LincRNA-MSTRG.673.2 Promotes Chicken Intramuscular Adipocyte Differentiation by Sponging miR- 128-3p

LincRNA-MSTRG.673.2 Promotes Chicken Intramuscular Adipocyte Differentiation by Sponging miR- 128-3p | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article LincRNA-MSTRG.673.2 Promotes Chicken Intramuscular Adipocyte Differentiation by Sponging miR- 128-3p Shuaipeng Zhu#, Binbin Zhang, Yuehua He, Wenjie Liang, Tingqi Zhu, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4405250/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Intramuscular fat content is positively correlated with meat flavor and juiciness. Increasing the intramuscular fat (IMF) content of chickens while increasing their growth rate has become a hot topic in molecular breeding.The group's previous studies showed that miR-128-3p inhibited chicken intramuscular adipocyte differentiation and lipogenesis. However, the regulatory mechanism of miR-128-3p in intramuscular preadipocytes is currently unknown. In this study, we investigated the mechanism of miR-128-3p regulation of chicken intramuscular adipocyte differentiation and deposition. Methods RNA-seq was performed to screen for long non-coding RNAs (lncRNAs) that bind to miR-128-3p. Dual luciferase reporter system was used to verify the targeting relationship between miR-128-3p and LincRNA-MSTRG.673.2; nucleoplasmic localization analysis and fluorescence in situ hybridization were used to investigate the localization of LincRNA-MSTRG.673.2 in the cells; a series of experiments such as Q-PCR, Oil O Red staining and triglyceride assay were used to explore the effect of interference with LincRNA-MSTRG.673.2 on the differentiation of intramuscular preadipocytes; co-transfection experiments were used to validate the regulatory patterns of miR-128-3p and LincRNA-MSTRG.673.2 in intramuscular adipocytes. Results Transcriptome data analysis of differential LincRNAs indicated that, compared to the NC group, the mimics-treated group had 17 significantly differentially expressed LincRNAs ( P < 0.05), including 6 upregulated and 11 downregulated ones; the inhibitor-treated group had 17 differentially expressed LincRNAs ( P < 0.05), including 8 upregulated and 9 downregulated ones; and 24 differentially expressed LincRNAs ( P < 0.05) were observed when comparing the mimics-treated group to the inhibitor-treated group, with 14 upregulated and 10 downregulated ones. Functional enrichment analysis revealed that DELincRNAs from the overexpression group (M group) and interference group (SI group) were involved in negative regulation of metabolic processes, response to steroid hormones, regulation of actin cytoskeleton. Furthermore, target gene prediction analysis showed that miR-128-3p can target many of the DELincRNAs, such as LincRNA-MSTRG.673.2, LincRNA-MSTRG.39.2, LincRNA-MSTRG.39.3, and LincRNA-MSTRG.14270.2. LincRNA-MSTRG.673.2 was predominantly expressed in cytoplasm of intramuscular adipocytes. Dual luciferase reporter identified the targeting relationship between miR-128-3p and LincRNA-MSTRG.673.2. The results of subsequent functional assays demonstrated that Interfering with MSTRG.673.2 has been shown to inhibit lipid deposition in intramuscular preadipocytes. Transfection experiments have shown that LincR-MSTRG.673.2 can affect the expression of miR-128-3p. Conclusion This study found that LincRNA-MSTRG.673.2 promoted chicken intramuscular adipocytes differentiation by down regulating miR-128-3p. The results are noteworthy for improving chicken meat quality, molecular breeding, and lipid metabolism research. miR-128-3p Intramuscular adipocytes LincRNA-MSTRG.673.2 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Chicken is currently a major player in the market, with consumers increasingly demanding better taste. In chicken production, intramuscular fat is a crucial factor that positively impacts the quality and flavor of the meat [ 1 , 2 ]. The amount of intramuscular fat is determined by the capacity of intramuscular adipocytes to synthesize and store lipids [ 3 , 4 ]. Numerous research studies have unveiled a plethora of molecular mechanisms that control the differentiation of intramuscular adipocytes. These mechanisms encompass various aspects such as transcription factors, signaling pathways, and non-coding RNAs. These mechanisms include transcription factors [ 5 , 6 ], cell cycle regulators, non-coding RNA, signaling pathways [ 7 ] and others. Micro-RNAs (miRNAs), a subclass of small non-coding RNAs with an 18–25 nucleotide length, are essential regulators of many biological processes, including cell division, proliferation, differentiation and apoptosis [ 8 ]. For example, miR-425-5p [ 9 ] and miR-125a-5p [ 10 ] inhibit differentiation in Porcine intramuscular preadipocytes. bta-miR-210 positively regulates the adipogenesis of PDGFRα + cells in Bovine [ 11 ]. miR-340-5p inhibits sheep adipocyte differentiation [ 12 ]. miR-223 [ 13 ], gga-miRNA-18b-3p [ 14 ] and miR-24-3p [ 15 ] inhibits intramuscular adipocytes differentiation, while miR-15a [ 16 ] and gga-miR-140-5p [ 17 ] significantly promoted intramuscular adipogenic differentiation. miR-128-3p is involved in many physiological processes, including follicular development, gastric cancer cell development [ 18 , 19 ], and lung cancer cell development [ 20 ]. In addition, it is also a biomarker for detecting breast cancer in high-risk benign breast tumors [ 21 ], the diagnosis of non-small cell lung cancer [ 22 ] and lymphoblastic leukemiav [ 23 ]. This suggests that miR-128-3p plays an important role in the regulation of various physiological and pathological processes in animals. miR-128-3p promoted GC apoptosis through 14-3-3β/FoxO pathway via repressing YWHAB, and inhibited lipid synthesis by impeding the PPAR-γ/LPL pathway, as well as reduced the secretion of progesterone and estrogen [ 18 ]. And it impeded 3T3-L1 adipogenesis by targeting Pparg and Sertad2, resulting in the obstruction of preadipocyte differentiation and promotion of lipolysis [ 24 ]. Our previous research also showed that miR-128-3p inhibits intramuscular adipocytes differentiation in chickens by downregulating FDPS [ 25 ]. Therefore, we speculated that miR-128-3p might be a core miRNA regulated by intramuscular fat differentiation. Similar to miRNAs and long non-coding RNAs (lncRNAs) have also been found to regulate the transcription and translation processes of genes by interacting with DNA, RNA, or proteins. For example, LncRNA PVT1 Facilitates Tumorigenesis and Progression of Glioma via Regulation of MiR-128-3p/GREM1 Axis and BMP Signaling Pathway [ 26 ]. LncRNA IMFNCR Promotes Intramuscular Adipocyte Differentiation by Sponging miR-128-3p and miR-27b-3p [ 27 ]. However, LincRNA-MSTRG.673.2 is a newly identified lncRNA whose role in chicken intramuscular adipocyte differentiation has not been fully investigated. In this study, We identified several key LincRNAs that play a role in the regulation of lipid metabolism pathways via miR-128-3p using transcriptome data and functional prediction. We explored the interaction between LincRNA-MSTRG.673.2 and miR-128-3p. In addition, the results of our cell functional assays confirmed that LincRNA-MSTRG.673.2 regulates the differentiation of intramuscular adipocytes in chickens.This will provide new ideas for a better understanding of the molecular mechanisms of chicken intramuscular adipocyte differentiation and new strategies and methods for improving chicken meat quality. Result Identification and Characterization of LincRNAs in Chicken Intramuscular Adipocytes Groups Post miR-128-3p Overexpression and Interference To identify the major LincRNAs that interacted with miR-128-3p and involved in the regulation of intramuscular fat deposition. A model of miR-128-3p differentiation in chicken intramuscular adipocytes was constructed, including mock-treated and inhibitor-treated groups, and transcriptome sequencing was performed. After additional filtering and removal of potential coding transcripts that were identified using CNCI, CPC, PFAM and CPAT (Fig. 1 A), a total of 1665 lnicRNAs were obtained. Comparative analyses of gene structure, expression, and sequence conservation are used to explore differences between LincRNAs and protein codes genes. lncRNAs were typically shorter than mRNAs (Fig. 1 B) and tended to contain only two or three exons in contrast to the mRNAs (Fig. 3 C, D). lncRNAs also appeared to be expressed at lower levels than mRNAs (Fig. 3 C). This suggested that the LincRNA obtained by sequencing conformed to the characteristics of a non-coding RNA. To confirm the reliability of the RNA-seq results, 5 DELincRNAs (MSTRG.2230.3, MSTRG.2262.4, MSTRG.39.2, MSTRG.673.2, MSTRG.7049.1) were randomly selected, and their expression levels were measured by RT‒qPCR (Fig. 1 E). As expected, the expression levels of all 5 candidate LincRNAs showed