MiR-1434 Regulates Abdominal Preadipocytes Differentiation and Adipogenesis by Targeting IGFBP2 in Chickens

doi:10.21203/rs.3.rs-3231780/v1

Download PDF

Research Article

MiR-1434 Regulates Abdominal Preadipocytes Differentiation and Adipogenesis by Targeting IGFBP2 in Chickens

https://doi.org/10.21203/rs.3.rs-3231780/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

High-intensity selection has dramatically increased growth rate and daily weight gain in broilers, but the accompanying problem is the excessive deposition of abdominal fat. According to our previous transcriptome analysis, insulin-like growth factor binding protein 2 (IGFBP2) and miR-1434 were identified as involved in abdominal fat. In this study, we further investigated their function in the proliferation and differentiation of chicken preadipocytes. The results indicated that overexpression of IGFBP2 promoted the proliferation and differentiation of preadipocytes, while interference of IGFBP2 inhibited cell proliferation and lipogenic differentiation. The regulatory effect of miR-1434 on the proliferation and differentiation of preadipocytes was opposite to that of IGFBP2. Dual-luciferase reporter assay proved that miR-1434 directly binds to the 3'-untranslated region (3'UTR) of IGFBP2. As expected, the miR-1434 mimics eliminated the impact of the overexpression vector of IGFBP2 on preadipocytes. In brief, we revealed that miR-1434 promoted the proliferation and differentiation of preadipocytes by blocking IGFBP2expression, thus impacting deposition in broilers. These findings may provide a novel target for improving chicken meat quality.

abdominal fat

abdominal preadipocytes

IGFBP2 gene

miR-1434

As an important economic animal in the livestock farming industry, broilers have high production performance, which can be suitable for large-scale collective feeding models with high demand in the market [1]. In the last 50 years, with the intensive selection and breeding of broiler chickens for intensive production, their growth rate has increased substantially, but this has been accompanied by excessive deposition of abdominal fat, therefore reducing feed conversion, increasing farming costs, and impacting consumer health as well [2]. In addition, chickens are regarded as an ideal experimental animal model for biomedical research, helping to study the biological mechanisms and genetic regulation of fat metabolism [3]. Therefore, studying the mechanisms of adipogenesis in chickens will provide theoretical information for the genetic selection and breeding of quality broilers to reduce abdominal fat deposition.

The process of fat formation in chickens consists of three distinct stages, whereby adipocytes proliferate, adipocytes differentiate, and lipids accumulate within mature adipocytes [4]. Mesenchymal stem cells in connective tissue can differentiate into preadipocytes, which can continue to proliferate and eventually differentiate into adipocytes, a process characteristic of lipid accumulation [5]. The growth of adipose tissue is primarily through the proliferation and hypertrophy of adipocytes [6]. Consequently, expanded adipose tissue can result from increased fat formation and cytoplasmic triglyceride deposition in lipid droplets [7]. Stages of fat production in chickens involve multiple genes whose expression is affected by the regulation of different microRNAs (miRNAs) [8], transcription factors [9], and hormones [10]. Investigating the molecular regulatory mechanisms of fat formation at the transcriptional and post-transcriptional regulatory levels will help us to understand the effects of genes, regulatory factors, and their interplay networks on chicken fat deposition and carcass weight.

MiRNAs are non-coding single-stranded RNA molecules with a length of 18–25 nucleotides, encoded by endogenous genes [11]. MiRNAs have the function of regulating gene expression at the translational level or the transcriptional level[12]. The regulation of target genes by mature miRNAs relies on supplementation between the seed sequences of miRNAs and the 3'-untranslated region (3'UTR) regulatory regions of target genes, thereby inhibiting translation or gene shearing [13]. Several studies have shown that miRNAs are involved in the biological processes of adipose tissue development, including adipocyte proliferation, differentiation, adipogenesis, and lipid metabolism [14–17]. For example, miRNAs to promote adipogenesis have proven for miR-143, miR-17-92 cluster, miR-21, miR-204, miR-378, and miR-375, in addition to miR-27b, miR-22, and miR-130, which have previously reported to affect adipocyte differentiation [18–21]. Thus, miRNAs have emerged as new targets for studying the molecular mechanisms associated with animal adipose tissue development [22]. Over the past decade, there has been extensive research into miRNAs in adipose tissue. These studies have focused on the identification, expression, and function of miRNAs in adipose tissue from different anatomical pools, the findings of which have helped to elucidate the molecular mechanisms underlying adipose tissue development in animals [23–25]. However, previous studies have focused on humans, mice, and a few agricultural animals, alongside metabolic diseases such as obesity. There are few studies on miRNAs in chicken abdominal fat.

The insulin-like growth factor binding protein 2 (IGFBP2) belongs to Insulin-like growth factors (IGFs), which are peptides with high similarity to insulin and play an essential role in individual development and cell fate processes [26, 27]. DNA polymorphisms of IGFBP2 are associated with growth, body composition, and carcass traits in chickens [28–31], but little information is available on the role of IGFBP2 in adipogenesis. Recent studies in human disease and mouse models suggest a strong link between mRNA and serum IGFBP2 and metabolic disorders involving obesity, metabolic syndrome, insulin resistance, and type 2 diabetes [32–35]. However, the mechanism of IGFBP2 involvement in the development of obesity-related diseases is still controversial.

Guangxi Partridge chicken is an excellent breed in Guangxi province, China. Although it has many favorable characteristics, the accumulation of abdominal fat is still a problem that needs to be solved. Our previous transcriptome analysis revealed that IGFBP2 was expressed differentially in the abdominal fat tissue of Guangxi Partridge chickens between high and low abdominal fat percentage group [36]. Meanwhile, miR-1434 was predicted to bind to the 3'UTR region of IGFBP2. Consequently, in this study, we investigated the effects of IGFBP2 and miR-1434 on the proliferation and differentiation of chicken abdominal preadipocytes. These results will provide novel insight into abdominal deposition.

