The Role And Possible Mechanism Of LncRNA U90926 In Modulating 3T3-L1 Preadipocyte Differentiation

Obesity is a disease characterized by abnormal or excessive fat accumulation in the body.Previous studies showed that obesity is a major risk factor for many chronic diseases,such as type 2 diabetes,cardiovascular disease and some cancers.The number of obese people increased dramatically in recent years,especially in developed countries,as well as developing countries with rapid economic growth.The study of obesity can provide valuable theoretical basis for the prevention and treatment of obesity and improve the quality of life for people.At the cellular level,obesity occurs when the number and/or size of adipocyte increases.In another word,adipocytes are closely related with the occurrence and development of obesity.The process from preadipocyte to mature adipocyte is known as adipogenesis,which is a critical process for adipose tissue mass accumulation and development of obesity.Therefore,the process of adipocyte differentiation and its underlying mechanism have become the focus of obesity research and its related diseases.Long non-coding RNAs(IncRNAs)are a unique class of transcripts that have more than 200 nucleotides,often polyadenylated in 3’ terminal and have the following characteristics:1)lower expression abundance than protein encoding genes;2)often less conservative among different species;3)do not encode protein due to lack of open reading frame(ORF);4)tissue specific expression.Since little was known about IncRNAs before 1990s,they were considered to be"noise" of transcription process,a transcription by-product of RNA polymerase Ⅱ.But with the development of molecular biology technology,attentions are growing gradually on the correlation between IncRNA and a variety of complex diseases in recent years.Previous studies have indicated that IncRNAs play regulatory roles in a variety of biological processes,such as mRNA degradation,X chromosomal inactivation,chromatin remodeling,transcriptional repression,through the above ways eventually involved in the process of cell proliferation,differentiation,apoptosis,metabolism,tumor and other life activities.It has been reported that IncRNA can regulate the differentiation of adipocytes,which provides a new way to study the pathogenesis of obesity.This work is presented in two parts as follows:Part I The correlation between IncRNA U90926 expression and the occurrence of obesityAim:The aim of this study was to explore the expression of IncRNA U90926 in adipogenesis and the correlation between IncRNA U90926 and obesity.Methods:1.Mouse 3T3-L1 preadipocytes is a well-established cell line commonly used to study adipocyte differentiation in vitro.We exploited this system with differentiation inducer to induce 3T3-L1 preadipocytes differentiate to adipocytes.Oil red O staining was conducted to identify the formation of lipid droplets.Total RNA was isolated from 3T3-L1 cells with Trizol at indicated time point(day 0,2,4 and 13)in the process of differentiation.Global transcription profiling including coding and noncoding genes namely microarrays was performed to seek the genes differently expressed.qPCR was performed to verify microarray results at different time points(day 0,4,8,12).2.RNA fluorescent in situ hybridization(FISH)was performed to determine the localization of IncRNA U90926 in 3T3-L1 preadipocytes.3.Obese animal model:Six-week old male C57BL/6J mice,ob/ob mice and db/db mice were adaptively fed for 1 week(C57BL/6J mice were fed with basal diet containing 5%fat,ob/ob mice and db/db mice high-fat diet containing 20%fat)in laboratory cages in a temperature-controlled room under a 12 hr light-dark cycle with free access to water and food for a week.Then,C57BL/6J mice were divided into 3 groups randomly,the mice in one group were fed with a basal diet and the other 2 groups were fed with a high-fat diet(HFD).One group fed with high-fat diet was selected as a control for ob/ob mice and db/ab mice.These ob/ob mice and db/ab mice were kept feeding with HFD all the time