miR-27b-5p regulates chicken liver disease via targeting lRS2 to suppress the Pl3K/AKT signal pathway

ZHAO Jing, WU Ya-mei, ZHANG Yao, TANG Shu-yue, HAN Shun-shun, CUl Can, TAN Bo, YU Jie, KANG Hou-yang, CHEN Guang-deng, MA Meng-gen, ZHU Qing, YlN Hua-dong

1 Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, Sichuan Agricultural University,Chengdu 611130, P.R.China

2 College of Forestry, Sichuan Agricultural University, Chengdu 611130, P.R.China

3 Key Laboratory for Animal Disease Resistance Nutrition of China, Institute of Animal Nutrition, Sichuan Agricultural University,Chengdu 611130, P.R.China

4 Triticeae Research Institute, Sichuan Agricultural University, Chengdu 611130, P.R.China

5 College of Resources, Sichuan Agricultural University, Chengdu 611130, P.R.China

Abstract The liver is a vital organ in chickens that performs a number of crucial physiological functions, including the storage of hepatic glycogen, protein synthesis, detoxification, and deoxidation.The growth and metabolism of the liver are complex processes influenced by factors such as environment, diet, and genetics.MicroRNAs (miRNAs), as posttranscriptional regulatory molecules, play a role in various biological processes.There is growing evidence that miR-27b-5p plays a key role in the regulation of liver development and metabolism in various species.However, its role in chicken livers has yet to be determined.In our experiment, we found that chickens with fatty livers had significantly higher levels of serum triglyceride (TG) and total cholesterol (TC) compared to the normal chickens, while the control group had significantly higher levels of very low-density lipoprotein (VLDL) and serum hormones.Further research showed that the mRNA of miR-27b-5p was highly expressed in fatty livers.By exploring the function of miR-27b-5p in chicken livers, we discovered that it promotes lipogenesis, oxidative stress, and inflammatory responses, leading to hepatocyte apoptosis.Our study also established the mechanism by which miR-27b-5p interacts with its target gene, and found that miR-27b-5p targets insulin receptor substrate 2 (IRS2) and modulates the PI3K/AKT signaling pathway.Additionally, our investigation of IRS2 in chicken hepatocytes revealed that knocking down IRS2 has the same effects as overexpressing miR-27b-5p.In conclusion, our study revealed that miR-27b-5p directly binds to IRS2, inhibiting the PI3K/AKT signaling pathway and causing steatosis, oxidative stress, inflammation, and apoptosis in chicken liver.

Keywords: miR-27b-5p, IRS2, lipogenesis, oxidative stress, inflammation, apoptosis

1.lntroduction

The liver is one of the most critical organs in chickens and is responsible for synthesizing more than 90% of the new fat in the body (Bensadoun and Rothfeld 1972).The bulk of synthesized fat is assembled into very lowdensity lipoprotein (VLDL) and released into the blood circulation, where it transports to the yolk and influences egg formation in chicken (Caiet al.2009).However, the rate of fat oxidation and utilization in the liver is lower than the rate of fat synthesis, resulting in a significant accumulation of lipid droplet in the hepatocytes, which can lead to fatty liver disease (Day and James 1998).This disease is an unavoidable problem in modern intensive poultry production and can cause lipid metabolic disorders, liver rupture, blood clots, and negatively impact production performance (Trottet al.2014; Zhanget al.2018).The most important cause of fatty liver disease is the accumulation of large amounts of fat in the liver,which is accompanied by the occurrence of liver oxidative stress, cellular inflammation and apoptosis (Cherian and Goeger 2004; Yilmaz and Younossi 2014).

The formation of fatty liver is a complex and tightly regulated biological process, and numerous functional genes and transcription factors have been shown to play a role in its regulation.Non-coding RNAs, particularly microRNAs (miRNAs), have been found to be critical for liver metabolism and development.MiRNAs are a class of small non-coding RNA molecules, composed of 18–25 nucleotides, that exert their functions by interfering with target messenger RNA (mRNA).MiRNAs play a crucial role in many fundamental biological processes,including metabolism, proliferation, differentiation,apoptosis, and carcinogenesis (Giordano and Columbano 2013).Recently, miRNAs have been widely reported to participate in the pathogenesis of fatty liver disease,and for example, miR-27b-5p has been extensively studied and found to regulate fat metabolism and liver development in mice and humans (Vickerset al.2013;Teimouriet al.2020).However, its potential role in the chicken liver is still unknown.Hence, the main aim of this study was to investigate whether miR-27b-5p could directly regulate chicken fatty liver.