2.1 Dynamic expression patterns of IGFBP2 in vivo and in vitro

Previous transcriptome sequencing experiments showed that the mRNA expression levels of IGFBP2 were significantly different between HF and LF. To determine the expression of IGFBP2 in vivo and in vitro, we performed QRT-PCR on abdominal fat and other tissues from HF and LF chickens, as well as abdominal preadipocytes at different stages of differentiation. The relative expression of the IGFBP2 in 10 tissue samples from 120 days Guangxi Partridge chicken showed its high expression in abdominal fat tissue (Fig. 1A) In addition, Compared to LF chickens, IGFBP2 was significantly upregulated in the abdominal fat of HF broilers (Fig. 1B). Subsequently, we examined the expression of IGFBP2 during the differentiation from preadipocytes to mature adipocytes. With the days of induced differentiation of preadipocytes increased, an increasing trend of IGFBP2 expression was observed (Fig. 1C) These results suggest that IGFBP2 may be essential in adipogenesis of chickens.

2.2 IGFBP2 promotes the proliferation of chicken abdominal preadipocytes

To verify the function of IGFBP2 in chicken adipogenesis, we monitored the proliferation of abdominal preadipocytes tranfected with IGFBP2 overexpression and IGFBP2 interference. The mRNA expression levels of marker genes related to cell proliferation-including proliferating cell nuclear antigen (PCNA), cyclin D1 (CCND1), cyclin D2 (CCND2), cyclin B1 (CCNB1)-were significantly higher in the IGFBP2 overexpression group than that in the pEGFP-N1 control group (Fig. 2A). In contrast, the mRNA expression levels of IGFBP2 and marker genes involved in cell proliferation were significantly lower in the si-IGFBP2 group than that in the si-NC control group (Fig. 2B). EdU assay showed a higher number of EdU-positive cells in the IGFBP2 overexpression and a lower number of EdU-positive cells in the IGFBP2 interference (Fig. 2C-D). In addition, flow cytometry revealed that overexpression of IGFBP2 significantly decreased the number of cells in G0/G1 phase and dramatically increased the number of cells in S phase compared with the control group. In contrast, interference with IGFBP2 expression showed a significant increase in the number of cells in G0/G1 phase and significantly decreased the number of cells in S phase compared to the control group (Fig. 2E-F). These results suggest that IGFBP2 has a positive regulatory effect on the proliferation of abdominal preadipocytes in chickens.

2.3 IGFBP2 promotes the differentiation of chicken abdominal preadipocytes

To further assess the regulatory role of IGFBP2 in chicken adipogenesis, we monitored changes in droplet accumulation and cell differentiation-related genes in preadipocyte differentiation. Differentiation -related genes, including peroxisome proliferator-activated receptors gamma (PPARγ), fatty acid binding protein 4(FABP4), lipoprotein lipase (LPL), and perilipin 1 (PLIN1), were significantly upregulated in response to IGFBP2 overexpression (Fig. 3A). The mRNA expression of PPARγ, FABP4, LPL, and PLIN1 showed a significant decrease in the si-IGFBP2 group compared with the si-NC control group (Fig. 3B). After preadipocyte differentiation for 5 days, overexpression of IGFBP2 induced a significant increase in lipid droplet content (Fig. 3C). In contrast, the lipid droplet content of the si-IGFBP2 group was remarkably decreased than that of the si-NC control group (Fig. 3D). These results suggest that IGFBP2 may accelerate the differentiation of chicken abdominal preadipocytes.

2.4 The IGFBP2 is a direct target of miR-1434

The results of site prediction indicated that miR-1434 can bind to the 3'UTR region of the IGFBP2 (Fig. 4A). To confirm the interaction between miR-1434 and IGFBP2, we constructed wild-type and mutant plasmids containing and lacking the miR-1434 binding site of the 3'UTR of the IGFBP2, respectively. These plasmids were successfully co-transfected with miR-1434 mimics or NC into preadipocytes (Fig. 4B). As expected, the dual luciferase reporter assay showed that miR-1434 significantly inhibited the relative luciferase activity of the wild-type IGFBP2 reporter vector (miR-1434-IGFBP2-WT), but did not affect the relative luciferase activity of the mutant IGFBP2 reporter vector (miR-1434-IGFBP2-Mt) in preadipocytes (Fig. 4C). These results suggest that the IGFBP2is a direct target of miR-1434.

2.5 miR-1434 inhibits the proliferation of chicken abdominal preadipocytes

To confirm the effect of miR-1434 during cell proliferation, we transfected preadipocytes with miR-1434 mimics to overexpress miR-1434. QRT-PCR detected that overexpression of miR-1434 resulted in reduced mRNA expression of cell proliferation critical genes CCND1, CCND2, CCNB1, and PCNA (Fig. 5A ), while Interfering with miR-1434 showed opposite results (Fig. 5B). EdU assay showed a lower number of EdU-positive cells in miR-1434 mimics and a higher number of EdU-positive cells in the miR-1434 inhibitor (Fig. 5C-D). Meanwhile, the cell cycle was detected by flow cytometry, which showed that the number of cells in S phase in miR-1434 mimics group was significantly decreased than that in NC group (Fig. 5E-F). Therefore, it was suggested that miR-1434 inhibited the proliferation of abdominal preadipocytes in chickens.

