The Molecular Mechanism Of MSTN Regulating The Differentiation Of Porcine Preadipocytes Through MMP-2/7/9

Objective: With the improvement of people's quality of life,people have higher and higher requirements for pork quality.Therefore,improving the quality of pork and increasing the lean meat percentage are considered to be the main directions of livestock breeding.Intramuscular fat(IMF)has been reported to be a key factor affecting pork quality.Other studies have shown that excessive intake of fat can cause obesity in the human body,and induce diseases such as fatty liver and diabetes.Intramuscular fat deposition is regulated by multiple genes.Myostatin(MSTN)plays an important role in the regulation of intramuscular fat deposition.Studies have reported that MSTN can regulate C/EBP? through the MAPK/JNK signaling pathway to affect the differentiation of adipocyte precursors.Combining with the previous experiments of the research group: Through transcriptome and proteomic analysis of MSTN geneknockout pig backfat tissue,differential expression of C/EBP? and MMP-2/7genes was found.In addition,multiple binding sites were detected in the promoter regions of the C/EBP? and MMP-2/7 genes.In this study,we first detected the expression of MMP-2/7/9 in PK cells that have the MSTN gene knocked out;Then up-regulate the expression of MSTN and interfere with the expression of MSTN in porcine intramuscular adipose precursor cells,further verify the correlation between MSTN and MMP-2/7/9,in order to enrich and improve the basic research of MSTN regulating pig fat deposition,and provide new targets for improving pork quality.The research results are as follows:1.The genotypes of the three PK cells were verified by PCR,and the results showed that the PCR product of the wild-type PK15 cell was 1563 bp single band;the PCR product of PK3108 cell with MSTN single allele knockout was 1563 bp and 4389 bp two bands;The product of L18 cells with MSTN double allele knockout was 1359 bp and 4593 bp two bands.The results prove that the genotypes of the three cells are stable and can be used in subsequent experiments.2.The genotypes of the three PK cells were verified to be stable and can be used in subsequent experiments.3.The expression levels of MMP-2/7/9 in the three PK cells were detected,and the results showed that the loss of MSTN caused the m RNA and protein expression levels of MMP-2/7/9 to decrease significantly(p<0.01).4.The observation of cell morphology and identification of oil red Oproved that this experiment successfully isolated adipose precursor cells in pig muscle,and the cells had the ability to induce differentiation into mature adipocytes.5.Firstly,q RT-PCR and Western Blot were used to verify that the use of recombinant proteins to overexpress MSTN and RNAi to interfere with MSTN in adipose precursor cells had a significant effect(p<0.01).Secondly,q RT-PCR and Western Blot detection showed that As the expression of MSTN increased,the expression of MMP-2/7/9 also increased significantly(p<0.01).As the expression of MSTN decreased,the expression of MMP-2/7/9 also decreased significantly(p<0.01).Conclusion: The expression level of MMP-2/7/9 is significantly down-regulated in PK cells with the MSTN gene knocked out;Over-expressing MSTN or interfering with MSTN in intramuscular adipose precursor cells,MMP-2/7/9 expression levels will be up-regulated and down-regulated accordingly.In summary,it is concluded that there is a certain regulatory relationship between MSTN and MMP-2/7/9.