Effect Of Pig PLIN1 Gene On Skeletal Muscle Satellite Cell Metabolism And Mitochondrial Function

Lipid droplet coating protein 1(Perilipinl,PLIN1)is a surface protein located on lipid droplets of adipocytes,and has a regulatory effect on the metabolism of lipolytic cells and the function of mitochondria.In the early stage,our group used iTRAQ technology to detect differential proteins in muscle tissues of different pig breeds,and screened out a number of differential genes including PLIN1 gene.We speculated that PLIN1 may be related to differences in muscle and fat development of different breeds of pigs.In order to explore the mechanism of the effect of the porcine PLIN1 gene on the metabolism and mitochondrial function of skeletal muscle satellite cells,this study constructed and used the CRISPR/Cas9 gene knock-out system to prepare and screen the porcine PLIN1 gene knock-out vector,and to have myogenesis and growth.Porcine skeletal muscle satellite cells with adipogenic differentiation potential were used as the research object.By regulating the expression of PLIN1 gene,the effect of PLIN1 gene on the metabolism of porcine skeletal muscle satellite cells and the function of mitochondria was detected.This research mainly includes the following parts:Study 1 Constructing pig PLIN1 gene knockout vector and knockout efficiency test by Crispr/Cas9 systemPLIN1 is a protein that regulates basal lipolysis and promotes the interaction between mitochondria and lipid droplets.In this study,based on the porcine PLIN1 gene sequence,the CRISPR/Cas9 system was used to design 3 sgRNAs that can target the PLIN1 gene,and the corresponding gene knockout vectors were constructed.The knockdown efficiency was determined at the DNA level by sequence detection.The total RNA and protein of each group of cells were extracted,and the levels of PLIN1 RNA and protein were determined by qRT-PCR and Western Blot.The results showed that base mutations or fragment deletions were detected in the PLIN1 DNA of the cells in each group.The mutation rates of the PLIN1-KOl,PLIN1-KO2,and PLIN1-KO3 cell groups were 8%,30%,and 18%,respectively.The PLIN1 RNA expression in the three groups Compared with the normal cell group(CON group),PLIN1 protein expression decreased by 66.30%,78.26%,and 28.26%,respectively.Compared with the CON group,the PLIN1 protein expression in the three groups decreased by 55.00%,74.00%,and 63.00%,and the PLIN1-KO2 mutation efficiency was the highest,The difference is extremely significant(P<0.01).Study 2 The effect of porcine PLIN1 gene on the metabolism of porcine skeletal muscle satellite cells and mitochondrial functionIn this study,one-day-old Jiangquhai pig longissimus muscle was used to extract porcine skeletal muscle satellite cells by double enzyme digestion method,and the expression of skeletal muscle satellite cell marker genes MyoD1 and PAX7 were.detected by cell immunofluorescence labeling method for cell purity identification;Porcine skeletal muscle satellite cells were transfected with PLIN1 empty vector(PLIN1-NC),PLIN1 knock-out vector(PLIN1-KO),PLIN1 overexpression vector(PLIN1-EX),and selected by puromycin and bleomycin respectively,Enrich PLIN1 knockdown and overexpression cell populations,and use qRT-PCR and Western Blot.to measure RNA and protein levels to detect PLIN1 expression.In order to study the effect of porcine PLIN1 gene on cell metabolism and mitochondrial function,Western Blot was used to detect cell metabolism and the expression of mitochondrial function-related proteins in each cell group,and cytometric flow cytometry was used to detect the content of reactive oxygen species,apoptosis rate,and cell apoptosis rate in each group of cells.For the level of mitochondrial membrane potential,use the ATP detection box to detect the intracellular ATP level,and use the transmission electron microscope to observe the changes of mitochondrial morphology and structure.The results showed that the isolated cells detected the marker genes MyoD1 and Pax7,and the cell morphology was also consistent with the reported porcine skeletal muscle satellite cells.This study successfully isolated porcine skeletal muscle satellite cells.The PLIN1 mRNA and protein levels in the cells of the experimental groups were determined.The results showed that compared with the NC group,the mRNA of the PLIN1-KO group decreased by 47.9%,and the mRNA in the EX group increased by 82.7%.Compared with the NC group,the expression of PLIN1-EX histone increased by 29.87%,and the expression of PLIN1-KO histone decreased by 40.85%,which proved that this study successfully enriched the PLIN1-KO and PLIN1-EX porcine skeletal muscle satellite cell population.WB was used to detect the cell metabolism and mitochondrial index function-related protein levels in the cells of each group.Compared with the NC group,the ratio of Bax/Bcl2 in the cells of the PLIN1-KO and PLIN1-EX groups decreased by 91%and 59%,respectively(P<0.01);Compared with the NC group,the expression levels of metabolism-related proteins,Mfn2 and OPA1 protein in the cells of the PLIN1-KO group increased by 7%and 9%,respectively(P<0.05),and the expression level of DRP1 protein decreased by 24%(P<0.05);the expression levels of metabolism-related proteins Mfn2 and OPA1 protein in the cells of the PLIN1-KO group increased by 11%and 81%,respectively(P<0.05),and the expression level of DRP1 protein decreased by 15%(P<0.05).At the same time,compared with the NC group,the cell apoptosis rate in the PLIN1-KO and PLIN1-EX groups decreased by 45%and 14%(P<0.01),and the cell membrane potential decreased by 14%and 4%(P<0.05),respectively.The production of internal ATP was reduced by 60%and 32%,respectively(P<0.01).The content of reactive oxygen species in the PLIN1-KO group did not decrease significantly,and the PLIN1-EX group decreased by 28%;the cell cycle test results showed that the cells of the PLIN1-KO group were blocked In the G2 cell phase,cells in the PLIN1-EX group were blocked in the S phase.Compared with the NC group,the mitochondria in the PLIN1-EX group and PLIN1-KO group had mitochondrial cristae structure obscured,the mitochondrial morphology was swollen or shrunken,and the number of mitochondria in the cells was significantly reduced.The above results indicate that the stable expression of PLIN1 gene in porcine skeletal muscle satellite cells is very important for maintaining cell metabolism and normal mitochondrial function.Both knockdown and overexpression of PLIN1 gene can enhance the cell’s anti-apoptotic ability to a certain extent,and Speed up cell metabolism,but at the cost of damaging the structure of mitochondria,reducing the number of mitochondria and affecting mitochondrial function.This study preliminarily explored the effect of porcine PLIN1 gene on the cell metabolism and mitochondria of porcine skeletal muscle satellite cells,and provided a theoretical basis for the follow-up study of the effect of PLIN1 on porcine muscle tissue development and meat quality.