Mechanisms Underlying How Long Non-coding RNA BADLNCR Regulates Bovine Adipocyte Differentiation

Adipose tissue is very important for human growth and development,endocrine balance,and energy metabolism,since it helps to store energy and is an endocrine organ.To cattle breeding,in addition to its importance for growth and development,the content of adipose in beef is an important criterion for beef quality and taste.Therefore,researches about the growth and development of adipose tissue have been a hot issue in researches about beef improvement.Adipogenesis is very complex,more and more regulators have been identified to regulate adipogenesis,such as CEBP families and PPARγ.Recently,studies showed that micro RNA takes an essential part in adipogenesis.As the most abundant and complex functional non-coding RNA,lnc RNA takes a part in growth and development.Also,it can regulate adipogenesis.Hence,the aim of this study is to explore the molecular mechanisms underlying bovine adipocyte differentiation.Firstly,bioinformatics analysis and q RT-q PCR were applied to select the candidate lnc RNA.Secondly,gain-and loss-of-function experiments were performed to detect if overexpression and knocking down of candidate lnc RNA could regulate bovine adipocyte differentiation and/or proliferation.Finally,CHIRP-seq,CHIP-PCR,RNA pull-down assay,and dual-luciferase activity assay were applied to explore how this lnc RNA regulate adipocyte development.These results could enrich the researches about the role of lnc RNA in bovine adipoenesis,and provide useful information for Chinese cattle breeding and improvements.Main results obtained in this study were as follow:1.Based on the RNA-seq data,two candidate lnc RNAs were selected using bioinformatics analysis and RT-q PCR.They were then named as adipocyte differentiation related lnc RNA,BADLNCR1 and BADLNCR2.RACE method was carried out to obtain the full-length sequences of them.Then lengths of BADLNCR1 and BADLNCR2 are 1029 nt and 631 nt,respectively.Cell fractionation extraction and FISH experiments showed that BADLNCR1 and BADLNCR2 mainly expressed in nuclear.2.BADLNCR1 indicates a decreasing expression trendency during bovine adipocyte differentiation.In bovine adipocyte,BADLNCR1 was overexpressed or knocked down,and Oil Red O staining was used to detect the formation of lipid droplets.Expression of adipogenic genes was detected by RT-q PCR.As a result,overexpression of BADLNCR1 inhibited formation of lipid droplets and expression of adipogenic genes.Oppositely,after BADLNCR1 was knocked down,lipid droplets formation and adipogenic gene expression were reduced.Therefore,BADLNCR1 represses bovine adipocyte differentiation.The conversation of BADLNCR1 among different species is very low.3.A GC-box was found at 85 bp upstream of BADLNCR1 transcription start site(TSS).A series binding sites of transcriptional factors were found in the promoter region of BADLNCR1,including PPARγ,CEBPα,AP-2,NF-1,and Sp1.Dual-luciferase activity assay was used and found that KLF2 enhanced the relative luciferase activity of BADLNCR2 promoter.CHIRP-seq data indicated that BADLNCR1 could competitively binding on the DNA motif of SREBP1 and SREBP2.4.BADLNCR1 negatively regulates m RNA expression of GLRX5.In addition,BADLNCR1 could bind on the promoter of GLRX5.Then GLRX5 was chosen as the target gene of BADLNCR1.Gain-and loss-of-function experiments displayed that GLRX5 is a gene of stimulator in regulation of bovine adipocyte differentiation.5.CEBPα could regulate the transcriptional activity of GLRX5 promoter,while BADLNCR1 negatively regulates its activity.And the enhancement of CEBPα on GLRX5 promoter significantly inhibits by overexpression of BADLNCR1.On the other hand,CHIRP-PCR and CHIP-PCR results indicated that the binding of BADLNCR1 on GLRX5 promoter could repress the binding of CEBPα.6.The expression patterns of BADLNCR2 during bovine adipogenic differentiation showed that expression level of BADLNCR2 goes down at the beginning of differentiation and rises at the terminal differentiation.In bovine adipocytes,overexpression of BADLNCR2 is benefit for the formation of lipid droplets along with the significant rising of expression levels of adipogenic genes.In addition,Edu detection assay showed that overexpression of BADLNCR2 significantly increases the ratio of proliferating cells and the expression of CCND1(P<0.05).These results states that BADLNCR2 might be a positive regulator for bovine adipocyte proliferation and differentiation.In a conclusion,this study indicated that BADLNCR1 inhibits bovine adipogenic differentiation by inhibiting the binding of transcriptional factors on GLRX5 promoter,which is a positive regulator in bovine adipocyte differentiation.BADLNCR2 could promote the proliferation and differentiation of bovine adipocytes.These results primarily state the molecular mechanisms underlying the role of ADLNCR in bovine adipogenic differentiation,which could contribute to the breeding and improvement of Chinese cattle.