Regulation Of Goat Chi＿circ＿0009659 On Intramuscular Adipocyte Differentiation

CircRNAs are a class of endogenous non-coding RNA molecules with covalently closed loop structures,which are widely involved in the proliferation,differentiation,migration and invasion of cancer cells,and studies have shown that circ RNAs regulate fat deposition by regulating the proliferation and differentiation of adipocytes.Whether circ RNA affects the differentiation of goat intramuscular adipocytes and by what mechanism have not been reported yet.Therefore,this study took the differentially expressed chi＿circ＿0009659 screened in goat intramuscular preadipocytes and mature sdipocytes as the research target,and confirmed the formation of chi＿circ＿0009659 by realtime quantitative real-time PCR(q PCR)and Sanger sequencing;Nucleocytoplasmic isolation,q PCR,cell culture,vector construction,overexpression,silencing,interference,rescue experiments,dual luciferase reporter experiments,Western blotting(WB),immunofluorescence(IF)and other molecular and cell biological methods were used to clarify the regulatory effect and possible mechanism of chi＿circ＿0009659 on the differentiation of goat intramuscular adipocytes.(1)Identification of Chi＿circ＿0009659 and its effect on the differentiation of goat intramuscular adipocytesThe goat chi＿circ＿0009659 was identified by RNase R digestion,qPCR,Sanger sequencing and FISH technology.The expression level of chi＿circ＿0009659 in the induced differentiated intramuscular adipocytes was higher than preadipocytes,and the expression level was the highest at 48 h,reaching a very significant level(P<0.01).Adenovirus were used to overexpress chi＿circ＿0009659 in goat intramuscular adipocyte,the generation of intracellular lipid droplets increased,and the m RNA expressions of adipocyte adipogenic differentiation marker genes C/EBPα,PPARγ,Pref1 and LPL were significantly up-regulated(P<0.01).However,C/EBPβ was significantly down-regulated(P<0.01).Silencing chi＿circ＿0009659 inhibited the accumulation of lipid droplets in cells,the m RNA expression levels of C/EBPα and LPL were significantly(P<0.05)and extremely significantly(P<0.01)decreased,respectively,while C/EBPβ,PPARγ and FASN also showed a downward trend,Pref1 was significantly up-regulated(P<0.01).Overexpression of chi＿circ＿0009659 did not affect the expression of its host gene CEMIP,while silencing of chi＿circ＿0009659 significantly down-regulated the expression of CEMIP(P<0.01).(2)Identification of Chi＿circ＿0009659 target miRNA and chi＿miR-3431-5p and its effect on goat intramuscular adipocyte differentiationAccording to the results of RNAhybrid and RNA pull down,chi＿miR-3431-5p was found to be a potential target miRNA of chi＿circ＿0009659.The chi＿circ＿0009659 dual-luciferase reporter experimental vector was successfully constructed,and the targeted binding relationship between chi＿circ＿0009659 and chi＿miR-3431-5p was confirmed.The expression of chi＿miR-3431-5p at each time point of induced differentiation of goat intramuscular adipocytes showed a trend of first decrease and then increase,and the highest expression level at 72 h,which was generally opposite to that of chi＿circ＿0009659,which further proved that results of the above predictions.chi＿miR-3431-5p mimics inhibited lipid droplet accumulation in goat intramuscular adipocytes,and significantly and extremely significantly inhibited the expression of C/EBPα(P<0.05)and FASN(P<0.01),C/EBPβ and PPARγ also showed a downward trend,and Pref1 was significantly increased(P<0.05).chi＿miR-3431-5p inhibitor increased the generation of lipid droplets in goat intramuscular adipocytes,and promoted the expression of C/EBPα and PPARγ(P<0.01),and the relative expression level of LPL was significantly up-regulated(P<0.05),C/EBPβ and FASN also showed an upregulated trend,while the relative expression level of Pref1 was significantly decreased(P<0.05).(3)Identification of the targeting relationship between chi＿miR-3431-5p and STEAP4 and the effect of STEAP4 on the differentiation of goat intramuscular adipocytesThe RNAHybrid prediction showed that STEAP4 was a potential target gene of chi＿miR-3431-5p,and chi＿miR-3431-5p negatively regulated the expression of STEAP4.The dual luciferase reporter assay confirmed the targeted binding relationship between chi＿miR-3431-5p and STEAP4.The coding region of STEAP4 gene was cloned,and the STEAP4 eukaryotic expression vector was successfully constructed.Overexpression of STEAP4 promoted the accumulation of lipid droplets in intramuscular adipocytes,and C/EBPα,C/EBPβ and LPL were significantly up-regulated(P<0.01),and FASN was significantly was up-regulated(P<0.05),while Pref1 was extremely significantly down-regulated(P<0.01).More autophagosomes were observed in OE group cells,while NC group cells had lower levels of autophagy.Cell immunofluorescence showed that the expression of LC3 was lower in the NC group,while LC3 was strongly expressed in the OE group.WB detection of autophagy markers p62 and LC3 showed that the protein expressions of p62 and LC3 I in the OE group were lower than those in the NC group,while LC3 II was higher than that in the NC group.