2.6 miR-1434 inhibits the differentiation of chicken abdominal preadipocytes

To elucidate the function of miR-1434 in preadipocyte differentiation in chickens, we treated preadipocytes with miR-1434 mimics or miR-1434 inhibitor. QRT-PCR analysis showed that, compared to NC, the miR-1434 mimics group showed significant downregulation of lipogenic marker genes LPL, FABP4, PPARγ, and CCAAT enhancer binding protein alpha (C/EBPα) (Fig. 6A). Conversely, mRNA expression of LPL, FABP4, PPARγ, and CEBPα was significantly upregulated in the miR-1434 inhibitor group compared with the control group (Fig. 6B). Staining with Oil Red O (Fig. 6C) verified that miR-1434 mimics-treated preadipocytes exhibited a significant reduction in lipid droplet content. In contrast, the lipid droplet content was significantly increased in the miR-1434 inhibitor group compared with NC (Fig. 6D). These results suggest that miR-1434 may inhibit the differentiation of chicken abdominal preadipocytes.

2.7 miR-1434 targets IGFBP2 to regulate the proliferation and differentiation of abdominal preadipocytes

To further verify the interaction effect of miR-1434 and IGFBP2, we first co-transfected miR-1434 mimics + pEGFP-N1, mimics NC + pEGFP-IGFBP2 and miR-1434 mimics + pEGFP-IGFBP2 into preadipocytes a. The QRT-PCR results showed that after transfection with miR-1434 mimic + pEGFP-N1 and miR-1434 mimic + pEGFP-IGFBP2, miR-1434 was able to counteract the promotion of IGFBP2 on CCND1, CCND2, CCNB1 and PCNA genes (Fig. 7A). In addition, EdU staining showed that miR-1434 mimics significantly counteracted the promotion of cell proliferation activity by IGFBP2 (Fig. 7C). The results suggest that miR-1434 affects preadipocyte proliferation by targeting IGFBP2.

To further verify the interaction effect of miR-1434 and IGFBP2 on preadipocyte differentiation, the induction medium was changed to induce differentiation for 5 days after the preadipocytes were co-transfected with miR-1434 mimics + pEGFP-N1, mimics NC + pEGFP-IGFBP2, and miR-1434 mimics + pEGFP-IGFBP2. The cells were examined for lipogenic differentiation using QRT-PCR and oil red staining. The QRT-PCR results showed that miR-1434 counteracted the promotive effect of IGFBP2 on PPARγ, FABP4, C/EBPβ, and LPL genes (Fig. 7B). In addition, oil red O staining and quantitative analysis showed that miR-1434 inhibited the promotion of intracellular lipid droplet accumulation by IGFBP2 (Fig. 7D). Thus, it is suggested that miR-1434 affects preadipocyte differentiation by targeting IGFBP2.

Broiler abdominal fat accumulates economically in the poultry farming industry, and extra fat not only hinders production, but also lead to consumer rejection of meat products [37]. Adipogenesis is mediated by multiple factors, including trophic, hormonal, and transcriptional factors, governed by multiple genes and regulatory factors through different signaling pathways [38].

In recent years, investigations into the regulatory mechanisms underlying abdominal fat deposition in mammals have increased considerably. miRNAs are key regulators of gene expression at the post-transcriptional level and are known to play important roles in a wide range of biological processes. Thus, their emerging regulatory role in adipogenesis has attracted much interest. In the present study, a candidate miRNA, miR-1434, is involved in the adipogenic differentiation of abdominal preadipocytes in chickens. In addition, miR-1434 was associated with an overall decrease in the proliferation and adipogenic differentiation of abdominal preadipocytes in chickens. The overexpression of miR-1434 in abdominal preadipocytes contributed to a conspicuous decrease in cell proliferation, which was accompanied by a significant decrease in lipid droplet content during abdominal preadipocyte differentiation. This indicates that miR-1434 may serve as an inhibitor of adipogenesis, resulting in the proliferation and differentiation of abdominal preadipocytes in chickens.

MiRNAs are known to exert their effects via posttranscriptional silencing of the downstream target genes. In the present study, the IGFBP2 gene was validated as a direct target of miR-1434, whose 2–8 nt seed region at the 5'ends could recognize and bind to IGFBP2 (as determined by a dual-luciferase reporter assay). IGFs is a growth peptide involved mainly in animal growth and development with cell proliferation and differentiation [39]. IGFBP2, as one of its family members, is crucial in regulating different physiological and metabolic processes. IGFBP2 inhibits IGF-mediated growth and developmental rates [31]. IGF-binding proteins prolong the half-life of the IGFs and have been shown to either inhibit or stimulate the growth-promoting effects of the IGFs on cell culture. They alter the interaction of IGFs with their cell surface receptors [40]. Thus, IGFBP2 participates in the insulin signaling pathway regulatory network by regulating the bioavailability of IGFs [39], and the insulin signaling pathway has important effects on the regulation of fat metabolism in chickens [41].

In our previous RNA sequencing data, we identified that IGFBP2 showed high expression in abdominal fat tissue in the high abdominal fat group [36]. In subsequent experiments, which confirmed this result, we assayed the mRNA levels of IGFBP2 during the differentiation of primary preadipocytes. To further investigate the biological functions of IGFBP2, we performed a series of cellular experiments and found that IGFBP2 promoted the proliferation, differentiation, and lipid accumulation of chicken preadipocytes.

