miR-128-3p reduces fat cell formation in chicken muscles by lowering FDPS levels

Intramuscular adipocytes were transfected with the miR-128-3p mimic or inhibitor or with the negative control (NC) for 24 h. RT‒qPCR analysis showed that the miR-128-3p expression level was significantly increased in intramuscular adipocytes transfected with the miR-128-3p mimic compared to the NC (P < 0.001) (Fig. S1A) and was significantly decreased by transfection of the miR-128-3p inhibitor compared to the NC (P < 0.001) (Fig. S1B). These results indicated that the transfection experiment was effective and guaranteed the reliability of the data obtained in follow-up investigations. In chicken intramuscular adipocytes, overexpression of miR-128-3p significantly increased the expression of the marker genes BCL2 and PCNA, which promote cell proliferation, and significantly decreased the expression of P21, which inhibits cell proliferation (P < 0.05) (Fig. 1A). In contrast, miR-128-3p inhibition significantly decreased the expression of BCL2 and PCNA and significantly increased the expression of P21 (P < 0.05) (Fig. 1B). The results of the CCK-8 assay showed that overexpression of miR-128-3p promoted the proliferation of intramuscular adipocytes, whereas inhibition of miR-128-3p inhibited the proliferation of intramuscular adipocytes (Fig. 1C). The results of the EdU incorporation assay showed that the proportion of EdU-positive cells increased and decreased correspondingly after miR-128-3p overexpression and interference, respectively (Fig. 1D, E). To further confirm the role of miR-128-3p in the proliferation of chicken intramuscular adipocytes, we conducted overexpression experiments. Upon miR-128-3p overexpression, the numbers of cells in S phase and G2 phase increased significantly (P < 0.05), while the number of cells in G1 phase decreased (Fig. 1F, Fig. S2). The results of the apoptosis assay showed that the apoptosis rate decreased dramatically after transfection with the miR-128-3p mimic (P < 0.05) (Fig. 1G, Fig. S3). The opposite effects were observed when the expression of miR-128-3p was downregulated (P < 0.05). All above indicate that miR-128-3p may promote the proliferation of chicken intramuscular adipocytes.

The expression of adipogenic genes (FABP4, FASN, and CEBPA) was measured in chicken intramuscular adipocytes following transfection. The expression of the adipocyte differentiation marker genes FABP4 and CEBPA, which promote fat deposition, was substantially decreased and increased after miR-128-3p overexpression and interference, respectively (P < 0.05) (Fig. 2A, B). We next sought to further investigate the effect of adipocyte differentiation on lipid droplet accumulation and intracellular triglyceride (TG) content in chickens. Oil Red O staining demonstrated that overexpression of miR-128-3p greatly decreased lipid accumulation in intramuscular adipocytes (P < 0.05) (Fig. 2C, D) and that miR-128-3p inhibitor transfection had the opposite effect on adipogenesis (P < 0.05) (Fig. 2C, E). In addition, the results of the triglyceride assay showed that overexpression of miR-128-3p decreased intracellular triglycerides (P < 0.01) (Fig. 2F) but interference with miR-128-3p increased intracellular triglycerides (Fig. 2G). Summarize the above, these data that indicate miR-128-3p has the ability to prevent chicken intramuscular adipocytes from differentiating.

To further explore the gene regulatory pattern associated with miR-128-3p in intramuscular adipocytes and screen for downstream regulated genes, we performed transcriptome sequencing of intramuscular adipocytes after transfection with the miR-128-3p mimic and inhibitor. The clean reads were compared with the reference genome (Gallus_gallus-7.0), and the results showed that the average mapping rate of the clean reads was higher than 97% (Supplementary Table 2–4). The distribution of reads aligning to the genome was determined, and the findings indicated that approximately 85% of the reads were distributed on genes, approximately 15% of the reads were distributed in intergenic regions, and more than 90% of the reads were distributed in exonic regions (Supplementary Table 5). To explore the mRNA changes and biological clustering across groups, we first performed principal component analysis (PCA). PCA for the three groups showed that 92% of the variance could be explained by the first two principal components (Fig. 3A), indicating the diverse mRNA profiles across these three groups. To confirm the reliability of the RNA-seq results, six DEGs (CNCNB3, CHRDL2, FDPS, FMO3, PDGFRA, and STK32C) were randomly selected, and their expression levels were measured by RT‒qPCR (Fig. 3B). As expected, the expression levels of all six candidate genes showed a consistent trend of expression, therefore validating our results.

