| Intramuscular fat content influences sensory quality traits of pork, for example, juiciness, tenderness, and flavor. Meanwhile, it is also associated with insulin resistance in humans. Adipose-derived stem cells (ADSCs) and muscle satellite cells (MSCs) are two kinds of stem cells in muscle tissue. Previous studies have shown that they all have the characteristics of differentiation into adipocyte.While the main source of intramuscular fat cells has not been confirmed. Myostatin (MSTN) is a secreted protein that is secreted by the muscle cells, belongs to the transforming growth factor-P (TGF-P) family. Our previous study has indicated that MSTN can inhibit adipogenesis in MSCs but not in ADSCs. It was concluded that intramuscular fat cells may be mainly derived from ADSCs. However, the mechanism by which MSTN differently regulates adipogenesis in these two celltypes remains unknown. The main source of intramuscular fat cells derived from ADSCs has not been confirmed. In order to solve the above problems, this study mainly explored from two parts:First, porcine ADSCs and MSCs were treatment with MSTN, and then the expression of myogenic differentiation factor 1 (MyoD) and peroxisome proliferator-activated receptor y (PPARy) were detected; explored the role of MyoD in the regulation of PPARy. Second, porcine ADSCs and MSCs were treatment with MSTN for 48 h, screening of differentially expressed genes before and after MSTN treatment using high-throughput sequencing. The main results were as follows:1. Effect of MSTN on the expression of PPARy and MyoD geneFirstly, porcine ADSCs and MSCs were isolated by enzyme digestion method. And then, cells were identified by immunocytochemistry (detected the expression of desmin and CD44) and Oil red O staining (identified the ability of cells adipogenesis differentiation). The results indicated that ADSCs and MSCs were successfully isolated.ADSCs and MSCs were treatment with 100 ng/mL MSTN for 0 h,24 h, and 48 h. Then the expression of PPARy and MyoD in mRNA, protein, and methylation levels were detected, respectively. The results shown that the expression of PPARy and MyoD mRNA levels were not significant changed between 24 h and 0 h, while the expression of MyoD had an uptrend in ADSCs. PPARγ and MyoD mRNA levels significantly increased by 2.76-fold and 8.33-fold, respectively, at 48 h compared with the 0 h time point in ADSCs; PPARγand MyoD protein expression levels increased from 0 h to 48 h, while the methylation levels of the two genes were decreased. In MSCs, PPARγ and MyoD mRNA levels were steadily declined from 0 h to 48 h; the protein expression of PPARγ and MyoD were decreased in 24 h and 48 h compared with 0 h, while the the methylation levels of PPARγ and MyoD were increased.Conclusion, PPARγ and MyoD expression were up-regulated in ADSCs after treatment with MSTN. Meanwhile, the expression of MyoD in mRNA level increased earlier (24 h) and increased degree higher (8.33>2.76) compared to PPARγ. However, the expression of PPARγ and MyoD were down-regulated in MSCs treated by MSTN. These results indicated that the different regulatory adipogenic roles of MSTN in ADSCs and MSCs act bydifferentially regulating PPARγand MyoD expression.2. Effect of transcription factor MyoD on the transcriptional activity of PPARγThe results showed:over-expression of MyoD up-regulated PPARγ expression in ADSCs. Meanwhile, the storage of lipid droplets and the mRNA expression leves of adipogenic marker genes (aP2 and C/EBPα) were higher in over-expression group compared to control group. These indicated that over-expression of MyoD enhanced porcine adipocyte differentiation.Whereas small interfering RNA (siRNA) against MyoD significantly attenuated PPARγ expression and inhibited porcine adipocyte differentiation in MSCs.Five deletion fragments of PPARγ promoter were constructed into pGL3 plasmids. Dual luciferase activity analysis showed the transcriptional activity of pGL3-PPARγP3 was highest. Over-expression MyoD significantly improved PPARγ promoter deletion fragments transcriptional activity. Wild-type pGL3-PPARγP3 plasmid as template, the poteintial MyoD-binding site and E-box site were mutated, and then co-transfected with pcDNA-MyoD. Result showed that the transcriptional activity of mutated plasmid was significantly lower than that of wild-type plasmid. The results of EMS A and ChIP assays confirmed that MyoD bound to the MyoD binding site and E-box region in the PPARy promoter.3. Screening and functional analysis of differentially expressed genes in porcine ADSCs and MSCs treatment with MSTNADSCs and MSCs were treatment with 100 ng/mL MSTN for 48 h and then total RNA was extracted by Trizol method. There are 3 biological replicates of each group, and 12 sequencing library were constructed and used to expression profiling of high throughput sequencing. It stipulated that AM was "ADSCs treatment with MSTN group"; AC was "ADSCs Control group"; MM was "MSCs treatment with MSTN group"; MC was "MSCs Control group". Sequencing showed that:there were 257 differentially expressed genes in AC and MC group (116 up-regulation expression genes and 141 down-regulation expression genes); 139 differentially expressed genes in AM and AC group (37 up-regulation expression genes and 102 down-regulation expression genes); 741 differentially expressed genes in AM and MM group (649 up-regulation expression genes and 92 down-regulation expression genes); 1640 differentially expressed genes in AC and MC group (60 up-regulation expression genes and 1580 down-regulation expression genes).GO function and KEGG pathway analysis of differentially expressed genes, obtained from MSTN treatment group compared with control group in ADSCs and MSCs, respectively.Screening differentially expressed genes related to fat development and had different expression patterns in ADSCs and MSCs. In this study, we selected two genes (WISP2 and KLF6) for follow-up research. The results of Oil red 0 staining showed that over-expression of WISP2 inhibited porcine adipocyte differentiation in ADSCs; while siRNA against W1SP2 promoted adipocyte differentiation. Over-expression of KLF6 promoted adipocyte differentiation, siRNA against KLF6 inhibited porcine adipocyte differentiation in ADSCs.Conclution: ① In ADSCs, MSTN induces the expression of transcription factor MyoD. MyoD can bind to the PPARy promoter and stimulate PPARy gene expression, thereby enhancing adipocyte differentiation. Whlie in MSCs, the expression of MyoD was inhibited, the promotion effect of MyoD on PPARy was interrupted, and the differentiation of cells into fat was also affected. ② The expression of PPARγ and MyoD were affected by promoter methylation. ③ WISP2 and KLF6 play a role in regulayion of adipocyte differentiation in the process of MSTN treatment. ④ Intramuscular fat cells mainly derived from ADSCs. |
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