Regulation Of Goat Chi＿circ＿0006511 On Intramuscular Adipocyte Differentiation

Adipose tissue is an important energy storage site in the living body.Excessive fat deposition will disrupt the body’s energy balance and increase the risk of developing type 2 diabetes,hyperlipidemia,fatty liver and other metabolic diseases.The regulation of lipid metabolism during livestock and poultry farming can increase carcass leanness and improve meat tenderness and flavor.As a domestic animal,goat is one of the main sources of meat.Compared with beef and pork,mutton has a unique flavor and is widely respected by consumers.The level of fat content not only affects the meat quality of mutton,but also directly affects the economic value of mutton.Therefore,elucidating the molecular mechanism of fat deposition in goat is one of the key links in molecular breeding.Studies have shown that animal adipocyte differentiation is not only regulated by functional genes,but also regulated by non-coding RNA(nc RNA).Circular RNAs(circRNAs),as a new type of endogenous non-coding RNAs,play a unique regulatory role in important cellular biological processes from the post-transcriptional level.At present,studies on circRNAs in fat deposition have been carried out in livestock such as pigs and cattle,but there are no reports on key circRNAs regulating intramuscular adipocyte differentiation in meat goats.Therefore,in this study,chi＿circ＿0006511,which was differentially expressed during the differentiation of goat intramuscular adipocytes,was screened by RNA-seq as the research object.Hybridization and dualluciferase reporter gene detection system were used to explore the regulatory effect and possible molecular mechanism of goat chi＿circ＿0006511 on intramuscular adipocyte differentiation.The regulatory effect and possible molecular mechanism of goat chi＿circ＿0006511 on intramuscular adipocyte differentiation were investigated by cell culture,transfection,RT-PCR,real-time quantitative PCR,fluorescence in situ hybridization and dual-luciferase reporter gene detection system.(1)Identification of goat chi＿circ＿0006511 and its effect on intramuscular adipocyte differentiationMethods such as RNase R and RT-PCR and comprehensive Sanger sequencing results were utilized to identify that chi＿circ＿0006511 was expressed and circularized in goat intramuscular adipocytes;Subcellular localization found that goat chi＿circ＿0006511 was distributed in both nucleus and cytoplasm;chi＿circ＿0006511 gradually increased from 0 h to 84 h of goat intramuscular adipocyte differentiation,reached a peak at 84 h,and gradually decreased from 84 h to 120 h;Overexpression of chi＿circ＿0006511 promoted lipid accumulation in goat intramuscular adipocytes,and the m RNA expression levels of adipogenic differentiation-related genes PPARγ,C/EBPα,C/EBPβ and other genes were significantly up-regulated(P<0.01);interference with chi＿circ＿0006511 inhibited goat The lipid accumulation in intramuscular adipocytes was significantly decreased,and the m RNA expression levels of adipogenic differentiation-related genes PPARγ,C/EBPα,C/EBPβ were significantly down-regulated(P<0.01).(2)Identification of chi＿circ＿0006511/novel-mi R-87/CD36 targeting relationshipUsing the dual-luciferase reporter gene detection system,the dual-luciferase activity of transfected mi R-87-mim NC + chi＿circ＿0006511WT,mi R-87-mim NC + chi＿circ＿0006511MT,mi R-87-mim + chi＿circ＿0006511WT,mi R-87-mim + chi＿circ＿0006511MT Compared with the mi R-87-mim NC + chi＿circ＿0006511MT group,there was no significant change in the mi R-87-mim NC + chi＿circ＿0006511WT group(P>0.05),and the mi R-87-mim NC + chi＿circ＿0006511WT group was extremely significantly down-regulated(P<0.01).-87-mim + chi＿circ＿0006511MT had no significant change(P>0.05),indicating that chi＿circ＿0006511 targets and binds to novel-mi R-87;The results of rescue experiments further demonstrated that novel-mi R-87 inhibitor could rescue the inhibitory effect of interfering with chi＿circ＿0006511 alone on goat intramuscular adipocyte differentiation.Detection of transfection mi R-87-mim NC + CD36-3’WT,mi R-87-mim NC + CD36-3’MT,mi R-87-mim + CD36-3’WT,mi R-87-mim + CD36-3’MT dual luciferase activity,compared with mi R-87-mim NC + circ＿CD36-3’MT,mi R-87-mim NC + CD36-3’WT had no significant change(P>0.05),the dual luciferase activity in mi R-87-mim + CD36-3’WT was extremely significant downregulation(P<0.01),and no significant change in mi R-87-mim + CD36-3’MT(P>0.05),indicating that novel-mi R-87 and CD36 have a targeted binding relationship.(3)Goat chi＿circ＿0006511 regulates goat intramuscular adipocyte differentiation through the novel-mi R-87/CD36 axisSimulating the expression of novel-mi R-87 in goat intramuscular adipocytes inhibited lipid accumulation in intramuscular adipocytes,and the m RNA expression levels of adipogenic differentiation marker genes PPARγ,C/EBPα,C/EBPβ and other genes were significantly downregulated(P<0.01);Inhibiting the expression of novel-mi R-87 in goat intramuscular adipocytes,the lipid accumulation in intramuscular adipocytes was significantly increased,the adipogenic differentiation gene PPARγ was significantly up-regulated(P<0.05),the m RNAs of C/EBPα,C/EBPβ,etc.The expression levels were significantly up-regulated(P<0.01).The expression of CD36 was significantly down-regulated by novel-mi R-87 mimics(P<0.01),and the expression of CD36 was significantly up-regulated by novel-mi R-87 inhibitor(P<0.01).The goat CD36 knockout vector was constructed by CRISPR/Cas9,and the CD36-KD1 knockout efficiency was about 69%.Oil red O and Bodipy staining results showed that,lipid deposition in goat intramuscular adipocytes was significantly reduced after CD36 knockout,and adipogenicity was significantly reduced.The m RNA expression levels of PPARγ,C/EBPα,C/EBPβ and LPL were significantly down-regulated(P<0.01).The above results show that chi＿circ＿0006511 was a positive regulator of goat intramuscular adipocyte differentiation,and chi＿circ＿0006511 could sponge novel-mi R-87,thereby releasing the inhibitory effect of novel-mi R-87 on CD36 and promoting the differentiation of goat intramuscular adipocytes.That is,chi＿circ＿0006511 promotes the differentiation of goat intramuscular adipocytes through the novel-mi R-87/CD36 axis.This study provides a new theoretical and experimental basis for constructing the regulation network of intramuscular adipocyte differentiation in goat,lays a foundation for finally revealing the regulatory mechanism of intramuscular fat deposition in goat,and provides a theoretical basis for molecular breeding and elite breeding.