2.Materials and methods

2.1.Animals and sample gathering

A total of 100 (200-day-old) Tianfu broiler breeder hens were obtained from the poultry breeding farm at Sichuan Agricultural University in Ya’an (Sichuan, China).The hens were divided randomly into two groups: a control group and an experimental group.The experimental group had free access to a diet that met their nutritional requirements, as detailed in Appendix A, while the control group was given 120 g of feed per day.At 300 days of age, three chickens from each group were selected at random and had blood collected from their sub-wing vein.After euthanasia, the liver, breast muscle, leg muscle, spleen, intestine, brain,lung, kidney, and abdominal fat tissues were collected and stored at –80°C.In addition, egg production was measured every day from day 200 to 300.

2.2.ELlSA

The serum was separated from the blood, and the levels of follicle-stimulating hormone (FSH), luteinizing hormone(LH), and estradiol (E2) in the serum were then evaluated using enzyme-linked immunosorbent assay (ELISA)following the manufacturer’s guidance (Newgeorge,Shanghai, China).The supernatant was collected from chicken hepatocytes culture medium after exogenous miRNA treatment for 48 h.The concentrations of interleukin 1 beta (IL-1β) and interleukin 6 (IL-6) were also tested with the special ELISA kits (Newgeorge).

2.3.Liver histomorphology (HE) and Oil Red O stain

The liver sample was fixed with 4% paraformaldehyde(Beyotime, Shanghai, China) and embedded in paraffin wax.The slices were stained with Hematoxylin-Eosin and Oil Red, and the stained images were captured randomly using an electronic microscope (Olympus, Tokyo, Japan).

2.4.Chicken hepatocyte isolation and culture

The chicken hepatocytes were isolated from the 200-dayold broilers as described previously (Fraslinet al.1992;Tachibanaet al.2002).Briefly, the chickens were starved for 3 h, then anesthetized with 4% chloral hydrate (50 mg kg–1) and treated with heparin (1 750 U kg–1) as an anticoagulant.The abdominal cavity was opened and the entire liver was collected.The liver was perfused for 15 min with 1 000 mL of free-calcium HEPES buffer(pH 7.5), then washed with a buffer containing calcium chloride for 15 min.The cleaned portion of the liver was fragmented and digested with type II collagenase(Biofroxx, Einhausen, Germany).The cell suspensions were then filtered through cell strainers with 40 and 70 μm pore sizes (Biologix, Shandong, China).The resulting hepatocytes were collected and cultured in M199 medium(Gibco, Grand Island, NY, USA) with 1% penicillinstreptomycin (Solarbio, Beijing, China) and 10% fetal bovine serum (Gibco).The hepatocytes were grown in a 5% CO2-humid environment at 37°C and the culture medium was refreshed every 24 h.

2.5.Synthesis of small interfering RNAs and plasmids

The inhibitor of miR-27b-5p and negative control(NC), as well as the mimics of miR-27b-5p and NC,were constructed by GenePharma (Shanghai, China).Additionally, three siRNAs and siRNA NC (Si-NC) for chickenIRS2were also designed and synthesized by GenePharma.The sequences are outlined in Appendix B.Furthermore, the pmirGLO dual luciferase reporter vector ofIRS2, pmirGLO-IRS2-wild type (WT) and pmirGLO-IRS2-mutated type (MT) were constructed and synthesized by Sangon Biotech (Shanghai, China).

2.6.Cells treatment and transfection

Oil acid (OA, Sigma, St.Louis, MO, USA) was diluted with dimethyl sulfoxide (DMSO, Solarbio), then 0.6 mmol L–1OA was added to treat the cells.After 24 h of treatment,the cells were transfected with siRNA or mimics.When the hepatocytes reached 60–70% confluence, they were transfected with miR-27b-5p inhibitor and inhibitor NC,miR-27b-5p mimics and mimics NC, or Si-IRS2 and Si-NC using Lipofectamine 3000 and Opti-MEM?medium, as described in a previous report by Zhaoet al.(2022).The hepatocytes were collected for follow-up experiments at a designated time after transfection, as needed for the experiment.

2.7.RNA extraction, cDNA synthesis and qPCR

Total RNA was isolated from tissue and hepatocytes using TRIzol Reagent (Invitrogen, Carlsbad, USA).Subsequently, the One Step miRNA cDNA Synthesis Kit(HaiGene, Harbin, China) was used for miRNA cDNA synthesis, and the PrimeScript? RT Reagent Kit (TaKaRa Biotechnology, Tokyo, Japan) was used for mRNA cDNA synthesis.The synthesized cDNA was subjected to qPCR according to a previous report (Zhaoet al.2022).The primers used for qPCR are listed in Appendix C.U6(for miRNA) andβ-actin(for mRNA) were used as internal reference genes.Each sample was repeated three times, and the qPCR data was calculated using the 2–ΔΔCtmethod.