IGFBP2, a major binding protein secreted by differentiated white preadipocytes [42], is hypothesized to play a significant regulatory role in adipogenesis. In chickens, numerous studies have shown that IGFBP2 gene variants are associated with fat deposition [28, 43]. Leng et al.[44] found that the 1196 C > A single nucleotide variants (SNP) locus of the IGFBP2 gene may be a quantitative trait locus affecting abdominal fat deposition in chickens, which was significantly associated with fat deposition, as determined by body weight and body composition traits in six different populations. In our study, the IGFBP2 gene was not only highly expressed in abdominal fat, but also significantly more expressed in adipose tissue in the high abdominal fat group than in the low abdominal fat group. However, mRNA levels of the IGFBP2 gene in adipose tissue are independent of the diet and are not affected by age[40], presumably affecting chicken fat deposition through other pathways. Currently, studies show that numerous transcription factors can bind the IGFBP2 promoter in vitro and activate its transcriptional activity [45]. Related studies have shown that IGFBP2 binds to multiple endocrine neoplasia type 1 (MEN1), specificity protein 1 (Sp1), PPARα, C/EBPα, or hypoxia- inducible factor 1 (HIF1) and nuclear factor kappa B (NFκB) binding sites [46], it is hypothesized that IGFBP2 may be an important mediator of lipid metabolism and inflammatory responses.

Furthermore, in a report by Mu et al. [47], IGFBP2 gene overexpression promoted the expression of critical lipogenic factors such as PPARγ in chicken hepatocytes and the accumulation of intracellular triglycerides, which are the essential sites for the de novo synthesis of lipids and have an important role in lipid synthesis in chickens, conjecturing that IGFBP2 is a crucial gene affecting fat deposition in chickens. Our findings revealed that IGFBP2 gene expression was significantly upregulated during abdominal preadipocyte differentiation in chickens. Thus, we speculated that IGFBP2 could positively regulate the differentiation of abdominal preadipocytes in chickens. Our experiments examining the gain- and loss-of-function of the IGFBP2 gene demonstrated that the IGFBP2 gene could also facilitate lipogenic differentiation, as evidenced by increased lipid droplet accumulation in the abdominal preadipocytes of chicken. This was consistent with the results of the above-mentioned research on chicken showing that IGFBP2 functions as an activator in adipogenesis.

The PPAR signaling pathway is an important regulatory pathway for lipid deposition in chickens. Genes involved in the PPAR signaling pathway include PPARγ, FABP3, FABP4, LPL, acyl-CoA synthetase family member 3 (ACSF3), acyl-CoA synthetase short-chain family member 2 (ACSS2), cluster of differentiation 36 (CD36), carnitine palmitoyl transferase 2 (CPT2), and polypeptide. ACSS2, CD36, CPT2, and Polypeptide 1 (CYP27A1), are functional genes in chicken functional genes in lipid metabolism [48]. Among these genes, PPARγ is a critical transcription factor for adipocyte differentiation which stimulates the expression of several genes related to adipogenesis, such as insulin-dependent glucose transporter 4 (GLUT4) [49], adipocyte fatty acid binding protein 10 adipocyte fatty acid binding protein (aP2) [50], LPL and CD36 [51]. Our result demonstrated that a significant increase in PPARγ expression was triggered by IGFBP2 overexpression in chicken abdominal fat, which was consistent with the previous study in chickens [52]. Additionally, the transcriptional activities of PLIN1 gene (responsible for de novo lipogenesis) were positively regulated by IGFBP2 in the abdominal fat of chicken. Beyond that, some genes associated with cell proliferation-including CCND1, CCND2, CCNB1, and PCNA-were positively regulated by IGFBP2. Therefore, we hypothesized that during adipogenesis in chickens, IGFBP2 may serve as an activator in a manner that affects the expression of genes related to lipogenesis and cell proliferation, thus influencing the hyperplasia of abdominal preadipocytes and their differentiation into mature adipocytes. These regulatory mechanisms of IGFBP2 underlying adipogenesis need to be further verified.

4.1 Animals and Sample Collection

A total of 150 female Guangxi Partridge chicken were bred according to the standard breeding program in the experimental farm of Guangxi University. Chickens were slaughtered at 120 days old and divided into two groups according to the abdominal fat rate, namely high (HF) and low (LF) abdominal fat group (Table S1), each group contained three chickens. Ten tissues, including heart, liver, lung, kidney, pectoral muscle, leg muscle, abdominal fat, brain, glandular stomach, and cecum, were sampled and rapidly frozen by liquid nitrogen, and subsequently stored in the − 80 ℃ refrigerator of Animal Genetic Breeding Laboratory, College of Animal Science and Technology, Guangxi University.

4.2 Preadipocyte Isolation, Culture, and Differentiation

Preadipocytes were isolated from the abdominal fat tissue of 10-day-old chickens referring to previously reported methods [53]. All cells were incubated in Dulbecco’s modified Eagle’s medium (DMEM)/F12 (Gibco, Beijing, China) medium with 10% fetal bovine serum (FBS, Gibco, Grand Island, NY, USA) in a humidified atmosphere of 5% CO₂ at 37 ◦C.

After the preadipocytes in the culture dish were growing 90%, the original medium was aspirated and discarded, and the cells were washed twice with pre-warmed 37 ℃ PBS (Gibco, Beijing, China). Subsequently, the induction differentiation medium was changed every two days and cultured until the desired number of days for downstream experiments. To induce differentiation of preadipocytes, the 10% FBA DMEM/F12 complete medium was supplemented with 160 µmol/L oleic acid. The differentiation medium was changed daily.

4.3 Plasmid construction

Construction of overexpression vector of IGFBP2. The amplification primers for the IGFBP2 sequence were as follows: Forward primer: 5’-tataagcttTTGAGGTGCTGTTCCGCTGCCCGC-3’, Reverse Primer: 5’-tatctgcagCTGGCTCCGCAGGGCGTGTGCT-3’. The target fragment was amplified from myocytes’ cDNA library of various tissue mixture pools of Guangxi Partridge chicken by standard PCR and high-fidelity DNA polymerase. The target gene fragment was ligated to the pEGFP-N1 expression vector and transformed into DH5α cells. The transformants were cultured on LB solid medium containing ampicillin overnight. The colonies were identified by PCR and positive clones were compared to verify the successful construction of the pEGFP-IGFBP2 plasmid by sequencing and positive cloning.

