reported to undertake various biological role, while its function and action mechanism in chicken hepatic lipid metabolism is unclear. Conservation analysis and tissue expression pattern of miR-19b-3p and its target gene were evaluated, respectively. Dual luciferase reporter system and Western blot technologies were adopted to validate miR-19b-3p target gene. Overexpression and knockdown assays were done to explore the biological functions of miR-19b-3p and target gene in Leghorn Male Hepatoma cell line (LMH). Regulatory approaches of estrogen on miR-19b-3p and target gene expressions are analyzed through site-directed mutation combined with estrogen receptors antagonist treatment assays. The results showed that chicken miR-19b-3p mature sequences are highly conserved among Capra hircus, Columba livia, Rattus norvegicus, Mus musculus, Cricetulus griseus, Danio rerio, Danio novaehollandiae, Orycodylus porosus, Crocodylus porosus, Gadus morhua, and widely expressed in lung, ovary, spleen, duodenum, kidney, heart, liver, leg muscle, and pectoral muscle tissues. miR-19b-3p could significantly increase intracellular triglyceride (TG) content and decrease intracellular cholesterol (TC) content via targeting methylsterol monooxygenase 1 (MSMO1) and elongase of very long chain fatty acids 5 (ELOVL5), which are highly conserved among species, in both mRNA and protein levels. Estrogen could inhibit miR-19b-3p expression, but directly promoted MSMO1 transcription via estrogen receptor α (ERα) and indirectly regulated ELOVL5 expression at the transcription level. Meanwhile, estrogen could also upregulate MSMO1 and ELOVL5 expression through inhibiting miR-19b-3p expression at the post-transcription level. Taken together, these results highlight the role and regulatory mechanism of miR-19b-3p in hepatic lipid metabolism in chicken, and might produce useful comparative information for human obesity studies and biomedical research.

CRISPR-Cas9 assisted non-homologous end joining genome editing system of Halomonas bluephagenesis for large DNA fragment deletion.

BACKGROUND: Halophiles possess several unique properties and have broad biotechnological applications including industrial biotechnology production. Halomonas spp., especially Halomonas bluephagenesis, have been engineered to produce various biopolyesters such as polyhydroxyalkanoates (PHA), some proteins, small molecular compounds, organic acids, and has the potential to become a chassis cell for the next-generation of industrial biotechnology (NGIB) owing to its simple culture, fast growth, contamination-resistant, low production cost, and high production value. An efficient genome editing system is the key for its engineering and application. However, the efficiency of the established CRISPR-Cas-homologous recombination (HR) gene editing tool for large DNA fragments was still relatively low. In this study, we firstly report a CRISPR-Cas9 gene editing system combined with a non-homologous end joining (NHEJ) repair system for efficient large DNA fragment deletion in Halomonas bluephagenesis. RESULTS: Three different NHEJ repair systems were selected and functionally identified in Halomonas bluephagenesis TD01. The NHEJ system from M. tuberculosis H37Rv (Mt-NHEJ) can functionally work in H. bluephagenesis TD01, resulting in base deletion of different lengths for different genes and some random base insertions. Factors affecting knockout efficiencies, such as the number and position of sgRNAs on the DNA double-strands, the Cas9 protein promoter, and the interaction between the HR and the NHEJ repair system, were further investigated. Finally, the optimized CRISPR-Cas9-NHEJ editing system was able to delete DNA fragments up to 50 kb rapidly with high efficiency of 31.3%, when three sgRNAs on the Crick/Watson/Watson DNA double-strands and the arabinose-induced promoter Para for Cas9 were used, along with the background expression of the HR repair system. CONCLUSIONS: This was the first report of CRISPR-Cas9 gene editing system combined with a non-homologous end joining (NHEJ) repair system for efficient large DNA fragment deletion in Halomonas spp. These results not only suggest that this editing system is a powerful genome engineering tool for constructing chassis cells in Halomonas, but also extend the application of the NHEJ repair system.

Functional and miRNA regulatory characteristics of INSIG genes highlight the key role of lipid synthesis in the liver of chicken (Gallus gallus).