until the body weights of these animals were 30%greater than that of control mice(C57BL/6J mice fed with HFD),then these transgenic obese mice and the control mice were analyzed.While another high-fat diet group(designated as HFD)were kept feeding in parallel with the group of C57BL/6J mice with basal diet(designated as BD)until the difference of body weight between HFD and BD reached 30%,then these obese mice(HFD)and the control mice(BD)were analyzed.The animals were sacrificed following deep anesthesia with ether.Subcutaneous and visceral adipose tissues,the heart,liver,spleen,kidney,lung,testis,brown adipose tissues were collected and snap frozen in liquid nitrogen.4.To determine lncRNA U90926 expression in different tissues,we examined its expression in heart,liver,spleen,kidney,lung,testis,brown adipose tissue by qRT-PCR besides epididymal adipose tissue.5.lncRNA U90926,PPARy2,FABP4 and AdipoQ expression was evaluated by qPCR with subcutaneous,perirenal and epididymal adipose tissues from C57BL/6J mice and obese mice including HFD obese mice,ob/ob mice and db/db mice.Results:1.3T3-L1 preadipocytes differentiated into mature adipocytes with differentiation inducers(’cocktail’ reagents)and obvious lipid droplets were observed by oil red O staining.Microarray analysis at different time points showed that lncRNA U90926 was obviously reduced during 3T3-L1 preadipocytes differentiation which was further confirmed by qPCR analysis.2.RNA fluorescent in situ hybridization(FISH)assay showed that IncRNA U90926 was mainly localized in the cytosol of 3T3-L1 preadipocytes while there was little in the nucleus.3.After 4 weeks,the body weight of ob/ob mice and db/db mice were increased more than 30%compared with control group;after 12 weeks,the body weight of high fat diet C57BL/6J mice were also increased more than 30%of C57BL/6J mice fed with basic diet.They were both successful obese mice model.4.To determine lncRNA U90926 expression in different tissues,we examined its expression in 7 other tissues by qRT-PCR besides epididymal adipose tissue.Results showed that IncRNA U90926 was predominantly expressed in white adipose tissue with extremely lower expression in other tissues.5.To explore the correlation between lncRNA U90926 expression and obesity,we collected subcutaneous,perirenal and epididymal adipose tissues from C57BL/6J mice and obese mice including HFD obese mice,ob/ob mice and db/db mice.Results showed that lncRNA U90926 levels were lower in adipose tissues from obese mice compared to non-obese control mice.The expression of PPARy2,FABP4 and AdipoQ in mice epididymal adipose tissues significantly increased in obese mice compared to non-obese control mice.Conclusions:1.LncRNAU90926 obviously reduces during 3T3-L1 preadipocytes differentiation.2.LncRNA U90926 is predominantly expressed in white adipose tissue,and mainly localized in the cytosol of fat cells while there is little in the nucleus.3.LncRNA U90926 expression is significantly lower in adipose tissues from obese mice than those of control mice,revealing a negative correlation of IncRNA U90926 expression with obesity.Part Ⅱ The role and possible mechanism of IncRNA U90926 inmodulating 3T3-L1 preadipocytes differentiationAim:The aim of this study was to explore the possible mechanism of IncRNA U90926 in modulating preadipocytes differentiation.Methods:1.3T3-L1 preadipocytes were infected with lentivirus encoding IncRNA U90926 or control lentivirus,IncRNA U90926 over-expression stable cell clones(designated as OY)and control cell clones(designated as NC)were selected under puromycin pressure.OV cells and NC cells were induced for adipogenesis in vitro with routinely used ’cocktail’ reagents.Oil red O staining was conducted to identify the formation of lipid droplets.The samples were collected on day 0 and day 6 to detect PPARγ2,FABP4 and AdipoQ mRNA expression by qPCR.The samples were collected on day 0,2,4,6,8,10,12 to detect PPARy and FABP4 protein expression by Western Blotting.2.We designed three shRNA sequences targeting to mouse IncRNA U90926 and then