Functional Study Of SIRT3 On Intramuscular Preadipocyte Proliferation And Lipid Deposition In Chickens

The demand for higher-quality chicken meat rises as chicken products,which are a common meat product around the world,are consumed more frequently.The amount of intramuscular fat(IMF)is a significant predictor of the flavor,softness,and juiciness of the muscle.By boosting the IMF content in chicken meat during the breeding of local hens,the flavor and taste of chicken meat can be further enhanced.Many variables,including the volume and density of adipocytes as well as lipid absorption anabolism and catabolism,influence the amount of intramuscular fat.Research have revealed that Sirtuin3(SIRT3)is essential for a number of metabolic processes in the mammalian body.This study examined the effects of SIRT3 overexpression and interference in chicken intramuscular preadipocytes on the proliferation and lipid deposition of intramuscular preadipocytes by overexpressing and interfering with SIRT3 in Gushi chicken intramuscular preadipocytes in order to examine the function and regulatory mechanism of SIRT3 in chicken intramuscular preadipocytes.1.SIRT3 bioinformatics and expression pattern analysisThe cloned SIRT3 coding sequence from Gushi chicken had 1 041 bp,8 exons,and encoded 346 amino acids.Bioinformatics software was used to evaluate the amino acid sequence of the SIRT3 coding region.The results revealed that the SIRT3 protein lacked a signal peptide and a transmembrane structure.It was an evolutionarily highly conserved weak hydrophilic protein.By using PCR,the 2000 bp sequence preceding the SIRT3 gene’s transcription start point was amplified.Promoter primers of various lengths were created using bioinformatics analysis to create truncated vectors with various lengths of the promoter region,and the dual fluorescence activity of several truncated vectors of the SIRT3 promoter was observed.The outcomes demonstrated that the transcription start point was 724 bp upstream of the SIRT3 core promoter region.C/EBP,ER,SPl,T3 R,and other transcription factors may be involved in the transcriptional control of SIRT3 according to bioinformatics analysis of the main promoter region of SIRT3.The Gushi chicken’s tissue expression profile revealed that the relative expression of the SIRT3 gene was higher in the heart,leg,and breast muscles than in the liver,abdomen,and subcutaneous fats(P<0.05).During various developmental stages,the SIRT3 gene expression in the breast muscle tissue of Gushi hens showed a pattern of first increase and then reduction,peaking at 30 weeks.Preadipocytes were induced,and the SIRT3 gene’s expression first rose and subsequently fell.2.Effect of SIRT3 on the proliferation and lipid deposition of intramuscular preadipocytesIn this study,a vector for SIRT3 overexpression and a SIRT3 interference fragment were created.In intramuscular preadipocytes,SIRT3 was overexpressed or interfered with,and the cells were subsequently grown for 48 hours.According to the q PCR results,SIRT3 expression was highly upregulated following SIRT3 overexpression(P<0.05)and significantly downregulated following SIRT3 interference(P<0.05).The genes associated with boosting proliferation were dramatically downregulated and the genes associated with preventing proliferation were significantly upregulated after SIRT3 overexpression in intramuscular preadipocytes(P<0.05).SIRT3 interference led to the opposite outcomes.The proliferation of intramuscular preadipocytes was decreased after SIRT3 overexpression,according to CCK-8data.Results from flow cytometry revealed a large increase in G1 phase cells and a significant decrease in G2 and S phase cells(P<0.05),demonstrating an inhibition of the cell growth cycle.Results from the Experiment revealed that overexpression of SIRT3 decreased preadipocyte proliferation after transfection of pc DNA3.1-SIRT3 into ICP1 cells(P<0.05).When SIRT3 was hampered,the reverse outcome was seen.Our findings show that SIRT3 restricts the growth of chicken intramuscular preadipocytes.After transfection of the SIRT3 overexpression vector and interference fragment,the expression levels of genes involved in lipid deposition were discovered.According to the q PCR data,SIRT3 overexpression significantly elevated the expression of CPT1 A,SLC27A1,and ATGL(P<0.05).The expression of SIRT3 decreased after SIRT3 was interfered with(P<0.05).Oil red O staining results revealed that SIRT3 overexpression decreased intramuscular adipocyte lipid droplet production whereas SIRT3 interference enhanced lipid droplet concentration.Examining the OD at 500 nm helped to further confirm the aforementioned findings.Triglyceride content findings revealed that SIRT3 overexpression considerably lowered and SIRT3 knockdown dramatically raised the triglyceride content in adipocytes.The extracellular glycerol level and mitochondrial membrane potential both significantly increased with SIRT3 overexpression in intramuscular preadipocytes(P<0.05).It dropped after interference(P<0.05).Our findings show that SIRT3 suppresses lipid synthesis and speeds up triglyceride breakdown.3.SIRT3-mediated lipid deposition critical gene and lnc RNA screening and coexpression network building in chicken intramuscular preadipocytesA Intramuscular preadipocytes overexpressed SIRT3.According to RNA-seq analysis,a total of 173 differentially expressed genes were found,of which 74 showed a significant upregulation and 99 a significant down-regulation.In the overexpression group,there were 44 lncrnas that were statistically different,of which 18 had significant up-regulation and 26 had significant down-regulation.The PPAR signaling pathway,amino acid biosynthesis,unsaturated fatty acid biosynthesis,metabolic pathway,and other processes were enriched in the target genes of differentially expressed SIRT3 overexpressed m RNA and differentially expressed lnc RNA.Six differentially expressed genes that are linked to fat deposition are involved in the control and metabolism of biological activities.For instance,SAT1 is crucial in controlling the system’s energy metabolism,HMOX1 influences lipid peroxidation,RGN encourages the hydrolysis of triglycerides in lipid droplets,ANGPTL4 deficiency increases triglyceride uptake in adipose tissue,and PPARG is a crucial transcription factor for preadipocyte differentiation and lipid metabolism.The relationship between these 6 target genes and 14 lnc RNAs with correlation coefficients of ≥ 0.9 or ≤-0.9 was further investigated,and a co-expression network with lnc RNA-m RNA was built as a result.In conclusion,this study demonstrated that SIRT3 inhibits proliferation and lipid deposition in chicken intramuscular preadipocytes through overexpression and interference assays.Additionally,a theoretical framework for further investigation of SIRT3’s regulatory mechanism for chicken intramuscular lipid deposition was developed through transcriptome sequencing screening of the network of lnc RNA and m RNA co-expression.