a consistent trend of expression, therefore validating our results. Analysis of Differential LincRNAs Expression and Screening for miR-128-3p Targeting LincRNAs Transcriptome data analysis of differential LincRNAs indicated that, compared to the NC group, the mimics-treated group had 17 significantly differentially expressed LincRNAs ( P < 0.05), including 6 upregulated and 11 downregulated ones (Fig. 2 A); the inhibitor-treated group had 17 differentially expressed LincRNAs ( P < 0.05), including 8 upregulated and 9 downregulated ones (Fig. 2 B); and 24 differentially expressed LincRNAs ( P < 0.05) were observed when comparing the mimics-treated group to the inhibitor-treated group, with 14 upregulated and 10 downregulated ones (Fig. 2 C). GO function enrichment analysis found revealed that DELincRNAs from the overexpression group (M group) and interference group (SI group) were involved in negative regulation of metabolic processes, response to steroid hormones and negative regulation of cellular metabolic processes (Fig. 2 D). KEGG pathway analysis showed that DELincRNAs were involved in Regulation of actin cytoskeleton, Focal adhesion, Tight junction and VEGF signaling pathway (Fig. 2 E). Upregulated miRNAs affect fat accumulation by downregulating their target LincRNAs. The differential LincRNAs potentially regulated by miR-128-3p, as obtained from the transcriptome data, were compared with the binding sites in the seed region of miR-128-3p. Subsequently, Cytoscape software was used to construct a network of targeting relationships centered on miR-128-3p. In this network, miR-128-3p can target 14 down regulated LincRNAs, such as MSTRG.673.2, MSTRG.2262.4, MSTRG.6216.1, and MSTRG.8694.1 (Fig. 2 F). To increase the probability that the selected LincRNAs regulate IMF deposition, we investigated whether these potential target LincRNAs overlapped among the 2 comparisons (NC vs. SI, and SI vs. M). miR-128-3p were targeted by 6 common lnicRNAs (Fig. 2 G). LincR-MSTRG.673.2 Targets Binding to miR-128-3p According to the online BLAST analysis conducted on the NCBI website, it was found that LincRNA-MSTRG.673.2 belongs to the non-coding sequence and does not overlap with any known genes (Fig. 3 A).. Further inquiry on the LncFinder website revealed that LincRNA-MSTRG.673.2 shares similar non-coding RNA coding characteristics with the known LncRNA-GHR-AS. The coding ability scores of both are much lower than the commonly used reference gene GAPDH, indicating that they are non-coding sequences (Fig. 3 B). RNA FISH and indicated that LincR-MSTRG.673.2 is predominantly localized in the cytoplasm of preadipocytes (Fig. 3 C). After separately extracting cytoplasmic RNA and nuclear RNA from intramuscular preadipocytes, the relative expression level of MSTRG.673.2 was analyzed. The results showed that MSTRG.673.2 was expressed in both the nucleus and cytoplasm (Fig. 3 D). we used online software to evaluate the protein coding ability of LincR-MSTRG.673.2. Analysis of the sequence by online software NCBI-BLAST Using dual-luciferase reporter assay, we further validated whether LincR-MSTRG.673.2 served as ceRNA of miR-128-3p. Compared to the cotransfection of psiCHECK2-LincR-MSTRG.673.2-WT and miR-128-3p-mimics groups, the luciferase activity was significantly upregulated in the cotransfection of psiCHECK2 and psiCHECK2-LincR-MSTRG.673.2-WT groups ( P < 0.01). Similarly, the cotransfection of psiCHECK2-MSTRG.673.2-MuT and miR-128-3p-mimics groups also showed a significant increase compared to the control group ( P < 0.05). This indicated the targeted binding relationship between LincR-MSTRG.673.2 and miR-128-3p (Fig. 3 E). To further investigate the regulatory effects of LincR-MSTRG.673.2 and miR-128-3p on intramuscular fat deposition in chickens, quantitative detection was used to examine the expression of miR-128-3p and LincR-MSTRG.673.2 at different time points (0, 2, 4, 6, 8 and 10 days) after induction of intramuscular fat differentiation. The results showed that LincR-MSTRG.673.2 and miR-128-3p exhibited opposite expression patterns during intramuscular adipocytes differentiation (Fig. 3 F). Interfering with MSTRG.673.2 Inhibits Lipid Deposition in Intramuscular Preadipocytes. To ascertain the effect of LincR-MSTRG.673.2 in intramuscular preadipocytes differentiation, LincR-MSTRG.673.2 inhibitor or NC was transfected into chicken intramuscular preadipocytes, respectively. The results showed that the interference effect could reach over 50%, meeting the experimental requirements (Fig. 4 A). The expression of adipogenic genes ( CEBPA 、 PPARG 、 FASN ) were remarkably reduced in LincR-MSTRG.673.2 inhibitor treated cells (Fig. 4 B). Subsequently, Oil red staining and intracellular triglyceride content detection were performed. The Oil red staining showed the lipid droplet accumulation in the LincR-MSTRG.673.2 interference group was significantly lower than that in the blank control group ( P < 0.01) (Fig. 4 C, D). The intracellular triglyceride content detection indicated that that interfering with LincR-MSTRG.673.2 decreased intracellular triglycerides ( P < 0.05) (Fig. 4 E). These results suggest that interfering with LincR-MSTRG.673.2 reduceed lipid deposition in intramuscular preadipocytes, thereby achieving an inhibitory effect on differentiation. LincR-MSTRG.673.2 Promoted Intramuscular Differentiation by Adsorbing miR-128-3p After cotransfection of LincR-MSTRG.673.2-Si with miR-128-3p-mimics/inhibitor, the expression levels of miR-128-3p wasmeasured.Compared to the blank control group, the expression level of miR-128-3p was significantly upregulated after interfering with LincR-MSTRG.673.2 ( P < 0.05). Simultaneously interfering with LincR-MSTRG.673.2 and overexpressing miR-128-3p led to a highly significant upregulation of miR-128-3p expression ( P < 0.01). Furthermore, when interfering with both LincR-MSTRG.673.2 and miR-128-3p, the expression level of miR-128-3p was significantly downregulated ( P < 0.01) (Fig. 5 A). It indicated that LincR-MSTRG.673.2 had an inhibitory effect on the expression of miR-128-3p. Additionally, compared with the blank control group, interfering with LincR-MSTRG.673.2 and simultaneously overexpressing miR-128-3p resulted in a significant downregulation of adipogenic marker genes such as FABP4 and PPARG ( P < 0.05). When interfering with both MSTRG.673.2 and miR-128-3p, CEBPA , FABP4 , PPARG , and FASN were significantly upregulated ( P < 0.05) (Fig. 5 B). DISSCUSSION Adipose tissue, with its highly active secretory capacity, can absorb or release non-coding RNAs, leading to a significant role for miRNAs and lncRNAs in the process of fat deposition[ 39 , 40 ]. In this experiment, we specifically selected samples of cells overexpressing/underexpressing miR-128-3p for transcriptome sequencing. We identified 14 upregulated and 10 downregulated differential LincRNAs. Functional enrichment analysis revealed that DELincRNAs were involved in Regulation of actin cytoskeleton, Focal adhesion, Tight junction and VEGF signaling pathway. A series of pathways related to cell junctions including tight junctions, focal adhesion and regulation of the actin cytoskeleton might contribute to the deposition of IMF [ 41 ]. Fatty acid metabolism-related candidate genes also were involved in these pathways [ 42 ]. VEGF inactivation may induce fat accumulation [ 43 ]. In addition, some studies have shown that VEGF inactivation induces fat accumulation. Through analysis of significantly differentially expressed LincRNAs following interference, we identified LincRNAs regulated by miR-128-3p, such as LincRNA-MSTRG.673.2, LincRNA-MSTRG.39.2, LincRNA-MSTRG.39.3, and LincRNA-MSTRG.14270.2. The specific mechanisms by which lncRNAs regulate miRNAs include: acting as potential pri-miRNAs to generate mature miRNAs, indirectly regulating target gene expression[ 44 ]; indirectly inhibiting miRNA-mediated negative regulation of target genes by competitively binding to the 3'-UTR of miRNA target mRNAs[ 45 ]; and serving as a "miRNA sponge" to inhibit miRNA expression [ 46 ]. On one hand, miRNAs can bind to the 3’-UTR region of lncRNAs, thereby reducing the stability and expression of lncRNAs [ 47 ]. On the other hand, miRNAs can return to the cell nucleus and regulate the transcription of lncRNAs, enhancing lncRNA expression. Long Noncoding RNA HCP5 Contributes to Nasopharyngeal Carcinoma Progression by Targeting MicroRNA-128-3p [ 48 ]. Knockdown of LncRNA DLEU2 Inhibits Cervical Cancer Progression via Targeting miR-128-3p [ 49 ]. In this study, LincRNA-MSTRG.673.2 was mainly localized in the cytoplasm. This study analyzed and verified the existence of a targeting relationship between LincRNA-MSTRG.673.2 and miR-128-3p. Also, LincR-MSTRG.673.2 and miR-128-3p exhibited opposite expression patterns during intramuscular adipocytes differentiation. Hence, the LincRNA-MSTRG.673.2 