The Effect And Mechanism Of LncIMF1 On Lipid Deposition In Porcine Intramuscular Preadipocytes

China is a major producing and consuming market of pork.With the improvement of people’s living standards,pork is the most commonly eaten meat,and its quality has received more and more attention.Intramuscualr Fat(IMF)content is one of the key factors that determine the quality of pork,and it affects the juiciness,flavor and tenderness of pork.The meat quality of local Chinese pigs is better than the imported ones.It is generally believed that high intramuscular fat content is one of the internal factors for the deliciousness of Chinese local pork.Therefore,changing the intramuscular fat content is an effective way to improve meat quality.Fat deposition includes processes such as adipocyte proliferation,differentiation,and apoptosis,which are regulated by a cascade of multiple transcription factors,fat secretion factors,and non-coding RNA.Non-coding RNA includes transfer RNA(t RNA),small RNA(micro RNA),circular RNA(circular RNA),and long noncoding RNA(lnc RNA).Among them,lnc RNA can regulate fat cell metabolism through a variety of mechanisms.Therefore,studying the effect of lnc RNA on the proliferation and differentiation of intramuscular precursor adipocytes and its mechanism are of great significance to the improvement of livestock meat quality.fatty-type Bamei pigs have excellent pork meat quality.One of the main internal factors is higher intramuscular fat content.The intramuscular fat content is affected by the number of intramuscular precursor adipocytes and the ability to collect fat,and lnc RNA is fat.It is an important metabolic regulatory factor in the cell.Therefore,we speculate that there are differences in the lnc RNAs transcribed during the differentiation of Bamei pig and Large White pig intramuscular preadipocytes.In order to explore the role of lnc RNAs in the process of intramuscular fat deposition in pigs,this experiment isolated and cultured Bamei pig and Large White pig intramuscular preadipocytes and collected the 0,2,4,and 8 d cell samples for sequencing and analysis.Differences in the transcriptome between the two varieties.In this experiment,the lnc IMF1 with the highest expression level and the highest abundance between the two varieties was selected as the follow-up test object.Flow cytometry,Edu staining,dual luciferase report analysis,oil red O staining,Bodipy staining,Real-Cell molecular biology techniques such as Time q PCR and Western blot were used to detect the expression of lnc IMF1 in the proliferation and differentiation of pig tissues and intramuscular preadipocytes,as well as the proliferation,differentiation and apoptosis of lnc IMF1 on intramuscular preadipocytes.The effect of splenic acid clarifies its mechanism of action in the differentiation process of pig intramuscular preadipocytes.The main results obtained are as follows:1.The intramuscular fat content of Bamei pig and Large White pig is 4.75% and 2.27%,respectively,and the lipid differentiation ability of intramuscular precursor fat cells derived from Bamei pig is significantly higher than that of intramuscular precursor derived from Large White pig Fat cells.2.760 unannotated lnc RNAs were newly discovered during the differentiation of intramuscular preadipocytes in Bamei pigs and large white pigs.Among them,compared with large white pigs,the intramuscular preadipocytes of Bamei pigs had 48,29,23,and 117 lnc RNAs that were significantly up-regulated at 0,2,4,and 8 days of differentiation;and 15,29,23,and 117 lnc RNAs were significantly down-regulated.10,26 and 56.These differentially expressed lnc RNAs are mainly enriched in PI3K-AKT and MAPK pathways.3.In this experiment,lnc IMF1 with the highest expression in Bamei pig intramuscular adipocytes was selected from differentially expressed lnc RNA as the research object.It showed an upward trend during the proliferation and differentiation of intramuscular preadipocytes.The results of nuclear and cytoplasmic separation showed that about 70% of lnc IMF1 was expressed in the cytoplasm and 30% in the nucleus.tissue level detection showed that lnc IMF1 expressed highest in adipose tissue.Pathway enrichment analysis showed that lnc IMF1 is related to cell biological processes such as cell proliferation,differentiation and apoptosis.4.Interference with lnc IMF1 si RNA during proliferation inhibited DNA replication of intramuscular adipocytes and arrested preadipocytes in G1 phase;Cyclin B,Cyclin D and Cyclin E were significantly reduced(P<0.05),while the anti-proliferation gene p21 was significantly increased(P<0.01).In the process of adipocyte differentiation,interference with lnc IMF1 si RNA significantly reduced intracellular lipid accumulation(P<0.01),and the expression of key adipogenic genes PPARγ,FABP4,C/EBPα and C/EBPβ were also reduced(P<0.05).Meanwhile,The expression level of lipolysis gene ATGL was increased(P<0.05).5.Overexpression of lnc IMF1 significantly increased the number of intramuscular preadipocytes(P<0.01),meanwhile,the expressions of Cyclin B,Cyclin D,Cyclin E and PCNA were elevated,and the expression of the anti-proliferation gene p21 was reduced.Overexpression of lnc IMF1 also promoted preadipocytes differentiation and lipid accumulation(P<0.01),the expressions of adipocyte differentiation markers,FABP4 and C/EBPa(P<0.05),were promoted.In addition,overexpression of lnc IMF1 also reduced the rate of apoptosis.At the same time,the pro-apoptotic genes Bax and Caspase3 were significantly reduced(P<0.05),while the anti-apoptotic gene Bcl-2 was increased.6.lnc IMF1 promoted the differentiation of intramuscular adipocytesby sponging mi R-187.Overexpression of mi R-187 significantly reduced the expression of the adipogenic marker gene FABP4 and promoted the expression of ATGL.When Inc IMF1 is co-expressed with mi R-187,mi R-187 weakened the promotion of lnc IMF1 on adipogenic differentiation,while overexpression of mi R-187.When overexpression of the SMAD1,a target gene of mi R-187,the inhibitory effect of mi R-187 on the differentiation of intramuscular preadipocytes is reduced.In summary,760 newly lnc RNAs were identified by RNA-seq analysis between the intramuscualr adipocytes of Bamei pig and Yorkshire pig.The transcriptome of intramuscular adipocytes of Bamei pig and Yorkshire pig had significant differences during adipogenesis.Among them,lnc IMF1 is the differentially expressed lnc RNA with the highest expression abundance,and its expression is on the rise during the proliferation and differentiation of pig intramuscular adipocytes.lnc IMF1 promotes intramuscular fat deposition by promoting the proliferation and differentiation of intramuscular preadipocytes and inhibiting apoptosis.lnc IMF1 promoted the differentiation of intramuscular adipocytes through mi R-187/SMAD1 pathway.These datas are helpful to complete the regulation mechanism of lnc RNA on IMF deposition between fatty type and lean type pigs,and expose a new aspect of improving meat quality traits.