The insulin-induced genes (INSIG1 and INSIG2) have been demonstrated to play a vital role in regulating lipid metabolism in mammals, however the function and regulation mechanism of them remains unknown in poultry. In this study, firstly the phylogenetic trees of INSIGs among various species were constructed and their subcellular locations were mapped in chicken LMH. Then the spatiotemporal expression profiles, over-expression and knockdown assays of chicken INSIGs were conducted. Furthermore, conservation of potential miRNA binding sites in INSIGs among species were analyzed, and the miRNA biological function and regulatory role were verified. The results showed that chicken INSIGs located in cellular endoplasmic reticulum, and were originated from the common ancestors of their mammalian counterparts. The INSIGs were widely expressed in all detected tissues, and their expression levels in the liver of chicken at 30 wk were significantly higher than that at 20 wk (P < 0.01). Over-expression of INSIGs led no significant increase in mRNA abundance of lipid metabolism-related genes and the contents of triacylglycerol (TG) and cholesterol (TC) in LMH cells. Knockdown of INSIG1 led to the decreased expressions of ACSL1, MTTP-L, ApoB, ApoVLDLII genes and TG, TC contents (P < 0.05). Knockdown of INSIG2 could significantly decrease the contents of TG and TC, and expressions of key genes related to the lipid metabolism (P < 0.05). Moreover, INSIG1 was directly targeted by both miR-130b-3p and miR-218-5p, and INSIG2 was directly targeted by miR-130b-3p. MiR-130b-3p mimic and miR-218-5p mimic treatment could significant decrease the mRNA and protein levels of INSIGs, mRNA levels of genes related to lipid metabolism, and the contents of TG and TC in LMH cells. The inhibition of miR-130b-3p and miR-218-5p on TG and TC contents could be restored by the overexpression of INSIGs, respectively. No significant alteration in expressions of sterol regulatory element binding protein (SREBPs) and SREBP cleavage-activating protein (SCAP) were observed when INSIGs were over-expressed. SCAP was down-regulated when INSIG1 was knocked down, while SREBP1 was down-regulated when INSIG2 was knocked down. Taken together, these results highlight the role of INSIG1 and INSIG2 in lipid metabolism and their regulatory mechanism in chicken.

Genome-Wide Analysis of the FABP Gene Family in Liver of Chicken (Gallus gallus): Identification,Dynamic Expression Profile, and RegulatoryMechanism.

The fatty acid-binding protein (FABP) gene family, which encodes a group of fatty acid-trafficking molecules that affect cellular functions, has been studied extensively in mammals. However, little is known about the gene structure, expression profile, and regulatory mechanism of the gene family in chickens. In the present study, bioinformatics-based methods were used to identify the family members and investigate their evolutionary history and features of gene structure. Real-time PCR combined with in vivo and in vitro experiments were used to examine the spatiotemporal expression pattern, and explore the regulatory mechanism of FABP genes. The results show that nine members of the FABP gene family, which branched into two clusters and shared a conserved FATTYACIDBP domain, exist in the genome of chickens. Of these, seven FABP genes, including FABP1, FABP3-7, and FABP10 were abundantly expressed in the liver of hens. The expression levels of FABP1, FABP3, and FABP10 were significantly increased, FABP5 and FABP7 were significantly decreased, and FABP4 and FABP6 remained unchanged in hens at the peak laying stage in comparison to those at the pre-laying stage. Transcription of FABP1 and FABP3 were activated by estrogen via estrogen receptor (ER) α, whilst FABP10 was activated by estrogen via ERß. Meanwhile, the expression of FABP1 was regulated by peroxisome proliferator activated receptor (PPAR) isoforms, of which tested PPARα and PPARß agonists significantly inhibited the expression of FABP1, while tested PPARγ agonists significantly increased the expression of FABP1, but downregulated it when the concentration of the PPARγ agonist reached 100 nM. The expression of FABP3 was upregulated via tested PPARß and PPARγ agonists, and the expression of FABP7 was selectively promoted via PPARγ. The expression of FABP10 was activated by all of the three tested PPAR agonists, but the expression of FABP4-6 was not affected by any of the PPAR agonists. In conclusion, members of the FABP gene family in chickens shared similar functional domains, gene structures, and evolutionary histories with mammalian species, but exhibited varying expression profiles and regulatory mechanisms. The results provide a valuable resource for better understanding the biological functions of individual FABP genes in chickens.

miR-34a-5p Increases Hepatic Triglycerides and Total Cholesterol Levels by Regulating ACSL1 Protein Expression in Laying Hens.

Accumulating evidence has shown that miR-34a serves as a posttranscriptional regulatory molecule of lipid metabolism in mammals. However, little studies about miR-34a on lipid metabolism in poultry have been reported until now. To gain insight into the biological functions and action mechanisms of miR-34a on hepatic lipid metabolism in poultry, we firstly investigated the expression pattern of miR-34a-5p, a member of miR-34a family, in liver of chicken, and determined its function in hepatocyte lipid metabolism by miR-34a-5p overexpression and inhibition, respectively. We then validated the interaction between miR-34a-5p and its target using dual-luciferase reporter assay, and explored the action mechanism of miR-34a-5p on its target by qPCR and Western blotting. Additionally, we looked into the function of the target gene on hepatocyte lipid metabolism by gain- and loss-of-function experiments. Our results indicated that miR-34a-5p showed a significantly higher expression level in livers in peak-laying hens than that in pre-laying hens. miR-34a-5p could increase the intracellular levels of triglycerides and total cholesterol in hepatocyte. Furthermore, miR-34a-5p functioned by inhibiting the translation of its target gene, long-chain acyl-CoA synthetase 1 (ACSL1), which negatively regulates hepatocyte lipid content. In conclusion, miR-34a-5p could increase intracellular lipid content by reducing the protein level, without influencing mRNA stability of the ACSL1 gene in chickens.