Conserved Function And Of The Long Noncoding RNA Blnc1 In Brown Adipogenesis And Thermogenesis

Adipose tissue plays an important role in regulating metabolism.It has been shown that there are two types of adipose tissue: White Adipose Tissue(WAT)and Brown Adipose Tissue(BAT).WAT mainly functions in energy storage;BAT is enriched with mitochondria and functions in thermogenesis through Uncoupling protein 1(UCP1)so that it could decrease plasma lipid content and the obesity incidence.[18F]-FDG-PET/CT result has shown that human also have BAT,which is similar with classic BAT in rodents.Long noncoding RNA(lncRNAs)have transcriprion functions which is the same with mRNA,but they do not have the protein coding potential because of lack of the ORF.Besides,lncRNAs functions in many biological process,such as transcriptional regulation,cell differentiation,tissue development and tumorigenesis.Blnc1 could promote brown and beige adipogenesis by a feedback loop with transcription factor EBF2.However,whether Blnc1 is enough conserved between mice and human and which domain plays an important role are still unknown.In addition,the further mechanism for Blnc1 is also not clear.To solve the problems above,it was evaluated by RACE,retrovial vectors and stable brown adipocyte cell line construction,Oil Red O staining,MitoTracker staining,Oxygen consumption detection,real-time qPCR,Western Blot and CoIP and so on.The specific results are as follows:1.Blnc1 is relatively conserved in sequence between mouse and human.hBlnc1 sequence was predicted by UCSC and has a high conservation with mice(71% and 76%,respectively).Then exact hBlnc1 sequence was got by RACE and it has two conserved sequence(83% and 73%,respectively).Coding potential analysis showed that hBlnc1 did not has the potential for protein coding.2.Blnc1 is relatively conserved in function between mouse and human during brown adipogenesis and thermogenesis.We got the hBlnc1 retrovial vector and stable brown adipocyte cell lines.Then the expression of key genes related to thermogenesis(such asUcp1,Ppar?,Prdm16,Elovl3 and Cox7a1)were detected by qPCR on base line and isoprenaline treatment,all of them were upregulated during brown adipogenesis.Similarly,Western Blot also showed that the expression of UCP1 and mitochondrial genes had a significant upregulation.However,lipogenesis genes like Ppar? and Fabp4 did not have dramatic changes.MitoTracker staining and DNA content detection demonstrated that hBlnc1 could increase the mitochondria content.In addition,oxygen consumption result showed that hBlnc1 could enhance the mitochondrial respiration.In order to further identify the conservation of Blnc1 in function,hBlnc1 was overexpressed in stable brown adipocyte cell line where m Blnc1 expression was inhibited.Compared with control,mBlnc1 knockdown inhibited the expression key genes related thermogenesis such as Ucp1,Elovl3,Dio2 and Ppar ?,while expression of hBlnc1 in the cells where the expression of m Blnc1 has been inhibited could partially rescue the function above.Western blot also showed the similar res?lts.3.Domain1 of Blnc1 plays an important role during Brown adipogenesis and thermogenesis.LncRNAs function mainly depending on RNA secondary structure.RNA Fold(RNA secondary structure prediction software)showed that mBlnc1 has 3 domains(RD1,RD2 and RD3)and RD1 is the conserved domain of Blnc1 between human and mouse).To identify which domain functions during brown adipogenesis,mBlnc1 truncations stable cell lines were constructed(T1/T2/T3).It showed that the function of T2 and T3 were similar with m Blnc1 full length which could induce brown adipogenesis and thermogenesis,while T1 was the same with control.The result above demonstrated that domain 1 of mBlnc1 played an important role during brown adipogenesis and thermogenesis.Also,research showed that the function of domain 1 between human and mouse is also conserved enough during the process which was comparable with Blnc1 full length.4.HnRNP U mediates the interaction between Blnc1 and EBF2.It has been demonstrated that Blnc1 is physically associated with EBF2,a transcription factor that has been implicated in the reg?lation of brown and beige adipocyte development.As EBF2 lacks a discernable RNA-binding domain,we post?lated that Blnc1 may interact with EBF2 via additional RNA-binding factors.CoIP experiment showed that Blnc1 interacted with EBF2 through hnRNP U.In addition,the interaction between Blnc1 and hnRNP U were mainly depended on domain was RD1(mBlnc)and RGG(hnRNP U),respectively.In addition,hnRNP U could not interact with EBF2 without RGG domain and there is no effect for the interaction without SAF.Interestingly,the deletion of SPRY domain could increase the interaction between hnRNP U and EBF2.5.HnRNP U is rather important for the function of Blnc1 during brown adipogenesis and thermogenesis.We got the stable brown adipocyte cell lines which Blnc1 or EBF2 overexpression and hnRNP U knockdown at the same time.Then they were induced into differentiation and some key genes were detected.It showed that the inhibition of hnRNP U in mBlnc1 brown adipocytes affected the functions of mBlnc1 on brown adipogenesis and thermogenesis.Similarly,hnRNP U knockdown also inhibited the function of EBF2 during the process above.However,it demonstrated that hnRNP U is a key factor for Blnc1 functions during brown adipogenesis and thermogenesis.In summary,Blnc1 is conserved in sequence between human and mouse;hBlnc1 also could induce thermogenesis which is similar with mBlnc1 and could partially rescue the effect m Blnc1 knockdown on brown adipogenesis and thermogenesis;RD1 was important during brown adipogenesis and thermogenesis;hnRNP U mediated the interaction between Blnc1 and EBF2.Also,RD1(Blnc1)and RGG domain(hnRNP U)play an important for the interaction;hnRNP U was important for the function of Blnc1 during brown adipogenesis and thermogenesis.