Functional And Regulatory Analysis Of TORCs In Adipocyte Proliferation And Differentiation Of Qinchuan Beef Cattle

Low intramuscular adipose tissue(marbling)continues to be challenge for improving beef quality in Chinese cattle.Highly marbled meat is very desirable;hence,methods to increase IMF content have become a key aspect of improving meat quality.Therefore,research on the mechanism of adipogenesis provides invaluable information for the improvement of meat quality.This study investigated the effect of TORCs and its underlying mechanism on lipid metabolism in bovine adipocytes of Qinchuan beef cattle.There are three members of TORC gene family: TORC1,TORC2 and TORC3,which is also known as CRTC [CREB(c AMP response element binding protein)-regulated transcription coactivator].The TORCs are required for CREB transcription,which is a versatile transcription factor that regulates the expression of more than 4000 genes,involved in the regulation of metabolism,proliferation,differentiation of cells,the immune response,as well as other physiological and pathological processes.The TORC2 gene is responsible for nutrient metabolism,gluconeogenesis,myogenesis and adipogenesis through the PI3K-Akt,AMPK,glucagon and insulin resistance signaling pathways.In the present study,we explored function of TORC2 gene in bovine adipocytes.We also explored transcriptional and post transcriptional regulation of both TORC1 and TORC2 gene through different experiments and confirmed the following findings.1.Sequencing of PCR amplicons explored three novel SNPs at loci g.16534694G>A,g.16535011C>T,and g.16535044A>T in the promoter region of the TORC2 gene in the Qinchuan breed of cattle.Allelic and genotypic frequencies of these SNPs deviated from Hardy-Weinberg equilibrium(HWE)(P < 0.05).SNP1 genotype GG,SNP2 genotype CT and SNP3 genotype AT showed significantly(P <0.05)larger body measurement and improved carcass quality traits.Haplotype H1(GCA)showed significantly(p<0.01)higher transcriptional activity(51.44%)followed by H4(ATT)(34.13%)in bovine preadipocytes.The diplotypes HI-H3(GG-CC-AT),H1-H2(GG-CT-AT)and H3-H4(GA-CT-TT)showed significant(P<0.01)associations with body measurement and improved carcass quality traits.2.In the present study,we confirmed the role of TORC2 in bovine preadipocyte proliferation through cell cycle staining flow cytometry,cell counting assay,5-ethynyl-2?-deoxyuridine staining(Ed U),and m RNA and protein expression analysis of proliferationrelated marker genes.In addition,Oil red O staining analysis,immunofluorescence of adiponectin,m RNA and protein level expression of lipid related marker genes confirmed the role of TORC2 in the regulation of bovine adipocyte differentiation.Furthermore,the transcription start site and sub-cellular localization of the TORC2 gene was identified in bovine adipocytes.To investigate the underlying regulatory mechanism of the bovine TORC2,we cloned a 1990 bp of the 5' untranslated region(5?UTR)promoter region into a luciferase reporter vector and seven vector fragments were constructed through serial deletion of the 5?UTR flanking region.The core promoter region of the TORC2 gene was identified at location-314 to-69 bp upstream of the transcription start site.Based on the results of the transcriptional activities of the promoter vector fragments,luciferase activities of mutated fragments and si RNAs interference,four transcription factors(CCAAT/enhancerbinding protein C/BEP?,X-box binding protein 1 XBP1,Insulinoma-associated 1 INSM1,and Zinc finger protein 263 ZNF263)were identified as the transcriptional regulators of TORC2 gene.These findings were further confirmed through Electrophoretic Mobility Shift Assay(EMSA)within nuclear extracts of bovine adipocytes.Furthermore,we also identified that C/EBP?,XBP1,INSM1 and ZNF263 regulate TORC2 gene as activators in the promoter region.3.The TORC2 gene was downregulated in bovine adipocytes by si RNA,and RNA sequencing was performed.Downregulation of TORC2 negatively affected bovine adipocyte differentiation.In addition,a total of 577 DEGs were found,containing 146 up-regulated and 376 down-regulated genes.KEGG pathway analysis revealed that the DEGs were linked with neuroactive ligand-receptor interaction pathway,calcium signaling pathway,c AMP pathway,chemokine signaling pathway and Wnt signaling pathway.Gene Ontology(GO)term analysis of the DEGs showed that down-regulation of TORC2 gene significantly suppressed the genes regulating important GO terms of adipogenesis-related processes in bovine adipocytes,especially regulation of biological activity,regulation of primary metabolic process,regulation of multicellular organismal process,cell adhesion,lipid metabolic process,secretion,chemical