The Effect And Mechanism Of Lnc-ORA On Animal Fat Deposition And Muscle Development

Adipose tissue is an important energy storage organ and a powerful endocrine organ,which plays an important role in animals energy metabolism,endocrine regulation,meat quality formation and the regulation of various physiological and pathological states.Skeletal muscle is the main protein source of animal food and the main executor of body movement,which is closely related to the formation of animal economic traits.Adipocytes and myoblasts are the basic components of adipose tissue and muscle tissue respectively,and their proliferation and differentiation ability have a close relationship with fat deposition and muscle development.Long non-coding RNAs(lncRNAs)are a new class of functionally regulatory RNAs discovered in recent years.Studies have shown that lncRNAs are involved in regulating the large number of biological processes at chromatin remodeling,transcription and translation multiple levels,including differentiation,reprogramming and genetic imprinting of pluripotent stem cells.However,the lncRNAs involved in the regulation of fat deposition and muscle development in animals need to be identified urgently,and the mechanism needs to be further explored.By using knockdown and overexpression,applicating the transcriptome sequencing,RACE,RNA-pulldown,RIP,FISH,Ed U staining,flow cytometry,half-life,immunofluorescence staining,dual luciferase report analysis and mass spectrum identification methods,in individual and cellular levels to study the effect and molecular mechanism of lnc-ORA on animal fat deposition and muscle development.The main results are as follows:1.Identification of a novel lnc-ORA by transcriptome sequencing.By transcriptome sequencing of adipose tissue between ob/ob and wild-type mouse,248 unannotated lncRNAs were screened.46 lncRNAs were significantly differentially expressed between obese mice and normal mice,among which one lncRNA was significantly upregulated 7 times in the adipose tissue of ob/ob mice and had the highest expression abundance in the adipose tissue of the two groups.Therefore,it was selected as a candidate lncRNA for subsequent functional studies and was named as obesity-related lncRNA(lnc-ORA).2.lnc-ORA promotes fat deposition and inhibits muscle development in vivo.AAV mediated lnc-ORA overexpression increased body weight and i WAT and e WAT adipose tissues weight,decreased GAS and TA muscle tissues weight,and upregulated the expression of adipogenic genes PPAR? and C/EBP? in adipose tissue,downregulated the expression of myogenic genes My HC and Myo D in muscle tissue.Meanwhile,overexpression of lnc-ORA lead to insulin resistance in vivo,increased the contents of TG,TC,LDL-c,leptin and adiponectin in serum,and decreased the content of HDL-c.3.lnc-ORA promotes adipogenesis through PI3K/AKT/m TOR signaling pathway.Knockdown of lnc-ORA decreased the number of EDU positive cells and S-phase cells and inhibited the DNA replication process.Meanwhile,lnc-ORA knockdown inhibited the levels of Cyclin B,Cyclin D1 Cyclin E and PCNA as well as promoted the level of negative proliferation regulator p27.Furthermore,knockdown of lnc-ORA inhibited adipogenic differentiation by downregulating the levels of adopogenic-related genes PPAR?,FASN and FABP4 through PI3K/AKT/m TOR signaling pathway.4.lnc-ORA inhibits myogenesis by interacting with miR-532-3p and IGF2BP2.lnc-ORA was significantly differentially expressed in muscle tissues of different weeks of age and the process of proliferation and differentiation of myoblasts.Overexpression of lnc-ORA increased the number and total number of EDU positive cells,promoted the cell cycle transition from G1 phase to S phase,and up-regulated the expression of proliferation marker genes Cyclin D1,Cyclin E and PCNA.Furthermore,overexpression of lnc-ORA decreased the number of My HC-positive cells,differentiation index and fusion index,and downregulated the expression of differentiation marker genes Myo D and My HC.In addition,knockdown experiment of lnc-ORA also further confirmed the function of lnc-ORA in proliferation and differentiation of myoblasts.Mechanically,lnc-ORA inhibited myogenesis by absorbing miR-532-3p to weaken its inhibitory effect on target gene PTEN.Meanwhile,lnc-ORA competitively bound with the protein IGF2BP2,enhancing its own RNA stability,weakening the RNA stability of Myo D and My HC to inhibit myoblast differentiation.5.Overexpression of lnc-ORA promotes the adipogenic differentiation of porcine intramuscular preadipocytes through PGC1-? and PI3K/Akt signaling pathway.Overexpression of lnc-ORA increased the number of EDU positive cells,the number of S-phase cells and promoted cell proliferation in porcine intramuscular preadipocytes.Meanwhile,lnc-ORA overexpression promoted adipogenesis of porcine intramuscular preadipocytes through absorbing miR-532-3p to target the key adipogenic factor PGC1-?and PI3K/Akt signaling pathway.Collectively,this study identified a novel key lncRNA,named lnc-ORA,which promoted fat deposition and inhibited muscle development by interacting with miR-532-3p and IGF2BP2.The research results will provide a new candidate lncRNA gene to increase meat production and improve intramuscular fat content of pigs.