Regulatory Effect Of Plnc1 On Adipogenic Differentiation Of Bone Marrow Mesenchymal Stem Cells And Its Mechanism

Purpose: LncRNA(Long non-coding RNA)is a class of non-coding RNA with a length of more than 200 nucleotides,which is commonly found in the mammalian genome.Lnc RNA is involved in various biological processes such as gene expression regulation,cell differentiation and metastasis of cancer cells.Plnc1 is a new type of Lnc RNA,which is transcribed from a position approximately 25,000 bp upstream of the mouse peroxisome proliferator-activated receptor ?2(PPAR?2)gene.Previous microarray analysis confirmed that the expression of Plnc1 was increased during adipogenesis of Bone marrow mesenchymal stem cells(BMSCs).Therefore,this study aims to analyze and determine its role in adipogenic differentiation of BMSCs and its possible molecular mechanism.Methods: 1.The distribution of Plnc1 in different tissues of mice was detected.Eight-week-old C57BL/6J normal mice,ob/ob obese mice,and KKAy obese mice were prepared,and then,the different tissues of normal mice(including brain,heart,lung,liver,kidney,skeletal muscle,epididymal fat,lnguinal fat,perirenal fat,and interscapular brown adipose tissue)and the adipose tissues of obese mice(including epididymal fat,lnguinal fat,and perirenal fat)were isolated.The expression level of Plnc1 in different tissues of normal mice and different adipose tissue of obese mice were detected by RT-q PCR.2.Functional study of Plnc1 in adipogenic differentiation of mouse mesenchymal stem cells.Bone marrow stromal cells(ST2 cells)were cultured and induced in adipocyte-inducing medium.Then,the cells were collected at different time points.Adipogenic differentiation of ST2 cells was confirmed by Oil Red O staining,and the Oil Red O quantification was further measured with light absorbance at 520 nm.Finally,the m RNA level of Plnc1,adipogenic differentiation marker genes a P2,and adipogenic differentiation related transcription factor PPAR? were detected by RT-q PCR.3.The Plnc1 overexpression plasmid was constructed,and Plnc1 si RNAs were synthesized.To identify the role of Plnc1 in adipocyte differentiation,the gain-of-function study was conducted with transfected Plnc1 overexpression plasmid,and the loss-of-function study was performed using Plnc1 si RNAs.After adipogenic treatment,Oil Red O staining was performed,and the expression of a P2,PPAR? and C/EBP? was tested with RT-q PCR and Western blotting.4.The primary bone marrow mesenchymal stem cells(BMSCs)were isolated from six-week-old C57BL/6J mice.For confirming overexpression and knockdown studies,Plnc1 overexpression and knockdown lentivirus were packaged.Then,the cells were infected with these lentiviruses and induced in the adipogenic medium.After that,the role of Plnc1 in adipogenic differentiation was verified with same methods as above.5.The molecular mechanism underlying the function of Plnc1 in vitro continued to be identified.3T3-L1 cells were cotransfected with the PPAR?1 or PPAR?2 promoter construct and Plnc1 si RNA or expression plasmid.After that,luciferase assay was done to make sure if Plnc1 exerted its function during adipocyte differentiation only through regulating PPAR?2.6.With loss-of-function and gain-of-function strategies,MSP(methylation-specific PCR)analysis was used to test whether Plnc1 influenced DNA methylation of the PPAR?2 promoter.7.3T3-L1 cells were transfected with Plnc1 overexpression plasmid and the methylated or nonmethylated PPAR?2 promoter constructs in presence or absence of adipogenic medium.Then,the luciferase activity of the constructs was detected to confirm the effect of Plnc1 on the transcriptional activity of PPAR?2 promoter.Results: 1.Plnc1 was plentifully expressed in adipose tissues and upregulated in obese mice.Plnc1 was highly expressed in epididymal and inguinal fat,moderately expressed in perirenal fat and brain,and relatively low in heart,kidney,liver,lung,skeletal muscle,and interscapular brown fat.And the expression of Plnc1 in adipose tissues of obese mice was upregulated comparing with wild type mice.2.Plnc1 was induced during adipocyte formation.The expression of Plnc1 was monitored during adipogenesis from ST2 cells.The results of RT-q PCR showed that adipogenic factors PPAR? and a P2 were upregulated and then downregulated after adipogenic treatment.And the expression of Plnc1 was also upregulated and then downregulated at all the indicated time points during adipogenic differentiation.3.Overexpression of Plnc1 induced adipocyte differentiation.A gain-of-function study was conducted to confirm the role of Plnc1 in adipocyte differentiation from ST2 cells and BMSCs.In the presence of adipocyte induced medium,Plnc1 enhanced expression led to a significant increase in the number of differentiated adipocytes.Accordingly,the m RNA and protein levels of PPAR?,C/EBP? and a P2 were increased.4.Knockdown of Plnc1 repressed adipogenic differentiation.To identify the role of Plnc1 in adipocyte differentiation,a loss-of-function study was performed with si RNAs in ST2 cells and sh RNA lentiviruses in primary BMSCs.After adipogenic treatment,Silencing of Plnc1 significantly suppressed adipocyte formation.RTq PCR and Western blotting further confirmed that Plnc1 si RNAs and sh RNA lentiviruses decreased expression levels of PPAR?,C/EBP? and a P2.5.Plnc1 suppressed methylation and promoted transcriptional activity of PPAR?2 promoter.Plnc1 knockdown repressed the transcriptional activity of the PPAR?2 promoter more than that of the PPAR?1 promoter in undifferentiated and differentiated 3T3-L1 cells.Conversely,enhanced expression of Plnc1 stimulated the PPAR?2 promoter activity more than the PPAR?1 promoter.Furthermore,Plnc1 si RNA resulted in a promoted methylation level of the PPAR?2 promoter,whereas Plnc1 overexpression led to a reduced methylation level of the PPAR?2 promoter.Next exploration revealed that Plnc1 overexpression further increased PPAR?2 promoter activity in nonmethylated and methylated status.Conclusions: 1.Plnc1 was highly expressed in fat tissues and was significantly up-regulated in adipose tissues from obese ob/ob and KKAy mice vs.nonobese control mice.2.Plnc1 was substantially up-regulated during adipocyte differentiation,and Plnc1 acts as a positive regulator in adipogenic differentiation.3.Plnc1 enhanced the promoter activity of PPAR?2 through attenuating the methylation status of the promoter,thereby increasing PPAR?2 transcripts.