acted as a sponge for miR-128-3p. lncRNAs are involved in a variety of bioregulatory processes, including muscle growth, fat deposition, and lipid metabolism [ 50 ]. For example, lncSAMM50 [ 51 ], lncRNA 1 [ 52 ] enhances adipocytes differentiation. We showed that interfering with LincRNA-MSTRG.673.2 inhibited intramuscular fat deposition. The group's previous research showed that miR-128-3p could inhibit the differentiation of intramuscular adipocytes, a result consistent with the interference of LincRNA-MSTRG.673.2. Additionally, after interfering with LincRNA-MSTRG.673.2, miR-128-3p was significantly upregulated, aligning with the mechanism of action between LincRNA and miRNA. For example, LincRNA TINCR promoted lipid deposition by absorbing miR-31-5p to enhance the expression of CEBP pathway genes [ 53 ]. PPARG is indeed a direct target of miR-128-3p [ 27 ]. Transfection experiments with LincRNA-MSTRG.673.2-Si and miR-128-3p-mimics showed that interfering with LincRNA-MSTRG.673.2 upregulated the expression of miR-128-3p and downregulated the expression of adipocytes differentiation marker genes ( CEBPA , FABP4 , PPARG 、 FASN) . This implied that lncRNA may have competitively adsorbed miR-128-3p, thereby affecting PPARG and potentially influencing intramuscular fat deposition. Our research identified LincRNA-MSTRG.673.2 targeting miR-128-3p to promote chicken intramuscular adipocytes differentiation. Determining miR-128-3p as a key regulatory factor for intramuscular preadipocytes fat deposition in chickens is of significant importance for breeding practices aimed at improving chicken meat quality. CONCLUSION In conclusion, this study demonstrated that LincR-MSTRG.673.2 could promote chicken intramuscular adipocytes differentiation by Sponging miR-128-3p. These findings may contribute to furnish new insights for improving chicken quality breeding. MATERIALS AND METHODS Collection of Sequencing Samples 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). The expression of miR-128-3p was detected by Q-PCR. Subsequently, samples with high overexpression efficiency (n = 3), high interference efficiency (n = 3), and samples from the blank group were sent to Nanjing Parsono Gene Technology Co., Ltd. for transcriptome sequencing. Transcriptome Data Analysis 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 [ 28 ]. Read count values were aligned to each gene using HTSeq statistics as the gene's original expression quantity [ 29 ]. CPC [ 30 ], CNCI [ 31 ], and PFAM[ 32 ] to distinguish the protein-coding genes from the noncoding genes. Differential LincRNAs expression analysis was conducted using DESeq [ 33 ], and differentially expressed LincRNAs were defined based on the following criteria: |log 2 fold change| > 1 and the P value < 0.05.Gene Ontology (GO) [ 34 ] and [ 35 ] (KEGG) analysis for predicting the function of the DE lncRNAs. Primary Intramuscular Adipocytes Isolation and Culture and Induced Differentiation Intramuscular adipocytes were isolated as previously described [ 36 ]. Breast muscle tissue was collected from 14-day-old Gushi chicken under sterile conditions. and then digested using 2 mg/ml collagenase type II (Sangon Biotech, Shanghai, China) with shaking for 2 h at 37°C. Then the tissue was was cut into 1 mm 3 pieces using surgical scissors and then digested performed by adding appropriate amounts of collagenase type I (Solaibao, Beijing, China) with shaking for 1 h at 37°C. The digested cell suspension was filtered through 45 and 75 mum filter collected by centrifugation at 1000 r/min for 10 min. Subsequently, the cells were kept in DMEM/F12 media (BI, Massachusetts, USA) supplemented with 10% fetal bovine serum (BI, Massachusetts, USA) and 1% penicillin/streptomycin (Solarbio) in an incubator that maintained a 5% CO 2 environment at 37°C. Cells were cultured for 2 hours, followed by a fluid change. Once the intramuscular adipocytes confluence reaches 90%, the media would be replaced entirely by the differentiation-inducing media composed of 0.5 mM of 3-isobutyl-1-methylxanthine (IBMX), 1 uM of dexamethasone (Sigma), and 10 g/l of insulin (Sigma) [ 37 ]. Transient Transfection of Intramuscular Adipocytes Cells were transfected with miR-128-3p mimics, inhibitors and LincR-MSTRG.673.2 with Liposome 3000 reagent (Invitgen, USA) according to the manufacturer's instructions. Localization Analysis of LincR-MSTRG.673.2 using Fluorescence In Situ Hybridization (FISH) and Nuclear/Cytoplasmic RNA Separation Fluorescence in situ hybridization was designed and synthesized by Wuhan Saviour Biotech Co., Ltd. The probe information was as follows: 5’-3’ GAGGAACAGGAGAGATATGCTACTCATT-TTGTATA. The intramuscular fat cells were attached to the glass slide and grew to 80% density. The operation was carried out according to the instructions of the in situ hybridization kit. 4% paraformaldehyde was used to fix the cells, DAPI was used to stain the cells, and images were captured under a fluorescence microscope. CY3 red light had an excitation wavelength of 510–560 nm and an emission wavelength of 590 nm, emitting red light. The primary intramuscular preadipocytes that were cultured were digested using trypsin, and the digestion was terminated with complete culture medium. Subsequently, PBS was added for preservation, and the subsequent operations were carried out according to the nuclear-cytoplasmic separation kit. The collected cytoplasmic mixture and cytoplasmic precipitate were separately subjected to RNA extraction using the Trizol method. Then, using U6 as the internal reference, the expression levels of MSTRG.673.2 in the cytoplasm and nucleus were quantitatively detected. Dual Luciferase Reporter Assay The laboratory psiCHECK2 vector was used to construct the psiCHECK2-LincR-MSTRG.673.2-3'UTR-WT and psiCHECK2- LincR-MSTRG.673.2-3'UTR-MuT plasmids. They were transfected with miRNA-128-3p-mimics and psiCHECK2 vector into DF1 cells. After 48h, the samples were collected, and the fluorescence activity was detected using the Dual-Glo Luciferase Assay Systemt (Promega, Madison, WI, USA). Oil-Red O Staining Triglyceride Assay Detection of Cell Differentiation The intramuscular adipocytes samples were fixed with 4% paraformaldehyde [ 17 ], and Oil Red O working solution (Sigma) was used for cell staining. Intramuscular fat cells were photographed under a microscope. After microscopic examination, isopropanol was used to dissolve the lipid droplets, and the absorbance value was calculated at 490nm. The triglyceride content in the cell homogenate was determined using a triglyceride content detection kit (APPLYGEN, Beijing, China). The protein concentration was measured using a BCA protein assay kit (EpiZyme, Shanghai, China) to standardize the triglyceride content. The absorbance values were calculated at 550nm using a microplate reader. Q-PCR Total RNA was extracted from intramuscular adipocytes using Trizol (Vazyme, Nanjing, China). mRNA was reverse transcribed using the HiScript II Q Select RT SuperMix for qPCR kit (Vazyme, Nanjing, China), and quantitative real-time PCR was performed using the ChamQ Universal SYBR qPCR Master Mix kit (Vazyme, Nanjing, China). The relative expression levels were calculated using the 2- ΔΔCt method [ 38 ]. The primer sequences were shown in Supplementary table 1 . Statistical Analysis 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 < 0.05, ** P < 0.01, and *** P < 0.001). Abbreviations CNCI Coding-noncoding index CPC Coding potential calculator IMF Intramuscular fat MiRNA MicroRNA NC group Blank treatment group M group Mimics-treated group SI group Inhibitor-treated group GO Gene ontology KEGG Kyoto encyclopedia of genes and genomes Q-PCR Quantitative PCR PBS Phosphate bufered saline PPARG Peroxisome proliferator-activated receptor γ FABP4 Fatty acid binding protein 4 FASN Fatty acid synthetase CEBPA CCAAT/enhancer-binding protein alpha FDPS Farnesyl Diphosphate Synthase Declarations 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. AUTHOR CONTRIBUTIONS Shuaipeng Zhu and Binbin Zhang conceived the project and designed the experiments. Yuehua He Wenjie Liang, and Tingqi Zhu, performed the animal experiments and sample collection. Wenting Li, Ruili Han, Donghua li, Fengbin Yan provided valuable suggestion and comments to improve the manuscript with contributions from all other authors. Yadong Tian, Guoxi Li, Xiangtao Kang, and Guirong Sun discussed the results. All authors have read and approved the manuscript. FUNDING INFORMATION This work was supported by grants from the National Natural Science Foundation of China (32072710), the Key Research Project of the Shennong Laboratory (SN01-2022-05) and the Scientific Studio of Zhongyuan Scholars (30601985). CONFLICT OF INTEREST STATEMENT The authors declare that there is no conflict of interest. AVAILABILITY OF DATA AND MATERIALS The RNA sequencing data used and analyzed during the current study are available from the NCBI (accession number: PRJNA986221). 