2.8.Western blot assay

After 48 h of transfection, the hepatocytes were collected and total protein was extracted.The protein concentrations were measured, and after standardization,the proteins were separated using SDS-PAGE.The protein bands were then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore Corporation,Billerica, USA), and then they were incubated overnight at 4°C with specific primary antibodies (listed in Appendix D).Afterwards, the PVDF membranes were incubated with the specific secondary antibody and protein bands were detected using enhanced chemiluminescence (ECL)luminous fluid (Beyotime).The relative protein levels were obtained using Image J Software with β-Tubulin as the reference gene.

2.9.Lipid assay

For BODIPY 493/503 staining of lipid droplets, the hepatocytes were fixed with 4% paraformaldehyde(Beyotime) for 30 min at room temperature.Afterward,the cells were treated with BODIPY staining solution(Maokangbio, Shanghai, China; diluted in PBS at 1 μg mL–1) for 15 minutes to label the lipid droplets.Hoechst 33342 (RiboBio, Guangzhou, China) was then used to stain the cell nuclei for 5 min.The images were then randomly captured.

The levels of serum triglyceride (TG), total cholesterol(TC) and VLDL in the different treatment groups were measured using commercial biochemistry kits (Jiancheng Bioengineering Institute, Nanjing, China), following the manufacturer's guidelines.

2.10.Determination of oxidative stress parameters

The superoxide dismutase (SOD), total antioxidant capacity (T-AOC), glutathione (GSH) and methane dicarboxylic aldehyde (MDA) assay kits were purchased from Jiancheng Biology Institute.The testing procedures were performed according to the manufacturer’s instructions.

2.11.Flow cytometric analysis

For measuring cell apoptosis, the transfected hepatocytes were collected and suspended with 500 μL of 1× binding buffer.The cell apoptosis was then measured using the Annexin V-FITC Apoptosis Detection Kit (Beyotime), and the data was analyzed using CytExpert.Additionally, the Reactive Oxygen Species (ROS) Assay Kit (Beyotime)was used to determine the intracellular ROS levels by flow cytometry.

2.12.miRNA target prediction and dual-luciferase reporter assay

The prediction of miR-27b-5p target genes was performed using TargetScan (http://www.targetscan.org/) and miRDB(http://mirdb.org/).The DF-1 cells were then plated in 48-well plates and co-transfected with pmirGLO-IRS2-WT or pmirGLO-IRS2-MT and either miR-27b-5p mimics or mimics NC.After a 48-h transfection period, the luminescent values of the Firefly and Renilla luciferase were evaluated using the Dual-GLO Luciferase Assay System Kits (Beyotime) on a Fluorescence/Multi-Detection Microplate Reader (Biotek, Winooski, VT, USA).

2.13.Statistical analysis

All data were analyzed using SPSS 19.0 Statistics Software (SPSS, Chicago, Illinois, USA).The results were represented by the mean±standard error of the mean(SEM).Each treatment had at least three replicates.The significance between two groups was determined using an unpaired Student’st-test.For multiple groups, a oneway ANOVA method was used, and the significant level was set at＊(P<0.05),＊＊(P<0.01), and the different letters(P<0.05).

3.Results

3.1.Comparison of the reproductive performance and phenotypic traits between chickens with control liver and fatty liver

The egg production of two groups from day 200 to 300 showed that there was a significant decline in the laying rate of chickens with fatty liver compared to the controls(Fig.1-A).Additionally, chickens with fatty liver disease had a considerably larger liver-to-body weight ratio(P<0.01; Appendix E).The serum TG and TC levels in chickens with fatty liver were significantly higher than those in normal chickens (P<0.01; Fig.1-B and C), but the content of VLDL and serum hormones (FSH, LH,and E2) were significantly higher in the controls (P<0.01;Fig.1-D and E).The phenotypic traits of normal livers and fatty livers showed that the fatty livers appeared yellow in color and swollen.In addition, Oil Red O staining results indicated that abundant lipid droplets were accumulated in the hepatocytes of the fatty liver, while the HE staining showed that the hepatocytes of the fatty liver had a paler color, loose cytoplasm, and local infiltration of inflammatory cells (Fig.1-F).