A total of 1,689 differentially expressed mRNAs, namely, 817 upregulated and 862 downregulated mRNAs, were identified by comparing the overexpression group (M group) with the interference group (SI group) (Fig. 3C). Functional enrichment analysis revealed that the DEGs were involved in lipid metabolism-related pathways, such as the MAPK and TGF-β signaling pathways (Fig. S4C, F). These DEGs, such as APOA1, FDPS, MSMO1, and HMGCS1, were identified to potentially regulate chicken preadipocyte differentiation (Supplementary Table 6). Upregulated miRNAs affect fat accumulation by downregulating their target mRNAs. Therefore, we performed network interaction analysis using Cytoscape software and plotted the regulatory network of miR-128-3p. In this network, miR-128-3p can target 56 down regulated DEGs, such as FDPS, GGT5, TMEM37, and ASL2, based on the comparison of binding sites in the miR-128-3p seed region (Fig. 3D). Enrichment analysis showed that these potential target genes were mainly involved in metabolic pathways, including steroid biosynthesis and terpenoid backbone and butanoate metabolism (Fig. 3E). To ensure that the selected target genes indeed play a role in IMF deposition, we investigated whether these potential target genes overlapped among the three comparisons (NC vs. M, NC vs. SI, and SI vs. M). The intersection of the overlapping genes indicated that 34 DEGs were potential target genes of miR-128-3p (Fig. 3F, Table S7).

We predicted the binding capacity of the gene 3'UTR region of miR-128-3p using an online website (https://bibiserv.cebitec.unibielefeld.de/rnahybrid/submission.html/↗). The results showed that FDPS had strong binding ability with both miR-128-3p (Fig. 4A, B). To validate this prediction, we then performed dual luciferase report assay, and the results supported that miR-128-3p mimics can significantly inhibit the luciferase activity of psiCHECK2-FDPS 3'UTR-WT. However, it has no effect on psiCHECK2-FDPS 3'UTR-MuT (Fig. 4C). It indicated that FDPS gene and miR-128-3p were in a targeted binding relationship.

To verify whether FDPS and miR-128-3p exert opposing regulatory effects on fat deposition, we conducted in vitro experiments to investigate the effect of FDPS on intramuscular adipocyte differentiation. During intramuscular preadipocyte differentiation, an increasing trend was observed in the expression of FDPS from 0 to 10 d, suggesting that FDPS may be related to adipose differentiation as a target gene (Fig. 5A). To further investigate the effect of FDPS interference on lipid deposition in intramuscular adipocytes, FDPS small interfering RNAs (siRNAs) were transfected into intramuscular adipocytes. Compared with that in the NC cells, the expression level of FDPS in transfected intramuscular adipocytes was decreased (Fig. 5B). Quantitative real-time PCR analysis showed that the expression of adipose differentiation marker genes (CEBPA and PPARG) was significantly reduced (P < 0.01), but there was no significant change in the expression level of FABP4 (Fig. 5C). The results of the triglyceride assay indicated that interfering with FDPS gene expression reduced the triglyceride content (Fig. 5D). Moreover, Oil Red O staining showed that interfering with FDPS gene expresison reduced lipid accumulation (Fig. 5E, F) (P < 0.001). Therefore, these findings further confirm that miR-128-3p can inhibit intramuscular adipocyte differentiation by regulating the expression of FDPS.

