Study On Regulating Mechanism Of CircFUT10 In Qinchuan Cattle Adipocyte Proliferation And Differentiation

Adipose tissue is the material basis of life.It can store energy,maintain constant body temperature,buffer external pressure,promote the absorption of fat-soluble vitamins,and provide essential fatty acids for the human body.At the same time,fat content is also one of the important factors for evaluating beef quality.Appropriate fat content can both improve the impact value of beef and ensure good taste.Fat development is regulated by a series of complex processes.Non-coding RNA(ncRNA),including circular RNA(circRNA),plays an important role in regulating the proliferation and differentiation of adipocytes.CircRNA can be used as mi RNA sponge to exert its biological effects.In this study,high-throughput sequencing technology was used to analyze the expression of circRNA in adipose tissue of Qinchuan cattle at different developmental stages.The differentially expressed circRNA in adipose tissue of calves and adult cattle was identified,and circFUT10 related to fat development was screened.Through overexpression and interference experiments,the functions of circFUT10 were identified using CCK-8,Ed U,flow cytometry,real-time fluorescence quantitative PCR(RT-qPCR),western blot and oil red O staining technology.At the same time,the dual luciferase reporting system,sensor experiment and RIP technology were used to verify that circFUT10 can be used as a competitive endogenous RNA to competitively bind mi RNA let-7c with PPARGC1 B gene,promoting the proliferation of adipose cells and inhibiting their differentiation.The mechanism of circFUT10 regulating the fat proliferation and differentiation of Qinchuan cattle was revealed,which provided a new theoretical basis for the breeding of Qinchuan cattle.1.Identification of circRNA in Qinchuan cattle adipose tissueHigh-throughput sequencing of subcutaneous adipose tissue in Qinchuan cattle found that a total of 14,274 circRNAs were detected in calves and adults,among which 8,809 and 9,937 circRNAs were expressed in calves and adults,respectively,and 4,427 circRNAs were detected in both periods.Most circRNAs consist of 1?4 exons,one host gene can produce 1?5 circRNAs,and a few genes can produce more than 10 circRNAs.A total of 307 circRNAs were differentially expressed in the two periods,among which 151 circRNAs were significantly down-regulated in the calf period and 156 circRNAs were significantly up-regulated in the calf period.GO analysis found a total of 339 functional groups,and KEGG analysis enriched 27 pathways.The high expression and differential expression of circFUT10 were quantitatively verified,and it was used as a candidate circRNA for further research.2.circFUT10 promotes adipocyte proliferation and inhibits its differentiationSanger sequencing verified the authenticity of circFUT10,amplified the full length of circFUT10,and constructed the overexpression vector p CD2.1-circFUT10.CCK-8,Ed U,flow cytometry,RT-qPCR,western blot,and oil red O staining results showed that overexpression of circFUT10 can significantly promote the vitality of adipocytes and promote the expression of the proliferation marker genes PCNA and CDK2,inhibit the expression of the adipose differentiation marker genes PPAR? and C / EBP? and reduce the number of lipid droplets,that is,circFUT10 can promote the proliferation of adipocytes and inhibit Differentiation of adipocytes.3.circFUT10 promotes adipocyte proliferation and inhibits differentiation by sponging let-7c.RNAhybrid software predicts that circFUT10 contains the let-7 family binding site.The dual luciferase reporter system found that let-7c and let-7e significantly inhibited the fluorescence activity of PCK-circFUT0.Let-7c inhibited the effect more significantly.Sensor experiments found that circFUT10 can rescue the decrease in fluorescence activity caused by overexpression of let-7c,and RIP experiments found that both circFUT10 and let-7c can bind to AGO2 protein,further indicating that circFUT10 can adsorb let-7c.RT-qPCR,Western blot,and oil red O staining experiments found that let-7c can inhibit the expression of adipocyte proliferation markers PCNA and CDK2,and promote the expression of adipocyte differentiation markers PPAR? and C / EBP?.Co-expression of circFUT10 can alleviate this effect.That is,circFUT10 promotes adipocyte proliferation and inhibits its differentiation by adsorbing let-7c.4.circFUT10 can competitively combine with PPARGC1 B gene to let-7c promote adipocyte proliferation and inhibit its differentiationThe dual luciferase reporter system found that PPARGC1 B is a target gene of let-7c.Synthesis of PPARGC1 B interfering si-PPARGC1 B transfected into bovine primary adipose cells.CCK-8,Ed U,flow cytometry,RT-qPCR,western blot and Oil red O staining experiments found that si-PPARGC1 B reduced the proliferation coefficient of adipocytes and the expression of proliferation marker genes PCNA and CDK2,promoted the expression of differentiation marker genes PPAR? and C / EBP?,and the formation of lipid droplets.Based on the above results,circFUT10 can release let-7c's inhibitory effect on the target gene PPARGC1 B gene expression by binding let-7c,thereby regulating the proliferation and differentiation of adipocytes.In this study,transcriptome sequencing was performed on the adipose tissue of Qinchuan cattle in different developmental stages,and circFUT10 related to adipose development was selected.The construction of circRNA-mi RNA-m RNA network reveals the mechanism of circFUT10 regulating the proliferation and differentiation of adipocytes in Qinchuan cattle.This study provided a new idea for the molecular development mechanism of cattle adipose tissue,and also provides a certain reference for molecular breeding in Qinchuan cattle.