3.2.The expression pattern of miR-27b-5p in chickens

The mRNA levels of miR-27b-5p in both the control liver and the fatty liver showed that miR-27b-5p was significantly more highly expressed in the fatty liver (P<0.01; Fig.2-A).The expression of miR-27b-5p was also found to be significantly higher in OA-treated hepatocytes compared to the control (P<0.01; Fig.2-B).The results of miR-27b-5p expression measurement in different chicken tissues showed that miR-27b-5p was highly expressed in muscle and also enriched in the liver (P<0.05; Fig.2-C).To determine the effect of miR-27b-5p on chicken liver, the expression of miR-27b-5p was examined after chicken hepatocytes were transfected with miR-27b-5p inhibitor,inhibitor NC, miR-27b-5p mimics, and mimics NC,respectively.The results showed that compared to the inhibitor NC transfection group, the miR-27b-5p inhibitor significantly decreased the expression of miR-27b-5p(P<0.01), meanwhile, the mimics miR-27b-5p dramatically enhanced the miR-27b-5p abundance (P<0.01; Fig.2-D).

3.3.MiR-27b-5p accelerates lipogenesis in hepatocytes

To analyze the regulatory function of miR-27b-5p in lipid metabolism in chicken hepatocytes, the mRNA expression levels of three lipid metabolism-related genes (peroxisome proliferator-activated receptor alpha (PPARα), fatty acid synthase (FASN), and acetyl-CoA carboxylase alpha(ACACA) were measured using qPCR.The results showed that treatment with the miR-27b-5p inhibitor significantly reduced the mRNA levels of these genes,while exogenous miR-27b-5p increased their mRNA expressions significantly (P<0.05; Fig.3-A and B).Lipid droplets are considered a marker of lipogenesis (Youet al.2013), so the activity of lipogenesis was determined using BODIPY 493/503 staining.The results indicated that decreased expression of miR-27b-5p significantly reduced lipid deposition in hepatocytes, while overexpression of miR-27b-5p induced cellular lipogenesis (P<0.05;Fig.3-C, D, and E).Additionally, exogenous miR-27b-5p significantly elevated the concentrations of TG and TC in cells, while interference with miR-27b-5p expression had the opposite effect (P<0.01; Fig.3-F and G).These findings suggest that miR-27b-5p promotes the accumulation of lipid droplets and disrupts the balance of fat metabolism in chicken hepatocytes.

Fig.1 Comparison of the reproductive performance and phenotypic traits between chickens with normal livers and fatty livers.A, egg production in chickens with normal livers (NL) and fatty livers (FL).n=50.B–D, serum triglyceride (TG), total cholesterol(TC), and very low-density lipoprotein (VLDL) levels in chickens with NL and FL.n=9.E, serum follicle-stimulating hormone (FSH),luteinizing hormone (LH), and estradiol (E2) levels in chickens with NL and FL.n=9.F, morphological characteristics of the NL and FL in chicken.The red arrow means lipid droplets, and the blue arrow means inflammatory cells.n=3.Values represent mean±SEM.＊, P<0.05; ＊＊, P<0.01.

Fig.2 The expression pattern of miR-27b-5p in chickens.A, the expression of miR-27b-5p in chickens with NL and FL.n=9.B,the expression of miR-27b-5p in the control hepatocytes (NC) and OA-treated hepatocytes of chicken.n=9.C, the expression of miR-27b-5p in different tissues of chicken.n=9.D, the expression level of miR-27b-5p after transfected with inhibitor miR-27b-5p and mimics miR-27b-5p in chicken hepatocytes.n=9.Values represent mean±SEM.Different letters mean P<0.05; ＊, P<0.05;＊＊, P<0.01.

3.4.MiR-27b-5p induces oxidative stress in hepatocytes

Fig.4 miR-27b-5p induces oxidative stress in hepatocytes.A and B, ROS levels in hepatocytes after knockdown of miR-27b-5p.n=3.C, anti-oxidative stress-related genes mRNA level in hepatocytes after transfected with inhibitor miR-27b-5p.n=9.SOD,superoxide dismutase; TRX, thioredoxin; GST, glutathione S-transferase alpha 3.D and E, ROS levels in hepatocytes after overexpression of miR-27b-5p.n=3.F, anti-oxidative stress-related genes mRNA level in hepatocytes after transfected with mimics miR-27b-5p.n=9.G, intracellular MDA level in hepatocytes after knockdown and overexpression of miR-27b-5p.n=3.H–J, the antioxidant enzyme activity in hepatocytes after knockdown and overexpression of miR-27b-5p.n=9.Values represent mean±SEM.＊, P<0.05; ＊＊, P<0.01.