The intramuscular adipocytes were from the pectoral muscle tissue of 14-day-old Gushi chicken under sterile conditions [53]. Pectoral muscle tissue was separated using a scalpel and then was digested with collagenase type I (solaibao, Beijing, China) at 37 °C for 60 min. Briefly, the digested cell fraction was filtered sequentially passed through cell strainers (Biologix, Jinan, China) with pore sizes of 70 µm and 45 µm. The cell precipitate was centrifugated at 1000 rpm for 10 min. Subsequently, these cells were maintained in DMEM/F12 medium (BI, Massachusetts, USA), supplemented with 10% fetal bovine serum (BI, Massachusetts, USA), and 1% penicillin/streptomycin (Solarbio)) in an incubator with a 5% CO₂ atmosphere at 37 °C. After 2 h, the medium was changed. Once upon the confluence of cells reaching 90%, according to the hormone "cocktail method" is used to induce differentiation [54], and the complete medium would be replaced with the differentiation inducing medium (0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 1 uM dexamethasone (Sigma), and 10 g/l insulin (Sigma)).

The miRNA mimic/inhibitor and siRNA interference sequences and their corresponding negative controls were purchased from Genechem (Shanghai, China). Cells were transfected with miRNA mimics, inhibitors and FDPS-SI with Liposome 2000 reagent (Invitgen, USA) according to the manufacturer's instructions. Transfection was carried out when the cell confluence was 60–70%, and the medium was changed to complete medium after 6 h.

The intramuscular adipocytes proliferation was detected after 12 h, 24 h, 36 h and 48 h of transfection using a Trans Detect CCK-8 kit (Toyohito, Japan) according to the manufacturer's protocol. 10 μL of CCK-8 solution was added to the cells and incubated at 37 ℃ constant temperature incubator for 2 h. Then, the absorbance value was measured at 450 nm using the microplate reader.

The intramuscular adipocytes was detected using a Cell-Light EdU Apollo567 In Vitro Kit (RiboBio, Guangzhou, China) according to the manufacturer’s protocol after transfection for 24 h. Briefly, 100 mL of 50 mm EdU reagent was added to each well and incubated for 2 h at 37 ℃. After the staining, the camera was photographed in the dark under the fluorescent microscope. Finally, the Imaje J software was used to count the number of new cells.

The intramuscular adipocytes were seeded in 6 well cell culture plates. The cells were transfected with miR-128-3p mimic, miR-128-3p inhibitor, and negative control. After 48 h, Cells were collected, washed with cold PBS, and fixed with 70% ethanol at 20 °C for 6 h. Then the cells were washed with PBS and added for a final mass concentration of 50 mg/mL to incubat for 30 min at 37 °C. Subsequently, 400 μl of 50 μg/mL propidium iodide (PI) solution was added, and the cells were stained in the dark for 30 min at 25 °C. Finally, the cell cycle was observed by flow cytometry (FACS Calibur, BD Biosciences).

The intramuscular adipocytes samples were washed three times with PBS (Gibco, Carlsbad, CA, USA) and fixed with 4% paraformaldehyde for 30 min [55]. Subsequently, cells were stained with Oil-Red O working solution (Sigma) for 20 min after being washed with PBS. Then the intramuscular adipocytes were photographed by microscope. Lipid droplets were dissolved by Isopropyl alcohol and the absorbance value was calculated at 490 nm using microplate reader.

The intramuscular adipocytes samples were treated with 0.25% trypsin until separation and centrifuged for 3 min at 1000 rpm. After all, TG content in cell homogenate was determined using a triglyceride content detection kit (APPLYGEN, Beijing, China) according to the manufacturer’s instructions. The protein concentrations were measured with the BCA Protein Assay Kit (EpiZyme, Shanghai, China) to normalize the TG content. The absorbance value was calculated at 550 nm using microplate reader.