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Stem cells translational Med. 2020;9(9):1053–67. Xie Z, Rahman I, Goniewicz ML, Li D. Perspectives on Epigenetics Alterations Associated with Smoking and Vaping. Function (Oxford England). 2021;2(3):zqab022. Yi X, He Z, Tian T, Kou Z, Pang W. LncIMF2 promotes adipogenesis in porcine intramuscular preadipocyte through sponging MiR-217. Animal Biotechnol. 2023;34(2):268–79. Zhou G, Zhang M, Zhang J, Feng Y, Xie Z, Liu S, Zhu D, Luo Y. The gene regulatory role of non-coding RNAs in non-obstructive azoospermia. Front Endocrinol. 2022;13:959487. Miao G, Liu B, Ling K, Peng T, Zhou E, Xie S, Tan Z. Long Noncoding RNA HCP5 Contributes to Nasopharyngeal Carcinoma Progression by Targeting MicroRNA-128-3p. Journal of oncology 2022, 2022:5740857. Wang B, Hang J, Li W, Yuan W. Knockdown of LncRNA DLEU2 Inhibits Cervical Cancer Progression via Targeting miR-128-3p. OncoTargets therapy. 2020;13:10173–84. Luo J, Shen L, Gan M, Jiang A, Chen L, Ma J, Jin L, Liu Y, Tang G, Jiang Y, et al. Profiling of skeletal muscle tissue for long non-coding RNAs related to muscle metabolism in the QingYu pig at the growth inflection point. Anim bioscience. 2021;34(8):1309–20. Zhu R, Feng X, Wei Y, Guo D, Li J, Liu Q, Jiang J, Shi D, Huang J. lncSAMM50 Enhances Adipogenic Differentiation of Buffalo Adipocytes With No Effect on Its Host Gene. Front Genet. 2021;12:626158. Zhao XY, Li S, DelProposto JL, Liu T, Mi L, Porsche C, Peng X, Lumeng CN, Lin JD. The long noncoding RNA Blnc1 orchestrates homeostatic adipose tissue remodeling to preserve metabolic health. Mol metabolism. 2018;14:60–70. Yang C, Luo M, Chen Y, You M, Chen Q. MicroRNAs as Important Regulators Mediate the Multiple Differentiation of Mesenchymal Stromal Cells. Front cell Dev biology. 2021;9:619842. Additional Declarations No competing interests reported. Supplementary Files AdditionalFile1Tables1.QPCRprimersequences..docx Supporting Information Additional File 1:Table s1. Q-PCR primer sequences. 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University","correspondingAuthor":true,"prefix":"","firstName":"Guirong","middleName":"","lastName":"Sun","suffix":""}],"badges":[],"createdAt":"2024-05-11 11:54:56","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4405250/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4405250/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":56998609,"identity":"e9357711-88be-4184-8d86-ff86887b441c","added_by":"auto","created_at":"2024-05-23 08:08:03","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2023073,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacterization of lincRNAs. (A). Venn diagram of lincRNAs predicted by CNCI, CPC2, PLEK, and PFAM. (B).\u003c/strong\u003e \u003cstrong\u003eExon number distribution of LincRNAs. (C).\u003c/strong\u003e \u003cstrong\u003eThe length distribution of lncRNAs. (D). The length distribution of known and novel lincRNAs. E.\u003c/strong\u003e \u003cstrong\u003eValidation of LincRNA sequencing data.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4405250/v1/6c34fe680c30dbfeeef8f8c4.png"},{"id":56998014,"identity":"49669ff2-8054-4784-9cf2-3e8673a98453","added_by":"auto","created_at":"2024-05-23 08:00:03","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2240473,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDifferential LincRNAs expression analysis. (A-C). Volcano plot of gene expression in the NC \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003evs\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. M, NC \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003evs\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. SI, and SI \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003evs\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. M comparisons. Numbers of upregulated and downregulated differentially expressed LincRNAs. The left blue bars represent the numbers. of upregulated genes; the orange bars represent the numbers of downregulated genes. (D). GO pathway enrichment analysis of the DELincRNAs in the M \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003evs\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. SI comparison. (E). KEGG pathway enrichment analysis of the DELincRNAs in the M \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003evs\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. SI comparison. (F). Based on the comparison of binding sites in the seed region of miR-128-3p, Cytoscape software was used for network interaction analysis, and the regulatory network of miR-128-3p was mapped. (G). Venn diagrams of DELincRNAs identified by RNA-seq in the NC \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003evs\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. M, NC \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003evs\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. SI, and SI \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003evs\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. M comparisons\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4405250/v1/6dd31b6a2bb1a3ba607786db.png"},{"id":56998019,"identity":"8c8f60b9-7721-45d7-93ef-d0af899ff415","added_by":"auto","created_at":"2024-05-23 08:00:03","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":5332976,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLincR-MSTRG.673.2 targets binding to miR-128-3p (A-B). LincR-MSTRG.673.2 coding ability evaluation. (C). LincR-MSTRG.673.2 in situhybridization analysis. (magnification 200). (D).Cytoplasm and nucleus RNA were extracted from intramuscular preadipocytes, respectively. LincR-MSTRG.673.2 cellular location was studied by qRT-PCR assay. (E). The DF1 cells were cotransfected with either psiCHECK2-LincR-MSTRG.673.2-WT or psiCHECK2-MSTRG.673.2-MuT, and miR-128-3p-mimics or psiCHECK2. Then, the relative luciferase activity was measured. (F). A The expression levels of LincR-MSTRG.673.2\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eduring the differentiation of chicken primary intramuscular adipocytes into mature adipocytes\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4405250/v1/e50ef70ee45847241e7a4e16.png"},{"id":56998015,"identity":"5ba31ad2-f9f0-4db1-b28d-236a82c6b9df","added_by":"auto","created_at":"2024-05-23 08:00:03","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":4617989,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInterference with \u0026nbsp;LincR-MSTRG.673.2 \u0026nbsp;expression inhibits intramuscular preadipocyte differentiation. (A). Detection of LincR-MSTRG.673.2 \u0026nbsp;interference efficiency. (B). Relative mRNA levels of adipocyte differentiation-related genes (\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCEBPA\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePPARG\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, and\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e FASN\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e) after interference with \u0026nbsp;LincR-MSTRG.673.2 \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eexpression. (C). Representative images of Oil Red O staining in intramuscular adipocytes transfected with LincR-MSTRG.673.2 siRNA or the corresponding NC (this image was acquired with a 20 × objective, and an enlarged image is shown in the bottom left corner of the image.). (D). Semiquantitative assessment of Oil Red O absorbance at 450 nm. (E). The triglyceride content was determined by measurement of the absorbance at 500 nm after interference with \u0026nbsp;LincR-MSTRG.673.2 \u0026nbsp;expression.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4405250/v1/320397a3d6db4b209377f8ce.png"},{"id":56998016,"identity":"6b7b8e5a-de94-4b61-9290-861fe60692b2","added_by":"auto","created_at":"2024-05-23 08:00:03","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":688804,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLincR-MSTRG.673.2 promoted intramuscular differentiation by adsorbing miR-128-3p. (A). Effects of cotransfection MSTRG.673.2-Si and miR-128-3p-mimics/inhibitor on the expression of miR-128-3p (B). Effect of cotransfection MSTRG.673.2-Si and miR-128-3p-mimics/inhibitor on the expression of adipose differentiation marker genes\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4405250/v1/2fe77624d3b0c91c6ef69b1e.png"},{"id":64155167,"identity":"c1132fe6-8137-4ca7-b8b8-e215d8188181","added_by":"auto","created_at":"2024-09-09 04:37:59","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":17573376,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4405250/v1/0964dc28-22fc-4b2f-b2ba-02efaef553d9.pdf"},{"id":56998018,"identity":"24d61d60-19f8-4e18-989c-3e7c8c2f1e1c","added_by":"auto","created_at":"2024-05-23 08:00:03","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":13709,"visible":true,"origin":"","legend":"\u003cp\u003eSupporting Information\u003c/p\u003e\n\u003cp\u003eAdditional File 1:Table s1. Q-PCR primer sequences.