To evaluate the role of miR-27b-5p in oxidative stress,the level of ROS was measured in hepatocytes.The results showed that interference with miR-27b-5p significantly decreased the level of ROS (P<0.05; Fig.4-A and B), while overexpression of miR-27b-5p significantly increased it (P<0.05; Fig.4-D and E).The qPCR results showed that anti-oxidative stress-related genes, such asSODand glutathioneS-transferase alpha 3 (GST) were significantly elevated in the miR-27b-5p interference group (P<0.05; Fig.4-C).However, the gene expression levels of these genes were lower in the miR-27b-5p overexpression group than in the normal group (P<0.05;Fig.4-F).MDA is a toxic end product of lipid peroxidation that can lead to cell structure and function damage, the level of which was therefore tested as an indicator of antioxidant capacity (Choudhariet al.2014).The results showed that knockdown of miR-27b-5p significantly decreased MDA accumulation in hepatocytes, while miR-27b-5p overexpression had the opposite effect (P<0.01;Fig.4-G).Additionally, overexpression of miR-27b-5p decreased the levels of antioxidant enzymes such as T-AOC, GSH, and SOD, while interference with miR-27b-5p significantly increased them (P<0.05; Fig.4-H, I,and J).These results suggest that miR-27b-5p induces oxidative damage and hinders antioxidant defense in chicken hepatocytes.

3.5.MiR-27b-5p aggravates hepatocellular inflammation in chicken

As shown in Fig.5-A, the mRNA levels of interleukin 1 beta(IL-1β), interleukin 6 (IL-6), interleukin 12 beta (IL-12β),interferon-gamma (IFN-γ), and tumor necrosis factor alphalike (TNF-α) were significantly downregulated after treatment with the miR-27b-5p inhibitor (P<0.01).In contrast, the expressions of inflammation-related genes in the miR-27b-5p overexpressed group were significantly higher than those in the control group (P<0.05; Fig.5-B).Western blot results also revealed that knockdown of miR-27b-5p significantly reduced the relative protein levels ofIL-6(P<0.05; Fig.5-C),while its protein abundance increased after miR-27b-5p overexpression (P<0.01; Fig.5-D).Additionally, the content of IL-1β and IL-6 was found to be significantly decreased in the miR-27b-5p inhibitor transfected group (P<0.01; Fig.5-E and F), but was markedly increased in the overexpression group (P<0.01; Fig.5-E and F).These results suggest that miR-27b-5p exacerbates the hepatocellular inflammatory response.

Fig.5 miR-27b-5p aggravates hepatocellular inflammation in chickens.A and B, the mRNA expression of inflammation-related genes after transfected with inhibitor and mimics miR-27b-5p in hepatocytes.n=9.C and D, the protein levels of IL-6 in hepatocytes after knockdown and overexpression of miR-27b-5p.n=3.E and F, the levels of IL-1β and IL-6 in the cell supernatant of hepatocytes after knockdown or overexpression of miR-27b-5p.n=9.Values represent mean±SEM.＊, P<0.05; ＊＊, P<0.01.

3.6.MiR-27b-5p induces hepatocytes apoptosis

In order to examine the role of miR-27b-5p in cell apoptosis, we evaluated the expression of apoptotic genes using qPCR and Western blot in transfected chicken hepatocytes.The qPCR results showed that knockdown of miR-27b-5p significantly reduced the relative mRNA levels ofCaspase3,Caspase8, andCaspase9(P<0.05; Fig.6-A), and similar results were seen in protein expression levels (P<0.05; Fig.6-C and D).Conversely, the opposite effects were observed in the miR-27b-5p mimic treatment (P<0.05; Fig.6-B, E, and F).Furthermore, flow cytometric analysis confirmed that cell apoptosis was noticeably reduced following decreased miR-27b-5p expression (P<0.05; Fig.6-G and H), but was significantly increased with miR-27b-5p overexpression(P<0.05; Fig.6-I and J).These results demonstrate that miR-27b-5p positively regulates chicken hepatocyte apoptosis.