homeostasis,regulation of transport,cell-cell signaling,c AMP metabolic process,cellular calcium ion homeostasis,fat cell differentiation,and cell maturation.4.In the present study,expression and sub-cellular localization of the TORC1 gene was analyzed in bovine preadipocytes.Bioinformatics tools were applied to characterize TORC1.To investigate the molecular mechanism of bovine TORC1 gene regulation,we cloned a 1008 bp of the 5'UTR regulatory region into a luciferase reporter vector.Different fragments were amplified using 5'UTR unidirectional deletion of the TORC1 promoter.Site directed mutation,dual luciferase reporter assay,RNAi interference and DNA-protein interaction(EMSA)were used to validate the regulatory roles of Smad3 and NRF1 in the regulation of TORC1 gene in bovine preadipocytes.The core promoter region of the TORC1 gene was identified at a location-410 to-155 bp upstream of transcription start site.Different vectors were constructed through serial deletion of the 5'UTR flanking region and in combination with site directed mutations and transcription interference through si RNA or sh RNA,two transcription factors of NRF1 and Smad3 were found to be repressors in the promoter of the TORC1 gene.These findings were further confirmed through Electrophoretic Mobility Shift Assay(EMSA)within nuclear extracts of bovine adipocytes.The core promoter region of the TORC1 gene,spanning from-410 to-155 bp upstream of the transcription start site was specified in this study and this information will provide opportunity for the improvement of intramuscular fat in cattle.5.Micro RNAs are small,single stranded,and non-coding RNAs that have been proven to be potent regulators of adipogenesis.However,the role of bta-mi R-149-5p in regulating bovine adipogenesis especially in the regulation of TORCs is still unclear.Expression profiling in different stages of adipogenesis revealed that bta-mi R-149-5p was enriched in the proliferation stage,and also on day 9 of differentiation in bovine adipocytes.Our gain of function study showed that bta-mi R-149-5p can negatively regulate both bovine adipocyte proliferation and differentiation.Overexpression of bta-mi R-149-5p suppressed the expression of proliferation marker genes at both the m RNA and protein levels,markedly decreased the percentage of S-Phase cells,decreased the number of Ed U stained cells,and substantially reduced adipocyte proliferation vitality in the cell count assay.Collectively,these findings elucidated that bta-mi R-149-5p inhibits adipocyte proliferation.Further,overexpression of bta-mi R-149-5p also suppressed the expression of adipogenic genes at both the m RNA and protein levels,inhibited lipid accumulation,and reduced the secretion of adiponectin in bovine adipocytes.Furthermore,a luciferase activity assay explored how btami R-149-5p targeted TORCs(TORC1 and TORC2)directly.This targeting was further validated by the m RNA and protein level expression of TORC1 and TORC2,which were down regulated by bta-mi R-149-5p overexpression.Moreover,bta-mi R-149-5p indirectly targeted TORC1 and TORC2 through regulating their key transcription factors.Overexpression of bta-mi R-149-5p suppressed the expression of SMAD3,while enriched the expression of NRF1,which are the key transcription factors and proven regulators of TORC1.Overexpression of bta-mi R-149-5p also repressed the expression of C/EBP?,XBP1,INSM1 and ZNF263,which are the key regulators of TORCs,at both the m RNA and protein levels.We can conclude that variants mapped within TORC2 can be used in marker-assisted selection for carcass quality and body measurement traits in breed improvement programs of Qinchuan cattle.Moreover,TORC2 gene is potentially a positive regulator of adipogenesis,we also identified that C/EBP?,XBP1,INSM1 and ZNF263 regulate TORC2 gene as activators in the promoter region.Additionally,the RNA-sequencing results also proved that TORC2 regulates lipid metabolism in bovine adipocytes,and provide a basis for studying the function and molecular mechanism of the TORC2 gene in regulating adipogenesis.Furthermore,two transcription factors of NRF1 and Smad3 were found to be repressors in the promoter of the TORC1 gene in bovine adipogenesis.In addition to that,we can conclude that bta-mi R-149-5p is a negative regulator of TORC1 and TORC2 both at transcriptional and post transcriptional levels,and that bta-mi R-149-5p can regulate adipogenesis,which implies that bta-mi R-149-5p could be a target for increasing intramuscular fat in beef cattle.These findings will not only provide an insight for the improvement of intramuscular fat in cattle,but will enhance our understanding regarding therapeutic intervention of metabolic syndrome and obesity in public health as well.