To determine whether miR-1434 targets the IGFBP2, the 3′UTR region of the IGFBP2containing a putative miR-1434 binding site and an insert containing the XhoI and SalI restriction enzyme sites was amplified via PCR using 2×San Taq PCR Mix (Thermo Fish, Shanghai, China) according to the manufacturer’s specifications. The fragments were then cloned into the XhoI and SalI (Takara, Kyoto, Japan) double-digested pmirGLO vector (Promega, Madison, WI, USA), and transformed into DH5α cells. The transformants were cultured on LB solid medium containing ampicillin overnight, to construct the wild-type plasmid miR-1434-IGFBP2-WT. Similarly, the miR-1434 binding site to the IGFBP2 gene was deleted from the 3′UTR region, which was then amplified via PCR using 2×Phanta Max Master Mix (Vazyme, Nanjing, China) and cloned to construct the mutant-type plasmid miR-1434-IGFBP2-MT. All primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. (Table S2).

4.4 RNA oligonucleotides synthesis

The oligonucleotides sequences used in the study are listed in Table S3. small interfering RNA (siRNA) were synthesized by Sangon Biotech (Shanghai) Co., Ltd. And miR-1434 mimics, which causes the overexpression of gga-miR-1434, as well as their corresponding negative controls (NC), were synthesized by RiboBio (Guangzhou) Co., Ltd.

4.5 Cell Transfection

When the cells reached 70–80% confluence, the cells were transfected. Plasmid was transfected with Lipofectamine™ 3000 (Thermo Fish, Shanghai, China), in the case of siRNA oligonucleotide, using Lipofectamine™ RNiMAX (Thermo Fish, Shanghai, China). Briefly, transfection reagents, OPTI-MEM (Gibco Beijing, China), plasmids, and RNA were proportionally diluted and mixed into DNA/RNA liposome complexes, adding to cell culture plates of different specifications in corresponding proportions as well as supplemented with complete medium.

4.6 RNA Extraction, cDNA Synthesis, and Quantitative Real-Time PCR (QRT-PCR)

Total RNA was extracted from tissues and cells using Trizol reagent (Thermo Fish, Shanghai, China) according to the manufacturer's protocol. RNA was reverse transcribed into cDNA using an RT kit (Thermo Fish, Shanghai, China). All primers were designed with Oligo 7.0 software and synthesized by Sangon Biotech (Shanghai) Co., Ltd. The primers used for QRT-PCR are listed in Supplementary Materials Table S3. The relative expression of genes was calculated by the 2-ΔΔCt method.

4.7 5-Ethynyl-2-deoxyuridine (EdU) assay

EdU assays were performed on preadipocytes in 96-well plates after 12 h of transfection with the Cell-Light EdU Apollo567 in vitro kit (RiboBio, Guangzhou, China) according to the manufacturer's protocol. Briefly, 50 µM EdU reagent was added to each well and incubated for 2 h at 37 ◦C. Randomly selected regions were trapped using a fluorescence microscope (Zen, Tokyo, Japan), to calculate the number of EdU-positive cells and the total number of cells using ImageJ software (Media Cybernetics, Rockville, MD, USA).

4.8 Oil Red O Staining

The treated cells were washed three times with PBS and fixed in 4% tissue cell fixative for 30 min. after three washes with PBS incubated with Oil Red O staining solution (Solarbio, Beijing, China) by incubation for 30 min in room temperature and protected from light. Lastly, after washing three times with PBS, we randomly selected three places in each well for imaging and quantified the Oil Red O staining area via the software ImageJ.

4.9 Target Prediction and Dual-Luciferase Reporter Assay

The 3'UTR sequence of the IGFBP2 was available from the Ensembl database to predict the binding site with miR-1434 using TargetScan 7.0. To determine the interaction between miR-1434 and its potential target gene (IGFBP2), cells were inoculated into 96 microtiter plates and co-transfected with miR-1434 mimics or mimic NC and wild-type or mutant plasmids as described above. The assay was performed 24 h after transfection according to the Dual-Glo® Luciferase Assay System (Promega, Madison, WI, USA) instructions. Firefly luciferase and Renilla luciferase signals were measured on an Infinite® 200 PRO ELISA (Tecan, Shanghai, China). The Renilla luciferase signal was normalized to the firefly luciferase signal Three biological replicates served as the basis for all reactions.

4.10 Statistical Analysis

Data collected were subjected to statistical analyses using Statistical Analysis System (SAS) software. The mean of three replicates was evaluated and then displayed as mean ± standard error (SD), * p < 0.05 and ** p < 0.01 were set as statistical significance. significance was determined using one-way ANOVA and Duncan's multiple-range tests. The results were illustrated using GraphPad Prism 8 (GraphPad Software, San Diego, CA, USA).

By examining the expression pattern, we discovered that IGFBP2 is strongly correlated with adipogenesis, which was highly expressed in abdominal fat tissue and significantly upregulated in HF. Cellular experiments demonstrated the promotional relationship of IGFBP2 on the proliferation/differentiation of preadipocytes, the inhibitory effect of miR-1434 on the proliferation/differentiation of preadipocytes, and confirmed that miR-1434 inhibited the proliferation/differentiation of preadipocytes by suppressing the activity of IGFBP2 (Fig. 8). The results of this study reveal that a novel miR-1434 inhibits adipogenesis by targeting IGFBP2.

Acknowledgements:

Not applicable

Author Contributions: Conceptualization, X.Y.; data curation, X.Y., Z.Y. and C.X.; formal analysis, X.Y. and Z.Y.; funding acquisition, X.Y.; investigation, T.S., Z.L.; methodology, Z.L.; project administration, X.Y.; resources, S.L. and Z.Z.; software, Z.Y. and C.X.; supervision, X.Y.; validation, Q.L. and R.C.; visualization, X.Y. and Z.Y.; writing, original draft, X.Y. All authors have read and agreed to the published version of the manuscript.