3.7.MiR-27b-5p targets the IRS2 gene

To study the regulatory mechanism of miR-27b-5p, we analyzed the miRNA sequences from various species,including human (hsa), chicken (gga), rat (rno), mouse(mmu), opossum (mdo), and rhesus (mml), and the results showed strong similarity among different species (Fig.7-A).We then screened the target genes of miR-27b-5p using two websites, miRDB and TargetScan.A total of 69 target genes were predicted by both websites (Fig.7-B).One of these genes,IRS2, has been reported to be highly expressed in the liver and involved in liver fat metabolism(Thironeet al.2006; Ecksteinet al.2017).To validate this prediction, we performed qPCR experiments and found that the mRNA abundance ofIRS2was upregulated after knockdown of miR-27b-5p and downregulated after overexpression of miR-27b-5p (P<0.01; Fig.7-D).We also constructed pmirGLO-IRS2-WT and pmirGLO-IRS2-MT vectors (Fig.7-C) and found that the relative luciferase activity of pmirGLO-IRS2-WT was downregulated by miR-27b-5p, while the activity of pmirGLO-IRS2-MT remained unchanged (P<0.01; Fig.7-E).These results suggest that miR-27b-5p is a highly conserved miRNA and thatIRS2is a target gene of miR-27b-5p.

Fig.7 miR-27b-5p targets the IRS2 gene.A, omparison of miR-27b-5p seed sequences of Gallus gallus (gga), Homo sapiens(hsa), Mus musculus (mmu), Rattus norvegicus (rno), Monodelphis domestica (mdo), and Macaca mulatta (mml).B, the target genes of miR-27b-5p.C, construction of pmirGLO-IRS2-wild type (WT) and pmirGLO-IRS2-mutated type (MT) dual-luciferase reporter genes.Red was used to label the seed sequences; the mutant sequences were marked in green.D, the mRNA level of IRS2 in hepatocytes after transfected with inhibitor miR-27b-5p and mimics miR-27b-5p.n=9.E, the dual-luciferase reporter analysis was implemented after co-transfected with pmirGLO-IRS2-WT or pmirGLO-IRS2-MT and mimics miR-27b-5p or mimics NC in DF-1 cells.n=3.Values represent mean±SEM.＊, P<0.05; ＊＊, P<0.01.

3.8.IRS2 alleviates lipogenesis, oxidative stress, inflammation and apoptosis in chicken hepatocytes

Next, we investigated the effects ofIRS2on the development and metabolism of chicken liver by altering its expression in hepatocytes (P<0.01; Appendix F).To evaluate its role in lipogenesis, we performed qPCR and BODIPY 493/503 staining.The results showed that the knockdown ofIRS2significantly upregulated the expression of three genes related to lipid metabolism(P<0.05; Fig.8-A), increased the intracellular content of TG and TC (P<0.01; Fig.8-B and C), and accelerated the accumulation of intracellular lipid droplets (P<0.05;Fig.8-D and E).These data reveal thatIRS2suppresses lipogenesis in chicken hepatocytes.

Furthermore, we observed that the content of ROS increased in the Si-IRS2 treatment group (P<0.05;Fig.8-F and G), and this resulted in a significant decrease in the mRNA level of anti-oxidative stress-related genes(P<0.05; Fig.8-I).The MDA content also increased(P<0.01; Fig.8-H) and the activities of antioxidant enzymes decreased afterIRS2interference (P<0.05;Fig.8-J, K, and L).These results suggest thatIRS2acts in opposition to miR-27b-5p by relieving oxidative stress in chicken livers

Additionally, we discovered that the mRNA expression ofIL-6,IL-1β,IL-12βandTNF-αwas significantly increased (P<0.05; Fig.9-A), and the protein level of IL-6 was also elevated followingIRS2interference (P<0.01;Fig.9-B).Similarly, the content of IL-6 and IL-1β in the Si-IRS2-transfected group was higher compared to the normal control group (P<0.01; Fig.9-C and D).These findings indicate thatIRS2has an inhibiting effect on hepatocellular inflammation in chickens.

Finally, the mRNA levels ofCaspase3,Caspase8,andCaspase9were significantly upregulated in the Si-IRS2 treatment group (P<0.05; Fig.9-E), and the protein levels were consistent with the qPCR results(P<0.05; Fig.9-F and G).Additionally, flow cytometric apoptosis analysis revealed that interference withIRS2significantly elevated the population of apoptotic cells(P<0.05; Fig.9-H and I).Taken together, these results demonstrate thatIRS2releases apoptosis in chicken hepatocytes.

Fig.10 miR-27b-5p regulates chicken hepatocytes development via IRS2/PI3K/Akt signal pathway.A and B, the Akt and P-Akt protein expression after knockdown of miR-27b-5p.n=3.C and D, the Akt and P-Akt protein expression after overexpression of miR-27b-5p.n=3.E and F, the protein expression of Akt and P-Akt after IRS2 knockdown.n=3.G–I, the protein expression level of Caspase 3, Caspase 9, IL-6, Akt and P-Akt in hepatocytes after co-transfected with inhibitor miR-27b-5p or inhibitor NC and Si-IRS2 or Si-NC.n=2.Values represent mean±SEM.Different letters mean P<0.05; ＊, P<0.05; ＊＊, P<0.01.