\u003c/p\u003e","description":"","filename":"AdditionalFile1Tables1.QPCRprimersequences..docx","url":"https://assets-eu.researchsquare.com/files/rs-4405250/v1/ba4e0e984616c43030547bf0.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"LincRNA-MSTRG.673.2 Promotes Chicken Intramuscular Adipocyte Differentiation by Sponging miR- 128-3p","fulltext":[{"header":"Introduction","content":"\u003cp\u003eChicken is currently a major player in the market, with consumers increasingly demanding better taste. In chicken production, intramuscular fat is a crucial factor that positively impacts the quality and flavor of the meat [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The amount of intramuscular fat is determined by the capacity of intramuscular adipocytes to synthesize and store lipids [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Numerous research studies have unveiled a plethora of molecular mechanisms that control the differentiation of intramuscular adipocytes. These mechanisms encompass various aspects such as transcription factors, signaling pathways, and non-coding RNAs. These mechanisms include transcription factors [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], cell cycle regulators, non-coding RNA, signaling pathways [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] and others.\u003c/p\u003e \u003cp\u003eMicro-RNAs (miRNAs), a subclass of small non-coding RNAs with an 18\u0026ndash;25 nucleotide length, are essential regulators of many biological processes, including cell division, proliferation, differentiation and apoptosis [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. For example, miR-425-5p [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] and miR-125a-5p [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] inhibit differentiation in Porcine intramuscular preadipocytes. bta-miR-210 positively regulates the adipogenesis of PDGFRα\u0026thinsp;+\u0026thinsp;cells in Bovine [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. miR-340-5p inhibits sheep adipocyte differentiation [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. miR-223 [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], gga-miRNA-18b-3p [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] and miR-24-3p [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] inhibits intramuscular adipocytes differentiation, while miR-15a [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] and gga-miR-140-5p [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] significantly promoted intramuscular adipogenic differentiation. miR-128-3p is involved in many physiological processes, including follicular development, gastric cancer cell development [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], and lung cancer cell development [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In addition, it is also a biomarker for detecting breast cancer in high-risk benign breast tumors [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], the diagnosis of non-small cell lung cancer [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] and lymphoblastic leukemiav [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. This suggests that miR-128-3p plays an important role in the regulation of various physiological and pathological processes in animals. miR-128-3p promoted GC apoptosis through 14-3-3β/FoxO pathway via repressing YWHAB, and inhibited lipid synthesis by impeding the PPAR-γ/LPL pathway, as well as reduced the secretion of progesterone and estrogen [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. And it impeded 3T3-L1 adipogenesis by targeting Pparg and Sertad2, resulting in the obstruction of preadipocyte differentiation and promotion of lipolysis [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Our previous research also showed that miR-128-3p inhibits intramuscular adipocytes differentiation in chickens by downregulating \u003cem\u003eFDPS\u003c/em\u003e [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Therefore, we speculated that miR-128-3p might be a core miRNA regulated by intramuscular fat differentiation. Similar to miRNAs and long non-coding RNAs (lncRNAs) have also been found to regulate the transcription and translation processes of genes by interacting with DNA, RNA, or proteins. For example, LncRNA PVT1 Facilitates Tumorigenesis and Progression of Glioma via Regulation of MiR-128-3p/GREM1 Axis and BMP Signaling Pathway [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. LncRNA IMFNCR Promotes Intramuscular Adipocyte Differentiation by Sponging miR-128-3p and miR-27b-3p [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. However, LincRNA-MSTRG.673.2 is a newly identified lncRNA whose role in chicken intramuscular adipocyte differentiation has not been fully investigated.\u003c/p\u003e \u003cp\u003eIn this study, We identified several key LincRNAs that play a role in the regulation of lipid metabolism pathways via miR-128-3p using transcriptome data and functional prediction. We explored the interaction between LincRNA-MSTRG.673.2 and miR-128-3p. In addition, the results of our cell functional assays confirmed that LincRNA-MSTRG.673.2 regulates the differentiation of intramuscular adipocytes in chickens.This will provide new ideas for a better understanding of the molecular mechanisms of chicken intramuscular adipocyte differentiation and new strategies and methods for improving chicken meat quality.\u003c/p\u003e"},{"header":"Result","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eIdentification and Characterization of LincRNAs in Chicken Intramuscular Adipocytes Groups Post miR-128-3p Overexpression and Interference\u003c/h2\u003e \u003cp\u003eTo identify the major LincRNAs that interacted with miR-128-3p and involved in the regulation of intramuscular fat deposition. A model of miR-128-3p differentiation in chicken intramuscular adipocytes was constructed, including mock-treated and inhibitor-treated groups, and transcriptome sequencing was performed. After additional filtering and removal of potential coding transcripts that were identified using CNCI, CPC, PFAM and CPAT (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), a total of 1665 lnicRNAs were obtained. Comparative analyses of gene structure, expression, and sequence conservation are used to explore differences between LincRNAs and protein codes genes. lncRNAs were typically shorter than mRNAs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) and tended to contain only two or three exons in contrast to the mRNAs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, D). lncRNAs also appeared to be expressed at lower levels than mRNAs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). This suggested that the LincRNA obtained by sequencing conformed to the characteristics of a non-coding RNA. To confirm the reliability of the RNA-seq results, 5 DELincRNAs (MSTRG.2230.3, MSTRG.2262.4, MSTRG.39.2, MSTRG.673.2, MSTRG.7049.1) were randomly selected, and their expression levels were measured by RT‒qPCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). As expected, the expression levels of all 5 candidate LincRNAs showed a consistent trend of expression, therefore validating our results.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of Differential LincRNAs Expression and Screening for miR-128-3p Targeting LincRNAs\u003c/h2\u003e \u003cp\u003eTranscriptome data analysis of differential LincRNAs indicated that, compared to the NC group, the mimics-treated group had 17 significantly differentially expressed LincRNAs (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), including 6 upregulated and 11 downregulated ones (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eA); the inhibitor-treated group had 17 differentially expressed LincRNAs (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), including 8 upregulated and 9 downregulated ones (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eB); and 24 differentially expressed LincRNAs (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) were observed when comparing the mimics-treated group to the inhibitor-treated group, with 14 upregulated and 10 downregulated ones (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). GO function enrichment analysis found revealed that DELincRNAs from the overexpression group (M group) and interference group (SI group) were involved in negative regulation of metabolic processes, response to steroid hormones and negative regulation of cellular metabolic processes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). KEGG pathway analysis showed that DELincRNAs were involved in Regulation of actin cytoskeleton, Focal adhesion, Tight junction and VEGF signaling pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Upregulated miRNAs affect fat accumulation by downregulating their target LincRNAs. The differential LincRNAs potentially regulated by miR-128-3p, as obtained from the transcriptome data, were compared with the binding sites in the seed region of miR-128-3p. Subsequently, Cytoscape software was used to construct a network of targeting relationships centered on miR-128-3p. In this network, miR-128-3p can target 14 down regulated LincRNAs, such as MSTRG.673.2, MSTRG.2262.4, MSTRG.6216.1, and MSTRG.8694.1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). To increase the probability that the selected LincRNAs regulate IMF deposition, we investigated whether these potential target LincRNAs overlapped among the 2 comparisons (NC vs. SI, and SI vs. M). miR-128-3p were targeted by 6 common lnicRNAs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eG).