constructed to vector GV118 to obtain GV118-U90926-shRNA(GV118-CON as control plasmid)for packaging lentivirus.3T3-L1 preadipocytes were infected with suitable titer of lentivirus,the effect of the shRNA on adipogenesis was determined after the ’cocktail’ reagents induction.Oil red O staining was conducted to identify the formation of lipid droplets.The samples were collected on day 0 and day 12 for RNA extraction to detect PPAR,y2,FABP4,C/EBPa and AdipoQ mRNA expression by qPCR.The cell samples were collected on day 0,2,4,6 to detect PPARy and FABP4 protein expression by Western Blotting.3.Dual luciferase assay was carried out to explore the trans-activation of target genes modulated by IncRNA U90926.The IncRNA U90926 sequence and the promoter sequence of C/EBPa,C/EBPβ and PPARγ2 were found in NCBI and Ensemble database.LncRNA U90926 overexpression plasmid and reporter vectors containing C/EBPα,C/EBPβ and PPARy2 full-length and truncated promoter were constructed,then the overexpression plasmid was co-transfected with a reporter plasmid into Hela cells.After determination of luciferase activity,the effect of IncRNA U90926 on C/EBPα,C/EBPβ and PPARγ2 transcriptional activity were analyzed.Results:1.To investigate the role of IncRNA U90926 in the process of 3T3-L1 preadipocyte differentiation,we generated IncRNA U90926 stably over-expressing 3T3-L1 preadipocyte cell line(OV)and control cell lines(NC)by infection with lentivirus encoding IncRNA U90926 or with control lentivirus.qRT-PCR analysis showed an 160-fold increase in IncRNA U90926 expression in OV cells over NC.Overexpression of IncRNA U90926 significantly prevented adipocytes differentiation,as evidenced by oil red O staining.In addition,qRT-PCR results showed that IncRNA U90926 overexpression significantly reduced the mRNA levels of PPARy2,FABP4 and AdipoQ on day 12 as compared to NC group.Meanwhile,PPARy2 and FABP4 were obviously decreased in OV compared with NC group on day 0.Accordingly,IncRNA U90926 also markedly reduced PPARy and FABP4 expression at protein level during adipocytes differentiation.2.To further verify the role of IncRNA U90926 in adipocytes differentiation,we conducted lentivirus-mediated loss of function(LOF)experiments.Three lentivirus containing shRNA sequence targeting IncRNA U90926 and one lentivirus containing scramble control sequence were used to infect 3T3-L1 preadipocytes.Knock-down IncRNA U90926 expression remarkably increased the mRNA levels of adipocyte markers including PPARy2,C/EBPa,FABP4,and AdipoQ on day 6 after adipogenesis induction as evaluated by qRT-PCR.Knock-down of lnc-U90926 also increased PPARy2,FABP4 and AdipoQ expression on day 0,and later on increased lipid accumulation as evaluated by oil red O staining.Moreover,the protein levels of PPARy and FABP4 were significantly elevated in the process of adipogenesis in IncRNA U90926 knockdown cells compared to control cells.3.Dual luciferase assay showed that lncRNA U90926 over-expression did not change C/EBPα and C/EBPβ transactivation while obviously inhibited PPARy2 promoter transactivation.The luciferase activity of full length PPAR-γ2 promoter(-2000 bp)was much lower in IncRNA U90926 overexpression cells than in vector control cells,but the other three shorter PPARγ2 reporters(-1500 bp,-1000 bp and-500 bp)displayed almost the same luciferase activity in IncRNA U90926 overexpression cells as in control cells.These results indicated that lncRNA U90926 was able to inhibit the transactivation of PPARy2 promoter probably through the region from-2000bp to-1500bp.Conclusions:1.Overexpression of IncRNA U90926 attenuates adipogenesis in 3T3-L1 preadipocytes.2.Knockdown of IncRNA U90926 in 3T3-L1 preadipocytes enhances adipogenesis.3.LncRNA U90926 plays an inhibitory role in adipogenesis in 3T3-L1 preadipocytes,and one mechanism is possibly through indirectly suppressing the transcriptional activity of PPARy2.4.LncRNA U90926 may modulate the expression of PPARy2 in both mRNA and protein level through other unknown ways,and eventually change the differentiation of preadipocytes.