3.9.MiR-27b-5p regulates hepatocytes development via lRS2/Pl3K/Akt signal pathway

In order to further understand the involvement of miR-27b-5p in the regulation process, we assessed the levels of total Akt (Akt) and phosphorylated Akt (P-Akt)in the transfected cells.Firstly, we found that miR-27b-5p knockdown significantly increased the protein level of P-Akt (P<0.05; Fig.10-A and B), but elevating its expression effectively mitigated this effect (P<0.05;Fig.10-C and D).Additionally, the impact ofIRS2interference on P-Akt was similar to that of the miR-27b-5p overexpression treatment group (P<0.01; Fig.10-E and F).Furthermore, when co-transfecting the cells with inhibitor miR-27b-5p and Si-NC, inhibitor miR-27b-5p and Si-IRS2, and inhibitor NC and Si-NC in hepatocytes,we found that the protein levels of Caspase 3, Caspase 9, and IL-6 were reduced in the co-transfection group of inhibitors miR-27b-5p and Si-NC.However, the addition of Si-IRS2 reversed this trend (P<0.05; Fig.10-G and I).Additionally, the phosphorylation level of Akt was increased after co-transfection with inhibitor miR-27b-5p and Si-NC, but this increase was alleviated in the cotransfection group of inhibitors miR-27b-5p and Si-IRS2(P<0.05; Fig.10-G and H).Taken together, these findings suggest that miR-27b-5p regulates the development and metabolism of the chicken liverviathe IRS2/PI3K/Akt signaling pathway.

4.Discussion

Chicken fatty liver is a type of metabolic disease that frequently affects well-fed laying hens during the peak laying period.The diseased chickens are often hypertrophic and susceptible to death (Bannisteret al.1975; Shiniet al.2019).Furthermore, the liver of hens with fatty livers produces less lipoprotein and yolk precursors, impacting follicle development and laying ability (Caiet al.2009).In our current study, we induced fatty lesions in the livers of laying hens during the peak period by unrestricted feeding.As the number of treatment days increased, the hormones and regulatory factors related to follicle development significantly decreased, leading to reduced egg production.It is therefore crucial to thoroughly investigate the molecular mechanism of liver fat metabolism to provide new ideas and strategies for addressing fatty liver in laying hens.

In recent years, numerous studies have reported that miRNAs are closely related to chicken liver metabolism and development processes.For example, miR-193b-3p was reported to regulate hepatocyte apoptosis by targetingMAML1in selenium-deficient broilers (Liuet al.2018); miR-187-5p involved in oxidative stressmediated apoptosis by targetingapaf-1in the livers of chickens for fattening (Xuet al.2020); and miR-34a-5p could accelerate the accumulation of lipid by acting as molecular sponge forACSL1in laying hens (Tianet al.2019).It has been widely observed that miR-27b participates in liver fat metabolism.For example, miR-27 regulates the hepatitis C virus (HCV) life cycle and has been seen as a novel mechanism by which the virus induces steatosis (Singaraveluet al.2014).Additionally,miR-27b regulates the insulin signaling pathway in human hepatoma cells by controlling post-transcriptional expression of the insulin receptor (Benito-Vicenteet al.2020).Furthermore, miR-27b participates in hepatic lipid metabolism by targetingANGPTL3andGPAMin mice(Vickerset al.2013).However, the function of miR-27b-5p in chicken liver metabolism remains unexplored.In our study, we primarily investigate the effect of miR-27b-5p on fat metabolism in the chicken liverviaknockdown and overexpression of miR-27b-5p.PPARα,ACACA, andFASNare known to be key factors that promote lipolysis(Clarke and Jump 2018), and the accumulation of TG and TC in the liver is often used as an indicator of lipid metabolic disorders (Nassiret al.2013).Our experiments showed that overexpression of miR-27b-5p significantly increased the abundances ofPPARα,ACACA, andFASNand also promoted the levels of TG and TC in chicken hepatocytes.