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eLincR-MSTRG.673.2 Targets Binding to miR-128-3p\u003c/h2\u003e \u003cp\u003eAccording to the online BLAST analysis conducted on the NCBI website, it was found that LincRNA-MSTRG.673.2 belongs to the non-coding sequence and does not overlap with any known genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eA).. Further inquiry on the LncFinder website revealed that LincRNA-MSTRG.673.2 shares similar non-coding RNA coding characteristics with the known LncRNA-GHR-AS. The coding ability scores of both are much lower than the commonly used reference gene GAPDH, indicating that they are non-coding sequences (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). RNA FISH and indicated that LincR-MSTRG.673.2 is predominantly localized in the cytoplasm of preadipocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). After separately extracting cytoplasmic RNA and nuclear RNA from intramuscular preadipocytes, the relative expression level of MSTRG.673.2 was analyzed. The results showed that MSTRG.673.2 was expressed in both the nucleus and cytoplasm (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). we used online software to evaluate the protein coding ability of LincR-MSTRG.673.2. Analysis of the sequence by online software NCBI-BLAST Using dual-luciferase reporter assay, we further validated whether LincR-MSTRG.673.2 served as ceRNA of miR-128-3p. Compared to the cotransfection of psiCHECK2-LincR-MSTRG.673.2-WT and miR-128-3p-mimics groups, the luciferase activity was significantly upregulated in the cotransfection of psiCHECK2 and psiCHECK2-LincR-MSTRG.673.2-WT groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Similarly, the cotransfection of psiCHECK2-MSTRG.673.2-MuT and miR-128-3p-mimics groups also showed a significant increase compared to the control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). This indicated the targeted binding relationship between LincR-MSTRG.673.2 and miR-128-3p (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). To further investigate the regulatory effects of LincR-MSTRG.673.2 and miR-128-3p on intramuscular fat deposition in chickens, quantitative detection was used to examine the expression of miR-128-3p and LincR-MSTRG.673.2 at different time points (0, 2, 4, 6, 8 and 10 days) after induction of intramuscular fat differentiation. The results showed that LincR-MSTRG.673.2 and miR-128-3p exhibited opposite expression patterns during intramuscular adipocytes differentiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003e \u003cb\u003eInterfering with MSTRG.673.2 Inhibits Lipid Deposition in Intramuscular Preadipocytes.\u003c/b\u003e To ascertain the effect of LincR-MSTRG.673.2 in intramuscular preadipocytes differentiation, LincR-MSTRG.673.2 inhibitor or NC was transfected into chicken intramuscular preadipocytes, respectively. The results showed that the interference effect could reach over 50%, meeting the experimental requirements (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The expression of adipogenic genes (\u003cem\u003eCEBPA\u003c/em\u003e、\u003cem\u003ePPARG\u003c/em\u003e、\u003cem\u003eFASN\u003c/em\u003e) were remarkably reduced in LincR-MSTRG.673.2 inhibitor treated cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Subsequently, Oil red staining and intracellular triglyceride content detection were performed. The Oil red staining showed the lipid droplet accumulation in the LincR-MSTRG.673.2 interference group was significantly lower than that in the blank control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, D). The intracellular triglyceride content detection indicated that that interfering with LincR-MSTRG.673.2 decreased intracellular triglycerides (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). These results suggest that interfering with LincR-MSTRG.673.2 reduceed lipid deposition in intramuscular preadipocytes, thereby achieving an inhibitory effect on differentiation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eLincR-MSTRG.673.2 Promoted Intramuscular Differentiation by Adsorbing miR-128-3p\u003c/h2\u003e \u003cp\u003eAfter cotransfection of LincR-MSTRG.673.2-Si with miR-128-3p-mimics/inhibitor, the expression levels of miR-128-3p wasmeasured.Compared to the blank control group, the expression level of miR-128-3p was significantly upregulated after interfering with LincR-MSTRG.673.2 ( P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Simultaneously interfering with LincR-MSTRG.673.2 and overexpressing miR-128-3p led to a highly significant upregulation of miR-128-3p expression (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Furthermore, when interfering with both LincR-MSTRG.673.2 and miR-128-3p, the expression level of miR-128-3p was significantly downregulated (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). It indicated that LincR-MSTRG.673.2 had an inhibitory effect on the expression of miR-128-3p. Additionally, compared with the blank control group, interfering with LincR-MSTRG.673.2 and simultaneously overexpressing miR-128-3p resulted in a significant downregulation of adipogenic marker genes such as \u003cem\u003eFABP4\u003c/em\u003e and \u003cem\u003ePPARG\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). When interfering with both MSTRG.673.2 and miR-128-3p, \u003cem\u003eCEBPA\u003c/em\u003e, \u003cem\u003eFABP4\u003c/em\u003e, \u003cem\u003ePPARG\u003c/em\u003e, and \u003cem\u003eFASN\u003c/em\u003e were significantly upregulated (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"DISSCUSSION","content":"\u003cp\u003eAdipose tissue, with its highly active secretory capacity, can absorb or release non-coding RNAs, leading to a significant role for miRNAs and lncRNAs in the process of fat deposition[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. In this experiment, we specifically selected samples of cells overexpressing/underexpressing miR-128-3p for transcriptome sequencing. We identified 14 upregulated and 10 downregulated differential LincRNAs. Functional enrichment analysis revealed that DELincRNAs were involved in Regulation of actin cytoskeleton, Focal adhesion, Tight junction and VEGF signaling pathway. A series of pathways related to cell junctions including tight junctions, focal adhesion and regulation of the actin cytoskeleton might contribute to the deposition of IMF [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Fatty acid metabolism-related candidate genes also were involved in these pathways [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. VEGF inactivation may induce fat accumulation [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. In addition, some studies have shown that VEGF inactivation induces fat accumulation. Through analysis of significantly differentially expressed LincRNAs following interference, we identified LincRNAs regulated by miR-128-3p, such as LincRNA-MSTRG.673.2, LincRNA-MSTRG.39.2, LincRNA-MSTRG.39.3, and LincRNA-MSTRG.14270.2.