The transfected miR-128-3p-mimics, miR-128-3p-inhibitor and miR-128-3p-NC were divided into overexpression group (M group), interference group (SI group), and blank treatment group (NC group), respectively. The total RNA was extracted using Trizol reagent (Vazyme, Nanjing, China). The expression of miR-128-3p was detected by fluorescent quantitative PCR. The sample with the highest overexpression efficiency (n = 3) and interference efficiency (n = 3) was collected, respectively, and sent to Nanjing Parsono Gene Technology Co., Ltd. for transcriptome sequencing.

After the removal of raw reads containing no insertion sequence, over 0.2% of poly-N, and low-quality paired reads, we obtained clean reads. The high-quality data (clean data) were mapped to the reference genome (GRCg7a) using TopHat2's upgraded HISAT2 software [56]. Read count values were aligned to each gene using HTSeq statistics as the gene's original expression quantity [57]. Differential gene expression analysis was conducted using DESeq [58], and differentially expressed genes (DEGs) were defined based on the following criteria: |log₂ fold change|> 1 and the P value < 0.05. The DEGs were subjected to functional annotation and pathway enrichment analysis using the KOBAS server [59].

Total RNA was extracted from tissues and harvested cells using Trizol (Vazyme, Nanjing, China). Reverse transcription of mRNA was performed using a HiScript II Q Select RT SuperMix for qPCR kit (Vazyme, Nanjing, China) according to manufacturer instructions. First step reaction system: 4 µl, 4 × gDNA wiper Mix, Total RNA 1000 ng, added RNase free ddH2O to a total system of 16 µl. The reaction procedure was set to 42 ℃ for 2 min and stored at 4 ℃. Second step reaction system: 4 µl, 5 × HiScript III qRT SuperMix was added to the previous product, resulting in a total system of 20 µl. The reaction procedure was set as 37 ℃ for 15 min and 8 ℃ for 5 s. Finally, the product cDNA was diluted twice for subsequent fluorescence quantitative testing.We designed the PCR primers using Primer Premier 5, and the primer sequences were shown in Supplementary table 1. The ChamQ Universal SYBR qPCR Master Mix kit (Vazyme, Nanjing, China) was used to conduct quantitative real-time PCR (Q-qPCR). The total reaction volume was 10 ul, and consisted of 1 μl cDNA, 0.5 μl reverse and forward primers (per gene), 5 ul SYBR, and 3 ul double-distilled water. The reaction procedure was performed under the following conditions:95 °C for 30 s, followed by 40 cycles at 95 °C for 15 s, and 60 °C for 34 s. Finally, the melting curve is collected at 60–95 °C. Relative expression level was quantified by the 2^−△△Ct [60] approach. β-actin and U6 was using as the normalization references for mRNA and miRNA, respectively.

The laboratory psiCHECK2 vector was used to construct the psiCHECK2-FDPS-3'UTR-WT and psiCHECK2-FDPS-3'UTR-MuT plasmids. They were transfected with miRNA-128-3p-mimics and psiCHECK2 vector into DF1 cells. After 48 h, the samples were collected, and the fluorescence activity was detected using the Dual-Glo Luciferase Assay Systemt (Promega, Madison, WI, USA).

Data analysis was performed with SPSS 26.0, All data was presented as “mean ± standard error (SEM)”. Significant differences between groups were analyzed using one-way ANOVA. Asterisks signify different significance levels (*P < 0.05, **P < 0.01, and ***P < 0.001).

All animal experiments were performed according to the Regulations for the Administration of Affairs Concerning Experimental Animals (Ministry of Science and Technology, China, 2004). The protocols and guidelines were approved by the Institutional Animal Care and Use Committee of Henan Agricultural University, China. All sections of this study adhere to the ARRIVE Guidelines for reporting animal research.

Not applicable.

The authors declare no competing interests.

The RNA sequencing data used and analyzed during the current study are available from the NCBI (accession number: PRJNA986221).