Mechanism Of ACSL1 And CAT Genes On Adipogenic Differentiation Of Sub-predecessal Fat Cells In Erhualian Pigs

As one of the main factors affecting pigs’ growth and meat quality,subcutaneous fat deposition has been focued of attention in pig production.In recent years,many important genes related to subcutaneous fat deposition in pigs have been discovered,which provided a foundation to further explore the genetic mechanism of subcutaneous fat formation in pigs,and this is a new way to accelerate the improvement of the meat quality of Erhualian.In current study,the long-chain acyl-COA synthetase 1(ACSL1)and catalase(CAT),which screened from the results of genome-wide transcriptome analysis of distinct differentiation stages of subcutaneous preadipocytes,selected as potential candidate gene related to subcutaneous adipogenesis.In this study,stromal vascular(SV)cells,separated from the subcutaneous adipose tissue of erhualian pigs,were used as model to study the differentiation mechanism of fat by using gene interfering,overexpression,real-time quantitative PCR(qRT-PCR)and oil red O staining methods,the function of candidate genes in subcutaneous adipogenesis further analyze and examine.The specific process and results of this paper were as followed:1.The effect of ACSL1 gene on the differentiation of porcine preadipocytes was studied.Firstly,we analyzed the expression of ACSL1 gene in distinct tissues of pigs and SV cells at 0,2,4 and 8 days of the differentiation process.The results showed that ACSL1 gene high express level in subcutaneous adipose tissue and the expression levels of ACSL1 gene increased significantly during the cell differentiation process.And then,siRNA could interfere with ACSL1 gene expression.The results showed that ACSL1 gene mRNA expression,protein level,and triglyceride synthesis ability in adipocytes significantly decreased(P<0.01);adipose differentiation marker genes PPARγ and C/EBPα did not significantly change the expression level(P>0.05);FABP4 and APOE gene expression significantly decreased(P<0.01);while lipolysis genes ATGL and HSL gene expression level significantly increased(P<0.05).Conversely,overexpression of the ACSL1 gene resultes in a significant(P<0.01)increased in the expression of ACSL1 gene mRNA,protein,and triglyceride in SV cells.There were not significant change in the early differentiation marker genes PPARy and C/EBPα expression level(P>0.05),but the lipid-binding genes FABP4 and APOE and the lipolysis genes ATGL and HSL significantly decreased(P<0.01,P<0.05).Moreover,miR-218-5p may target the ACSL1 gene by bioinformatics analysis.Further test results confirmed that miR-218-5p significantly reduced ACSL1 mRNA expression and protein level.Furthermore,miR-218-5p significantly inhibited triglyceride synthesis in SV cells via targeting A CSL1 gene.2.The influence of interfering CAT gene by siRNA on differentiation of porcine subcutaneous preadipocytes was researched.In this study,the expression patterns of CAT gene during the preadipocyte differentiation process and CAT gene mRNA level in various tissues of Erhualian pigs detect using qRT-PCR.Moreover,CAT gene was knockdowned by siRNA,and then,the ability of subcutaneous preadipocytes differentiation was evaluated by oil red O staining.At the same time,the expression levels of the key adipocyte differentiation-related genes such as PPARγ,C/EBPα,FABP4,APOE,ADIPOQ,PLIN1,and HSL,and the antioxidant-related genes such as SOD2,GSS,and PRDX3,were detected by qRT-PCR.The results indicated that the expression levels of CAT gene was the highest on the 4th day of the preadipocytes differentiation.Furthermore,the mRNA levels of CAT in subcutaneous fat tissues was significantly higher than that of intramuscular fat tissues(P<0.01).Decreasing the expression of CAT gene could significantly increase the H2O2 content in cells and inhibit the triglyceride levels in SV cells(P<0.05).The effect on the expression level of PPARγ,C/EBPα,FABP4 and APOE genes were not significant(P>0.05),but the expression level of ADIPOQ and PLIN1 gene were significantly decreased(P<0.05 or P<0.01)and the expression level of HSL gene was increased(P<0.05),while the expression of anti-oxidase-related genes SOD2,GSS and PRDX3 were not significantly changed(P>0.05).The result of the experiment showed that interfering CAT gene expression could inhibit the adipogenic differentiation of preadipocytes.In summary,this study found that interference of ACSL1 gene expression had an inhibitory effect on the subcutaneous proadipocyte differentiation of pigs,which resulted in reducing the expression of FABP4 and APOE genes in the late differentiation stage and promoting the expression levels of lipolysis genes of ATGL and HSL.On the other hand,overexpression of ACSL1 gene promoted the adipogenic differentiation of pig subcutaneous adipocytes by increasing the expression of FABP4 APOE gene and decreasing the expression of lipolysis genes ATGL and HSL.In addition,miR-218-5p could inhibit adipocyte differentiation by targeting ACSL1 gene.The CAT gene regulated the subcutaneous adipogenic differentiation of pigs by affecting the level of H2O2,while effected the expression levels of the adipogenic related gene of PLIN1 and ADIPOQ and the lipolytic gene of HSL.