\u003c/p\u003e \u003cp\u003eThe specific mechanisms by which lncRNAs regulate miRNAs include: acting as potential pri-miRNAs to generate mature miRNAs, indirectly regulating target gene expression[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]; indirectly inhibiting miRNA-mediated negative regulation of target genes by competitively binding to the 3'-UTR of miRNA target mRNAs[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]; and serving as a \"miRNA sponge\" to inhibit miRNA expression [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. On one hand, miRNAs can bind to the 3\u0026rsquo;-UTR region of lncRNAs, thereby reducing the stability and expression of lncRNAs [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. On the other hand, miRNAs can return to the cell nucleus and regulate the transcription of lncRNAs, enhancing lncRNA expression. Long Noncoding RNA HCP5 Contributes to Nasopharyngeal Carcinoma Progression by Targeting MicroRNA-128-3p [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Knockdown of LncRNA DLEU2 Inhibits Cervical Cancer Progression via Targeting miR-128-3p [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. In this study, LincRNA-MSTRG.673.2 was mainly localized in the cytoplasm. This study analyzed and verified the existence of a targeting relationship between LincRNA-MSTRG.673.2 and miR-128-3p. Also, LincR-MSTRG.673.2 and miR-128-3p exhibited opposite expression patterns during intramuscular adipocytes differentiation. Hence, the LincRNA-MSTRG.673.2 acted as a sponge for miR-128-3p. lncRNAs are involved in a variety of bioregulatory processes, including muscle growth, fat deposition, and lipid metabolism [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. For example, lncSAMM50 [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e], lncRNA 1 [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e] enhances adipocytes differentiation. We showed that interfering with LincRNA-MSTRG.673.2 inhibited intramuscular fat deposition. The group's previous research showed that miR-128-3p could inhibit the differentiation of intramuscular adipocytes, a result consistent with the interference of LincRNA-MSTRG.673.2. Additionally, after interfering with LincRNA-MSTRG.673.2, miR-128-3p was significantly upregulated, aligning with the mechanism of action between LincRNA and miRNA. For example, LincRNA TINCR promoted lipid deposition by absorbing miR-31-5p to enhance the expression of CEBP pathway genes [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. \u003cem\u003ePPARG\u003c/em\u003e is indeed a direct target of miR-128-3p [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Transfection experiments with LincRNA-MSTRG.673.2-Si and miR-128-3p-mimics showed that interfering with LincRNA-MSTRG.673.2 upregulated the expression of miR-128-3p and downregulated the expression of adipocytes differentiation marker genes (\u003cem\u003eCEBPA\u003c/em\u003e, \u003cem\u003eFABP4\u003c/em\u003e, \u003cem\u003ePPARG\u003c/em\u003e、\u003cem\u003eFASN)\u003c/em\u003e. This implied that lncRNA may have competitively adsorbed miR-128-3p, thereby affecting \u003cem\u003ePPARG\u003c/em\u003e and potentially influencing intramuscular fat deposition. Our research identified LincRNA-MSTRG.673.2 targeting miR-128-3p to promote chicken intramuscular adipocytes differentiation. Determining miR-128-3p as a key regulatory factor for intramuscular preadipocytes fat deposition in chickens is of significant importance for breeding practices aimed at improving chicken meat quality.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eIn conclusion, this study demonstrated that LincR-MSTRG.673.2 could promote chicken intramuscular adipocytes differentiation by Sponging miR-128-3p. These findings may contribute to furnish new insights for improving chicken quality breeding.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eCollection of Sequencing Samples\u003c/h2\u003e \u003cp\u003eThe 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). The expression of miR-128-3p was detected by Q-PCR. Subsequently, samples with high overexpression efficiency (n\u0026thinsp;=\u0026thinsp;3), high interference efficiency (n\u0026thinsp;=\u0026thinsp;3), and samples from the blank group were sent to Nanjing Parsono Gene Technology Co., Ltd. for transcriptome sequencing.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eTranscriptome Data Analysis\u003c/h2\u003e \u003cp\u003eAfter 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 [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Read count values were aligned to each gene using HTSeq statistics as the gene's original expression quantity [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. CPC [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], CNCI [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], and PFAM[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] to distinguish the protein-coding genes from the noncoding genes. Differential LincRNAs expression analysis was conducted using DESeq [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], and differentially expressed LincRNAs were defined based on the following criteria: |log\u003csub\u003e2\u003c/sub\u003e fold change| \u0026gt; 1 and the \u003cem\u003eP\u003c/em\u003e value\u0026thinsp;\u0026lt;\u0026thinsp;0.05.Gene Ontology (GO) [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] and [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] (KEGG) analysis for predicting the function of the DE lncRNAs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003ePrimary Intramuscular Adipocytes Isolation and Culture and Induced Differentiation\u003c/h2\u003e \u003cp\u003eIntramuscular adipocytes were isolated as previously described [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Breast muscle tissue was collected from 14-day-old Gushi chicken under sterile conditions. and then digested using 2 mg/ml collagenase type II (Sangon Biotech, Shanghai, China) with shaking for 2 h at 37\u0026deg;C. Then the tissue was was cut into 1 mm\u003csup\u003e3\u003c/sup\u003e pieces using surgical scissors and then digested performed by adding appropriate amounts of collagenase type I (Solaibao, Beijing, China) with shaking for 1 h at 37\u0026deg;C. The digested cell suspension was filtered through 45 and 75 mum filter collected by centrifugation at 1000 r/min for 10 min. Subsequently, the cells were kept in DMEM/F12 media (BI, Massachusetts, USA) supplemented with 10% fetal bovine serum (BI, Massachusetts, USA) and 1% penicillin/streptomycin (Solarbio) in an incubator that maintained a 5% CO\u003csub\u003e2\u003c/sub\u003e environment at 37\u0026deg;C. Cells were cultured for 2 hours, followed by a fluid change. Once the intramuscular adipocytes confluence reaches 90%, the media would be replaced entirely by the differentiation-inducing media composed of 0.5 mM of 3-isobutyl-1-methylxanthine (IBMX), 1 uM of dexamethasone (Sigma), and 10 g/l of insulin (Sigma) [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eTransient Transfection of Intramuscular Adipocytes\u003c/h2\u003e \u003cp\u003eCells were transfected with miR-128-3p mimics, inhibitors and LincR-MSTRG.673.2 with Liposome 3000 reagent (Invitgen, USA) according to the manufacturer's instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eLocalization Analysis of LincR-MSTRG.673.2 using Fluorescence In Situ Hybridization (FISH) and Nuclear/Cytoplasmic RNA Separation\u003c/h2\u003e \u003cp\u003eFluorescence in situ hybridization was designed and synthesized by Wuhan Saviour Biotech Co., Ltd. The probe information was as follows: 5\u0026rsquo;-3\u0026rsquo; GAGGAACAGGAGAGATATGCTACTCATT-TTGTATA. The intramuscular fat cells were attached to the glass slide and grew to 80% density. The operation was carried out according to the instructions of the in situ hybridization kit. 4% paraformaldehyde was used to fix the cells, DAPI was used to stain the cells, and images were captured under a fluorescence microscope. CY3 red light had an excitation wavelength of 510\u0026ndash;560 nm and an emission wavelength of 590 nm, emitting red light. The primary intramuscular preadipocytes that were cultured were digested using trypsin, and the digestion was terminated with complete culture medium. Subsequently, PBS was added for preservation, and the subsequent operations were carried out according to the nuclear-cytoplasmic separation kit. The collected cytoplasmic mixture and cytoplasmic precipitate were separately subjected to RNA extraction using the Trizol method. Then, using U6 as the internal reference, the expression levels of MSTRG.673.2 in the cytoplasm and nucleus were quantitatively detected.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eDual Luciferase Reporter Assay\u003c/h2\u003e \u003cp\u003eThe laboratory psiCHECK2 vector was used to construct the psiCHECK2-LincR-MSTRG.673.2-3'UTR-WT and psiCHECK2- LincR-MSTRG.673.2-3'UTR-MuT plasmids. They were transfected with miRNA-128-3p-mimics and psiCHECK2 vector into DF1 cells. After 48h, the samples were collected, and the fluorescence activity was detected using the Dual-Glo Luciferase Assay Systemt (Promega, Madison, WI, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eOil-Red O Staining Triglyceride Assay Detection of Cell Differentiation\u003c/h2\u003e \u003cp\u003eThe intramuscular adipocytes samples were fixed with 4% paraformaldehyde [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], and Oil Red O working solution (Sigma) was used for cell staining. Intramuscular fat cells were photographed under a microscope. After microscopic examination, isopropanol was used to dissolve the lipid droplets, and the absorbance value was calculated at 490nm. The triglyceride content in the cell homogenate was determined using a triglyceride content detection kit (APPLYGEN, Beijing, China). The protein concentration was measured using a BCA protein assay kit (EpiZyme, Shanghai, China) to standardize the triglyceride content. The absorbance values were calculated at 550nm using a microplate reader.