The occurrence of oxidative stress is a crucial physiological response in the progression of fatty liver disease (Videlaet al.2004).Numerous studies have reported that miRNAs play an important role in regulating oxidative stress in liver diseases.For example, miR-421 and miR-27a have been shown to increase the production of ROS by suppressingNrf2expression in HepG2 cells(Teimouriet al.2020), while miR-96-5p has been shown to reduce the accumulation of ROS in mice with hepatic steatosis (Zhanget al.2020).Our experiments have revealed that, like miR-421 and miR-27a, miR-27b-5p can induce oxidative stress in chicken hepatocytes.Inflammation is a common pathological phenomenon in most types of liver diseases (Pierantonelli and Svegliati-Baroni 2019), and miRNAs are deeply involved in regulating the inflammatory response of the liver.For instance, miR-223 is known for its anti-inflammatory properties and has been shown to relieve nonalcoholic steatohepatitis and malignancy by targeting multiple inflammatory pathways in mouse hepatocytes (Heet al.2019).Furthermore, miR-194 has been reported to be involved in the inflammation of nonalcoholic fatty liver disease (Wanget al.2021).Our current study has found that miR-27b-5p exacerbates the expression of inflammatory cytokines in hepatocytes, thus it can be considered a pro-inflammatory miRNA.Excessive fat accumulation in hepatocytes can cause an intensification of oxidative stress and inflammation, which can lead to cell apoptosis.Apoptosis is a common feature of liver injury and plays a crucial role in the evolution of liver diseases.MiRNAs are also important regulators of hepatocyte apoptosis.For example, miR-24 has been shown to inhibit hepatocyte apoptosis by targeting the proapoptoticBcl-2homolog in a lipopolysaccharideinduced mouse model of acute liver failure (Fenget al.2017), while miR-34a has been shown to induce apoptosis in rat liver cells and play a proapoptotic role in human fatty liver disease (Castroet al.2013; Ferreiraet al.2014; Shanet al.2015).Our study has found that miR-27b-5p can accelerate apoptosis in chicken hepatocytes.

The regulation of mRNA expression by miRNAs is well known.It has been reported that miR-27b-5p regulates the expression ofHSD3β1andWNT2Bto be involved in the progesterone synthesis in Bewo cells (Vermaet al.2021).In addition, miR-27b-5p controls the selfrenewal, invasion, and migration of ovarian cancer stem cells by regulatingSIRT5(Keet al.2022).Our study validatedIRS2as the target gene of miR-27b-5p in the regulation of chicken hepatocyte development.IRS2, a liver-specific protein similar in structure toIRS1(Thironeet al.2006), has been shown to play a role in insulin resistance.Deficiency of bothIRS1andIRS2has been reported to reduce AKT activity and cause insulin resistance in mouse liver (Kubotaet al.2008).The use of liraglutide has been shown to prevent nonalcoholic fatty liver disease in diabetic mice by activating the IRS2/PI3K/Akt signaling pathway (Yanget al.2019).Agriophyllumoligosaccharides have been found to improve hepatic damage in mice by triggering the IRS2/PI3K/AKT signaling pathway, which stimulates hepatocyte proliferation and reduces blood glucose levels (Baoet al.2020).Metformin has been confirmed to upregulate the activity of the IRS2-PI3K/AKT signaling pathway, leading to changes in liver histology and lipid peroxidation (Xuet al.2016).In our study, we found that miR-27b-5p is involved in the metabolism and development of the chicken liver by regulating the activity of the IRS2-PI3K/AKT signaling pathway (Fig.11).Additionally, Ayalaet al.(2009) have reported that the chicken model possesses several advantageous characteristics for studying steatohepatitis disease, including its simplicity, reproducibility, costeffectiveness, and technical advantages (Ayalaet al.2009).Therefore, our findings provide a new target for the prevention and treatment of human fatty liver disease.

Fig.11 Schematic diagram of miR-27b-5p regulating the metabolism and development of chicken hepatocytes.

5.Conclusion

In summary, our study has uncovered that miR-27b-5p influences lipogenesis, oxidative stress, inflammation, and apoptosis by targetingIRS2and suppressing the PI3K/AKT signaling pathway in chicken hepatocytes.

Acknowledgements

This experiment was financially supported by the National Key Research and Development Program of China(2021YFD1300600), the Sichuan Science and Technology Program, China (2021YFYZ0007 and 2022YFYZ0005),and the China Agriculture Research System of MOF and MARA (CARS-40-K06).

Declaration of competing interest

The authors declare that they have no conflict of interest.

Ethical approval

All the animals used for this experiment are approved by the Animal Welfare Committee of Sichuan Agricultural University, China, and the approval number is 2021102006.

Appendicesassociated with this paper are available on https://doi.org/10.1016/j.jia.2023.04.010