Funding: Science and Technology Key Project of Guangxi (GK AB21075010)

Availability of data and materials

All data generated or analyzed during the present study are available from the corresponding author on reasonable request. The datasets supporting the conclusions of this article are included in the main manuscript and supplemental materials.

Ethics approval and consent to participate

Ethics approval and consent to participate All experimental and sample collection procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the College of Animal Science and Technology of Guangxi University (Guangxi, China), with approval number GXU2018-058. All methods were carried out under relevant guide- lines and regulations. All methods are reported in accordance with ARRIVE guidelines for the reporting of animal experiments.

Consent for publication

Not applicable.

Conflicts of Interest

The authors declare no conflict of interests.

Knowles TG, Kestin SC, Haslam SM, Brown SN, Green LE, Butterworth A, Pope SJ, Pfeiffer D, Nicol CJ. Leg disorders in broiler chickens: prevalence, risk factors and prevention. PLoS ONE. 2008;3:e1545. 10.1371/journal.pone.0001545.
Wu GQ, Deng XM, Li JY, Li N, Yang N. A potential molecular marker for selection against abdominal fatness in chickens. Poult Sci. 2006;85:1896–9. 10.1093/ps/85.11.1896.
Abdalla BA, Chen J, Nie Q, Zhang X. Genomic Insights Into the Multiple Factors Controlling Abdominal Fat Deposition in a Chicken Model. Front Genet. 2018;9:262. 10.3389/fgene.2018.00262.
Morak M, Schmidinger H, Riesenhuber G, Rechberger GN, Kollroser M, Haemmerle G, Zechner R, Kronenberg F, Hermetter A. Adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) deficiencies affect expression of lipolytic activities in mouse adipose tissues. Mol Cell Proteomics. 2012;11:1777–89. 10.1074/mcp.M111.015743.
Zuk PA, Zhu M, Ashjian P, De Ugarte DA, Huang JI, Mizuno H, Alfonso ZC, Fraser JK, Benhaim P, Hedrick MH. Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell. 2002;13:4279–95. 10.1091/mbc.e02-02-0105.
Samulin J, Berget I, Lien S, Sundvold H. Differential gene expression of fatty acid binding proteins during porcine adipogenesis. Comp Biochem Physiol B Biochem Mol Biol. 2008;151:147–52. 10.1016/j.cbpb.2008.06.010.
Qi R, Feng M, Tan X, Gan L, Yan G, Sun C. FATP1 silence inhibits the differentiation and induces the apoptosis in chicken preadipocytes. Mol Biol Rep. 2013;40:2907–14. 10.1007/s11033-012-2306-4.
Chen Y, Zhao Y, Jin W, Li Y, Zhang Y, Ma X, Sun G, Han R, Tian Y, Li H, et al. MicroRNAs and their regulatory networks in Chinese Gushi chicken abdominal adipose tissue during postnatal late development. BMC Genomics. 2019;20:778. 10.1186/s12864-019-6094-2.
Wang Y, Viscarra J, Kim SJ, Sul HS. Transcriptional regulation of hepatic lipogenesis. Nat Rev Mol Cell Biol. 2015;16:678–89. 10.1038/nrm4074.
Hicks JA, Porter TE, Liu HC. Identification of microRNAs controlling hepatic mRNA levels for metabolic genes during the metabolic transition from embryonic to posthatch development in the chicken. BMC Genomics. 2017;18:687. 10.1186/s12864-017-4096-5.
Bartel DP. MicroRNAs: target recognition and regulatory functions. Cell. 2009;136:215–33. 10.1016/j.cell.2009.01.002.
Fu S, Zhao Y, Li Y, Li G, Chen Y, Li Z, Sun G, Li H, Kang X, Yan F. Characterization of miRNA transcriptome profiles related to breast muscle development and intramuscular fat deposition in chickens. J Cell Biochem. 2018;119:7063–79. 10.1002/jcb.27024.
Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell. 2004;116:281–97. 10.1016/s0092-8674(04)00045-5.
Carrington JC, Ambros V. Role of microRNAs in plant and animal development. Science. 2003;301:336–8. 10.1126/science.1085242.
Ye Y, Deng L, Liang M, Xu L, Zhang L, Ma Y, Li Y. MicroRNAs expression profiles in adipose tissues and liver from sex-linked dwarf and normal chickens. Acta Biochim Biophys Sin (Shanghai). 2014;46:723–6. 10.1093/abbs/gmu053.
Huang HY, Liu RR, Zhao GP, Li QH, Zheng MQ, Zhang JJ, Li SF, Liang Z, Wen J. Integrated analysis of microRNA and mRNA expression profiles in abdominal adipose tissues in chickens. Sci Rep. 2015;5:16132. 10.1038/srep16132.
Wang G, Kim WK, Cline MA, Gilbert ER. Factors affecting adipose tissue development in chickens: A review. Poult Sci. 2017;96:3687–99. 10.3382/ps/pex184.
Wang Y, Mu Y, Li H, Ding N, Wang Q, Wang Y, Wang S, Wang N. Peroxisome proliferator-activated receptor-gamma gene: a key regulator of adipocyte differentiation in chickens. Poult Sci. 2008;87:226–32. 10.3382/ps.2007-00329.
Price NL, Fernández-Hernando C. miRNA regulation of white and brown adipose tissue differentiation and function. Biochim Biophys Acta. 2016;1861:2104–10. 10.1016/j.bbalip.2016.02.010.
Esau C, Kang X, Peralta E, Hanson E, Marcusson EG, Ravichandran LV, Sun Y, Koo S, Perera RJ, Jain R, et al. MicroRNA-143 regulates adipocyte differentiation. J Biol Chem. 2004;279:52361–5. 10.1074/jbc.C400438200.
Kim YJ, Hwang SJ, Bae YC, Jung JS. MiR-21 regulates adipogenic differentiation through the modulation of TGF-beta signaling in mesenchymal stem cells derived from human adipose tissue. Stem Cells. 2009;27:3093–102. 10.1002/stem.235.
Guo Y, Zhang X, Huang W, Miao X. [Recent advances of miRNAs in adipose tissues]. Sheng Wu Gong Cheng Xue Bao. 2016;32:151–63.
Klöting N, Berthold S, Kovacs P, Schön MR, Fasshauer M, Ruschke K, Stumvoll M, Blüher M. MicroRNA expression in human omental and subcutaneous adipose tissue. PLoS ONE. 2009;4:e4699. 10.1371/journal.pone.0004699.
Zhang M, Li F, Ma XF, Li WT, Jiang RR, Han RL, Li GX, Wang YB, Li ZY, Tian YD, et al. Identification of differentially expressed genes and pathways between intramuscular and abdominal fat-derived preadipocyte differentiation of chickens in vitro. BMC Genomics. 2019;20. 10.1186/s12864-019-6116-0.
Li F, Li D, Zhang M, Sun J, Li W, Jiang R, Han R, Wang Y, Tian Y, Kang X, et al. miRNA-223 targets the GPAM gene and regulates the differentiation of intramuscular adipocytes. Gene. 2019;685:106–13. 10.1016/j.gene.2018.10.054.
Zhu M, Wang M, Shao Y, Nan Y, Blair HT, Morris ST, Zhao Z, Zhang H. Characterization of muscle development and gene expression in early embryos of chicken, quail, and their hybrids. Gene. 2021;768:145319. 10.1016/j.gene.2020.145319.