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eQ-PCR\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from intramuscular adipocytes using Trizol (Vazyme, Nanjing, China). mRNA was reverse transcribed using the HiScript II Q Select RT SuperMix for qPCR kit (Vazyme, Nanjing, China), and quantitative real-time PCR was performed using the ChamQ Universal SYBR qPCR Master Mix kit (Vazyme, Nanjing, China). The relative expression levels were calculated using the 2-\u003csup\u003eΔΔCt\u003c/sup\u003e method [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. The primer sequences were shown in Supplementary table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eData analysis was performed with SPSS 26.0, All data was presented as \u0026ldquo;mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error (SEM)\u0026rdquo;. Significant differences between groups were analyzed using one-way ANOVA. Asterisks signify different significance levels (#, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05, *\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, and ***\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e\u003cem\u003eCNCI \u003c/em\u003eCoding-noncoding index \u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCPC \u003c/em\u003eCoding potential calculator \u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIMF \u003c/em\u003eIntramuscular fat\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMiRNA \u003c/em\u003eMicroRNA\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eNC group \u003c/em\u003eBlank treatment group\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eM group \u003c/em\u003eMimics-treated group\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSI group \u003c/em\u003eInhibitor-treated group\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eGO \u003c/em\u003eGene ontology\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eKEGG \u003c/em\u003eKyoto encyclopedia of genes and genomes\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eQ-PCR \u003c/em\u003eQuantitative PCR\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePBS \u003c/em\u003ePhosphate bufered saline\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePPARG \u003c/em\u003ePeroxisome proliferator-activated receptor \u0026gamma;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eFABP4 \u003c/em\u003eFatty acid binding protein 4\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eFASN \u003c/em\u003eFatty acid synthetase\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCEBPA \u003c/em\u003eCCAAT/enhancer-binding protein alpha\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eFDPS \u003c/em\u003eFarnesyl Diphosphate Synthase\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAll 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.\u003c/p\u003e\n\u003cp\u003eAUTHOR CONTRIBUTIONS\u003c/p\u003e\n\u003cp\u003eShuaipeng Zhu and Binbin Zhang conceived the project and designed the experiments. Yuehua He Wenjie Liang, and Tingqi Zhu, \u0026nbsp;performed the animal experiments and sample collection. Wenting Li, Ruili Han, Donghua li, Fengbin Yan provided valuable suggestion and comments to improve the manuscript with contributions from all other authors. Yadong Tian, Guoxi Li, Xiangtao Kang, and Guirong Sun discussed the results. All authors have read and approved the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFUNDING INFORMATION\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants from the National Natural Science Foundation of China (32072710), the Key Research Project of the Shennong Laboratory (SN01-2022-05) and the Scientific Studio of Zhongyuan Scholars (30601985).\u003c/p\u003e\n\u003cp\u003eCONFLICT OF INTEREST STATEMENT\u003c/p\u003e\n\u003cp\u003eThe authors declare that there is no conflict of interest.\u003c/p\u003e\n\u003cp\u003eAVAILABILITY OF DATA AND MATERIALS\u003c/p\u003e\n\u003cp\u003eThe RNA sequencing data used and analyzed during the current study are available from the NCBI (accession number: PRJNA986221).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSun Y, Lin X, Zhang Q, Pang Y, Zhang X, Zhao X, Liu D, Yang X. Genome-wide characterization of lncRNAs and mRNAs in muscles with differential intramuscular fat contents. 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Front cell Dev biology. 2021;9:619842.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"miR-128-3p, Intramuscular adipocytes, LincRNA-MSTRG.673.2","lastPublishedDoi":"10.21203/rs.3.rs-4405250/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4405250/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eIntramuscular fat content is positively correlated with meat flavor and juiciness. Increasing the intramuscular fat (IMF) content of chickens while increasing their growth rate has become a hot topic in molecular breeding.The group's previous studies showed that miR-128-3p inhibited chicken intramuscular adipocyte differentiation and lipogenesis. However, the regulatory mechanism of miR-128-3p in intramuscular preadipocytes is currently unknown. In this study, we investigated the mechanism of miR-128-3p regulation of chicken intramuscular adipocyte differentiation and deposition.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eRNA-seq was performed to screen for long non-coding RNAs (lncRNAs) that bind to miR-128-3p. Dual luciferase reporter system was used to verify the targeting relationship between miR-128-3p and LincRNA-MSTRG.673.2; nucleoplasmic localization analysis and fluorescence in situ hybridization were used to investigate the localization of LincRNA-MSTRG.673.2 in the cells; a series of experiments such as Q-PCR, Oil O Red staining and triglyceride assay were used to explore the effect of interference with LincRNA-MSTRG.673.2 on the differentiation of intramuscular preadipocytes; co-transfection experiments were used to validate the regulatory patterns of miR-128-3p and LincRNA-MSTRG.673.2 in intramuscular adipocytes.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eTranscriptome data analysis of differential LincRNAs indicated that, compared to the NC group, the mimics-treated group had 17 significantly differentially expressed LincRNAs (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), including 6 upregulated and 11 downregulated ones; the inhibitor-treated group had 17 differentially expressed LincRNAs (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), including 8 upregulated and 9 downregulated ones; and 24 differentially expressed LincRNAs (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) were observed when comparing the mimics-treated group to the inhibitor-treated group, with 14 upregulated and 10 downregulated ones. Functional enrichment analysis revealed that DELincRNAs from the overexpression group (M group) and interference group (SI group) were involved in negative regulation of metabolic processes, response to steroid hormones, regulation of actin cytoskeleton. Furthermore, target gene prediction analysis showed that miR-128-3p can target many of the DELincRNAs, such as LincRNA-MSTRG.673.2, LincRNA-MSTRG.39.2, LincRNA-MSTRG.39.3, and LincRNA-MSTRG.14270.2. LincRNA-MSTRG.673.2 was predominantly expressed in cytoplasm of intramuscular adipocytes. Dual luciferase reporter identified the targeting relationship between miR-128-3p and LincRNA-MSTRG.673.2. The results of subsequent functional assays demonstrated that Interfering with MSTRG.673.2 has been shown to inhibit lipid deposition in intramuscular preadipocytes. Transfection experiments have shown that LincR-MSTRG.673.2 can affect the expression of miR-128-3p.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThis study found that LincRNA-MSTRG.673.2 promoted chicken intramuscular adipocytes differentiation by down regulating miR-128-3p. The results are noteworthy for improving chicken meat quality, molecular breeding, and lipid metabolism research.\u003c/p\u003e","manuscriptTitle":"LincRNA-MSTRG.673.2 Promotes Chicken Intramuscular Adipocyte Differentiation by Sponging miR- 128-3p","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-23 07:59:58","doi":"10.21203/rs.3.rs-4405250/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7e8ff8c6-db08-4b79-8321-c82e53fba320","owner":[],"postedDate":"May 23rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-09-09T04:29:43+00:00","versionOfRecord":[],"versionCreatedAt":"2024-05-23 07:59:58","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4405250","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4405250","identity":"rs-4405250","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}