The Effect Of LncIMF2 On Proliferation And Differentiation Of Porcine Intramuscular Preadipocytes

Intramuscular fat(IMF)content is an important economic trait to measure the quality characteristics of poultry meat.Pork,as the first meat consumer product on the Chinese people's table,is delicious because of its high intramuscular fat content,which is in line with Chinese taste.Intramuscular fat is a key factor that affects the juiciness,tenderness and flavor of pork.Chinese local breed pigs have higher intramuscular fat,but at the same time the back fat thickness is larger.With the improvement of living standards,people are increasingly pursuing carcasses with low back fat and high lean meat rate.Therefore,through genetic selection and crossbreeding,the carcass lean rate was increased,and the thickness of the backfat was reduced,but the fat content in the muscle was severely reduced,which also caused the quality of pork to fail to meet people's needs.It is of great significance to study the deposition of intramuscular fat in pork to improve pork quality and meet people's increasing dietary requirements.Intramuscular fat is located on the outer muscle membrane and the fascia,which is the basis for the formation of marbling,mainly composed of phospholipids and triglycerides.Among them,the main component phospholipids account for more than 50%,including C16 and C18 fatty acids,and the content of oleic acid in unsaturated fatty acids is up to about 50%.Phospholipids are important factors affecting the volatile flavor of pork.The content of intramuscular fat is mainly determined by the number of intramuscular precursor adipocytes and the adipogenic differentiation ability of adipocytes.Therefore,the proliferation,differentiation and apoptosis of intramuscular precursor adipocytes all affect the deposition of intramuscular fat.More and more studies have shown that in addition to functional genes,non-coding RNA is also involved in regulating fat formation.Long noncoding RNA(lncRNA)refers to an autonomously transcribed m RNA transcript that has no obvious ability to encode proteins and is longer than 200 nt.It has a complex structure,a large number,and different functions.Therefore,from a genetic point of view,by exploring the effect of lnc RNA on intramuscular fat production,we can increase the intramuscular fat content of pork and achieve the purpose of improving meat quality.In this study,we used siRNA and pcDNA3.1 overexpression vector to interfere and overexpress lncIMF2,combined with flow cytometry to detect changes in cell cycle,CCK-8 and Ed U staining to detect changes in the number of positive cells in the proliferation phase Oil red O and Adipo Red staining were used to observe the number of lipid droplets in order to explore the effect of lncIMF2 on the differentiation of porcine intramuscular adipocytes;at the same time,the genes corresponding to proliferation,differentiation and apoptosis were tested by Real-time q PCR and Western Blot.The main research results obtained are as follows:1.Numerous differentially expressed lnc RNAs were obtained from the sequencing results of intramuscular adipocytes of Bamei and Dabai pigs at different differentiation stages,in which the expression level of lncIMF2 increased first and then decreased and then increased again,and the expression level on the 9th day of differentiation The highest;the tissue expression profile of lncIMF2 shows that the expression level is higher in adipose tissue;the nucleoplasm separation experiment shows that lncIMF2 is mainly distributed in the nucleus;2.After interfering with lncIMF2,the cell cycle distribution was changed,the number of cells in S phase decreased,and the number of positive cells in the proliferation phase was reduced.At the same time,cell cycle related genes Cyclin B(P<0.01),Cyclin D(P<0.05)and Cyclin E(P<0.05)m RNA levels decreased significantly,and Cyclin D and Cyclin E protein levels also decreased significantly(P<0.05),indicating that interfering with lncIMF2 inhibited the proliferation of porcine intramuscular fat cells;3.After overexpressing lncIMF2,the number of cells in S phase and the number of positive cells in proliferation phase both increased,the m RNA levels of cell cycle related genes Cyclin B(P<0.05)and Cyclin D(P<0.01)increased significantly,and p21 decreased significantly(P<0.05),Cyclin D(P<0.05)and Cyclin E(P<0.01)increased protein expression,and the m RNA levels of apoptosis-related genes Bax,p53 and Caspase9(P<0.01)decreased significantly,indicating that overexpression of lncIMF2 promoted Proliferation of porcine intramuscular fat cells,4.After interfering with lncIMF2,the intracellular lipid droplets were significantly reduced,and the m RNA(P<0.01)and protein levels(P<0.01)and related genes that promote fat production were significantly reduced,indicating that interfering with lncIMF2 inhibited porcine intramuscular fat cells.Differentiation5.After overexpressing lncIMF2,the lipid droplets in the cells increased significantly,the protein level of the fat-generating gene PPAR? increased significantly(P<0.05),and the protein levels of the lipolysis-promoting genes ATGL and HSL decreased significantly(P<0.01),indicating that overexpression of lncIMF2 promotes the differentiation of intramuscular adipocytes.6.Through online software prediction and comparison,it is found that lncIMF2 can adsorb miR-217,and the target genes of miR-217 are PPARg,FTO and PGC-1? which are closely related to adipogenesis;the tissue expression of miR-217 shows that it is in There is a higher expression in adipose tissue;then overexpression of miR-217 found that it can inhibit the differentiation of pig intramuscular fat cells,while interference is the opposite.In summary,interfering with lncIMF2 inhibited the proliferation and differentiation of porcine intramuscular adipocytes,which were achieved by down-regulating the m RNA and protein expression of genes related to proliferation and differentiation;in addition,overexpressing lncIMF2 up-regulated the m RNA and protein expression of genes related to proliferation and the expression level of protein linking to adipogenesis.Finally,overexpression of lncIMF2 inhibited the apoptosis of porcine intramuscular fat cells by reducing the expression of apoptotic genes.And lncIMF2 may regulate the lipid deposition of pig intramuscular fat cells by adsorbing miR-217.This study explored the effect of long-chain non-coding RNA on the proliferation,differentiation and apoptosis of porcine intramuscular adipocytes,and clarified the role of lncIMF2 in regulating the proliferation,differentiation and apoptosis of porcine intramuscular adipocytes.