Kim JW. The Endocrine Regulation of Chicken Growth.
Li ZH, Li H, Zhang H, Wang SZ, Wang QG, Wang YX. Identification of a single nucleotide polymorphism of the insulin-like growth factor binding protein 2 gene and its association with growth and body composition traits in the chicken. J Anim Sci. 2006;84:2902–6. 10.2527/jas.2006-144.
Kita K, Nagao K, Taneda N, Inagaki Y, Hirano K, Shibata T, Yaman MA, Conlon MA, Okumura J. Insulin-like growth factor binding protein-2 gene expression can be regulated by diet manipulation in several tissues of young chickens. J Nutr. 2002;132:145–51. 10.1093/jn/132.2.145.
Rajaram S, Baylink DJ, Mohan S. Insulin-like growth factor-binding proteins in serum and other biological fluids: regulation and functions. Endocr Rev. 1997;18:801–31. 10.1210/edrv.18.6.0321.
Chesik D, De Keyser J, Wilczak N. Insulin-like growth factor binding protein-2 as a regulator of IGF actions in CNS: implications in multiple sclerosis. Cytokine Growth Factor Rev. 2007;18:267–78. 10.1016/j.cytogfr.2007.04.001.
Ong WK, Sugii S. Adipose-derived stem cells: fatty potentials for therapy. Int J Biochem Cell Biol. 2013;45:1083–6. 10.1016/j.biocel.2013.02.013.
Carter S, Li Z, Lemieux I, Alméras N, Tremblay A, Bergeron J, Poirier P, Deshaies Y, Després JP, Picard F. Circulating IGFBP-2 levels are incrementally linked to correlates of the metabolic syndrome and independently associated with VLDL triglycerides. Atherosclerosis. 2014;237:645–51. 10.1016/j.atherosclerosis.2014.09.022.
Li Z, Picard F. Modulation of IGFBP2 mRNA expression in white adipose tissue upon aging and obesity. Horm Metab Res. 2010;42:787–91. 10.1055/s-0030-1262854.
Chen X, Tang Y, Chen S, Ling W, Wang Q. IGFBP-2 as a biomarker in NAFLD improves hepatic steatosis: an integrated bioinformatics and experimental study. Endocr Connect. 2021;10:1315–25. 10.1530/ec-21-0353.
Xiao C, Sun T, Yang Z, Zou L, Deng J, Yang X. Whole-transcriptome RNA sequencing reveals the global molecular responses and circRNA/lncRNA-miRNA-mRNA ceRNA regulatory network in chicken fat deposition. Poult Sci. 2022;101:102121. 10.1016/j.psj.2022.102121.
Resnyk CW, Carré W, Wang X, Porter TE, Simon J, Le Bihan-Duval E, Duclos MJ, Aggrey SE, Cogburn LA. Transcriptional analysis of abdominal fat in genetically fat and lean chickens reveals adipokines, lipogenic genes and a link between hemostasis and leanness. BMC Genomics. 2013;14. 10.1186/1471-2164-14-557.
Zhang M, Li DH, Li F, Sun JW, Jiang RR, Li ZJ, Han RL, Li GX, Liu XJ, Kang XT, et al. Integrated Analysis of MiRNA and Genes Associated with Meat Quality Reveals that Gga-MiR-140-5p Affects Intramuscular Fat Deposition in Chickens. Cell Physiol Biochem. 2018;46:2421–33. 10.1159/000489649.
Choi YS, Cho HY, Hoyt KR, Naegele JR, Obrietan K. IGF-1 receptor-mediated ERK/MAPK signaling couples status epilepticus to progenitor cell proliferation in the subgranular layer of the dentate gyrus. Glia. 2008;56:791–800. 10.1002/glia.20653.
Bohannon-Stewart A, Kelley G, Kimathi B, Subramanya SH, Donkor J, Darris C, Tyus J, Payne A, Byers S, Hui D et al. Expression of Potential Regulatory Genes in Abdominal Adipose Tissue of Broiler Chickens during Early Development. Genet Res Int 2014, 2014, 318304, 10.1155/2014/318304.
Bibiloni P, Pomar CA, Palou A, Sánchez J, Serra F. miR-222 exerts negative regulation on insulin signaling pathway in 3T3-L1 adipocytes. BioFactors. 2022. 10.1002/biof.1914.
Wheatcroft SB, Kearney MT, Shah AM, Ezzat VA, Miell JR, Modo M, Williams SC, Cawthorn WP, Medina-Gomez G, Vidal-Puig A, et al. IGF-binding protein-2 protects against the development of obesity and insulin resistance. Diabetes. 2007;56:285–94. 10.2337/db06-0436.
Lei MM, Nie QH, Peng X, Zhang DX, Zhang XQ. Single nucleotide polymorphisms of the chicken insulin-like factor binding protein 2 gene associated with chicken growth and carcass traits. Poult Sci. 2005;84:1191–8. 10.1093/ps/84.8.1191.
Leng L, Wang S, Li Z, Wang Q, Li H. A polymorphism in the 3'-flanking region of insulin-like growth factor binding protein 2 gene associated with abdominal fat in chickens. Poult Sci. 2009;88:938–42. 10.3382/ps.2008-00453.
Badinga L, Song S, Simmen RC, Simmen FA. A distal regulatory region of the insulin-like growth factor binding protein-2 (IGFBP-2) gene interacts with the basic helix-loop-helix transcription factor, AP-4. Endocrine 1998, 8, 281–289, 10.1385/endo:8:3:281.
Wheatcroft SB, Kearney MT. IGF-dependent and IGF-independent actions of IGF-binding protein-1 and – 2: implications for metabolic homeostasis. Trends Endocrinol Metab. 2009;20:153–62. 10.1016/j.tem.2009.01.002.
Shen X, Bai X, Xu J, Zhou M, Xu H, Nie Q, Lu X, Zhang X. Transcriptome sequencing reveals genetic mechanisms underlying the transition between the laying and brooding phases and gene expression changes associated with divergent reproductive phenotypes in chickens. Mol Biol Rep. 2016;43:977–89. 10.1007/s11033-016-4033-8.
Cui H, Zheng M, Zhao G, Liu R, Wen J. Identification of differentially expressed genes and pathways for intramuscular fat metabolism between breast and thigh tissues of chickens. BMC Genomics. 2018;19:55. 10.1186/s12864-017-4292-3.
Wu Z, Xie Y, Morrison RF, Bucher NL, Farmer SR. PPARgamma induces the insulin-dependent glucose transporter GLUT4 in the absence of C/EBPalpha during the conversion of 3T3 fibroblasts into adipocytes. J Clin Invest. 1998;101:22–32. 10.1172/jci1244.
Tontonoz P, Hu E, Graves RA, Budavari AI, Spiegelman BM. mPPAR gamma 2: tissue-specific regulator of an adipocyte enhancer. Genes Dev. 1994;8:1224–34. 10.1101/gad.8.10.1224.
Schoonjans K, Staels B, Auwerx J. Role of the peroxisome proliferator-activated receptor (PPAR) in mediating the effects of fibrates and fatty acids on gene expression. J Lipid Res. 1996;37:907–25.
Mu Y, Sun Y, Leng L, Cao Z, Li Y, Li H, Wang Y. Effect of IGFBP2 Overexpression on the Expression of Fatty Acid Synthesis Genes in Primary Cultured Chicken Hepatocytes. J Poult Sci. 2019;56:177–85. 10.2141/jpsa.0180114.
Ramsay TG, Rosebrough RW. Hormonal regulation of postnatal chicken preadipocyte differentiation in vitro. Comp Biochem Physiol B Biochem Mol Biol. 2003;136:245–53. 10.1016/s1096-4959(02)00261-0.

No competing interests reported.

TableS1.pdf
Table S1. Summary statistics of abdominal fat in Guangxi Partridge chicken
TableS2.pdf
Table S2. Plasmid primer sequence information.
TableS3.pdf
Table S3. IGFBP2 siRNA sequence information.
TableS4.pdf
Table S4. Sequences of primers used for QRT-PCR.

Download PDF

Version 1

posted

You are reading this latest preprint version

MiR-1434 Regulates Abdominal Preadipocytes Differentiation and Adipogenesis by Targeting IGFBP2 in Chickens

Status:

Version 1

Abstract

Figures

1 Introduction

2 Result

2.1 Dynamic expression patterns of IGFBP2 in vivo and in vitro

2.2 IGFBP2 promotes the proliferation of chicken abdominal preadipocytes

2.3 IGFBP2 promotes the differentiation of chicken abdominal preadipocytes

2.4 The IGFBP2 is a direct target of miR-1434

2.5 miR-1434 inhibits the proliferation of chicken abdominal preadipocytes

2.6 miR-1434 inhibits the differentiation of chicken abdominal preadipocytes

2.7 miR-1434 targets IGFBP2 to regulate the proliferation and differentiation of abdominal preadipocytes

3 Discussion

4 Materials and Methods

4.1 Animals and Sample Collection

4.2 Preadipocyte Isolation, Culture, and Differentiation

4.3 Plasmid construction

4.4 RNA oligonucleotides synthesis

4.5 Cell Transfection

4.6 RNA Extraction, cDNA Synthesis, and Quantitative Real-Time PCR (QRT-PCR)

4.7 5-Ethynyl-2-deoxyuridine (EdU) assay

4.8 Oil Red O Staining

4.9 Target Prediction and Dual-Luciferase Reporter Assay

4.10 Statistical Analysis

5. Conclusion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1