Study On The Effect And Mechanism Of MiRNA-23b-5p On Thermogenesis Of Brown Adipocytes

Obesity is a nutritional disorder caused by the excessive accumulation of white adipose tissue in the body,which is caused by the disturbance of the body's energy metabolism.Obesity is a major risk factor for diseases such as type 2 diabetes,cardiovascular disease,hypertension,fatty liver and some malignant tumors.It not only seriously threatens personal health,but also causes huge economic losses to families and society.Brown adipose tissue(BAT)has a unique and powerful metabolic ability to consume fat with heat.In addition,the activity of brown adipose tissue is negatively correlated with body weight and BMI.Therefore,the activity of brown adipose tissue is the most promising breakthrough in the field of obesity research.Micro RNAs(miRNAs)are an abundant class of 21-24 nucleotide noncoding RNAs that control diverse biological processes,which negatively regulate gene expression at the post-transcriptional layer.Only in recent years several miRNAs were found to associate with transcription factors to switch on or off the brown adipogenesis and thermogenic program.The enhanced brown adipose tissue(BAT)activity by cold exposure has been demonstrated to promote the energy expenditure and protect from obesity.miRNAs are novel regulators of adipocytes differentiation and function.Our study is to characterize miRNAs responsive to cold stimulation and identify their role in brown adipogenesis.RNA-sequencing was performed to identify differential miRNAs in mouse BAT upon cold exposure.q PCR was used to validate miRNAs expression in the cold-stimulated BAT in vivo and CL-316,243-and forskolin-treated brown adipocytes in vitro.We found miR-23b-5p,miR-133a-3p,miR-135-5p,miR-491-5p and miR-150-3p expression were decreased and miR-455-5p expression was increased in the cold-stimulated BAT in vivo and the activated brown adipocytes in vitro.miR-23b-5p was screened and its expression was evaluated in the activated and aging mouse BAT in vivo.To reveal the potential function of miR-23b-5p in BAT.miR-23b-5p was over-expressed by lentivirus vectors in BAT precursor cells,and adipogenic and thermogenic capacities were detected.Luciferase reporter assays and thermogenic genes screening were adopted to reveal the mechanism of miR-23b-5p action.miR-23b-5p expression was regulated in the activated and aging BAT and negatively correlated with Ucp-1 expression.miR-23b-5p over-expression in the precursor cells revealed no significant effect on lipid accumulation,but impaired mitochondrial function and decreased BAT specific markers expression.Though luciferase reporter assays did not validate the association of miR-23b-5p with the 3'UTRs of the target Ern1,miR-23b-5p may affect thermogenic capacity through lipolysis and fatty acid ?-oxidation pathways.In summary,cold related miRNAs participatein the regulation of BAT function,in which miR-23b-5p may play an important role in the regulation of brown adipocyte heat related gene program.It is helpful to improve the functional regulation network of brown fat cells and to develop brown fat cells in order to develop brown fat cells.It will have a bright future for the treatment of obesityPart ? Screening and verification of miRNA under cold stimulation in BATObjective: The enhanced brown adipose tissue(BAT)activity by cold exposure has been demonstrated to promote the energy expenditure and protect from obesity.Our study is to characterize miRNAs responsive to cold stimulation through RNA-sequencing and identify their role in brown adipogenesis.Cold stimulation screening provides a theoretical and experimental basis for our study of the role of miRNA in brown adipocytes.Methods: With the usage of miRNA-seq technology,the differentially expressed miRNAs were identified in the i BAT between cold(4°C)and room temperature(RT,26±2?)exposure.Based on the selection criteria(|fold change| ?1,P < 0.05),a total of 82 miRNAs were detected significantly changed in the interscapular BAT after cold exposure.We defined a total of 20 miRNAs for further validation.Realtime PCR technology was used to detect IBAT to verify the differential expression of miRNA in brown adipocytes after cold stimulation.We employed ?3-ARs agonist CL 316,243(CL)and a kind of c AMP inducing agent forskolin(Fsk)to to mimic the regulatory role of ?3-adrenergic-c AMP signaling in miRNAs expression of BAT upon prolonged cold exposure to analyze the expression of cold-responsive miRNAs.Results: 1)The Realtime PCR results of 20 miRNAs differentially expressed by the cold stimulus defined by US showed that: miR-455-5p,miR-182-5p,miR-6715-5p,miR-3065-5p and miR-3073a-3p were upregulated in mouse BAT upon cold stimulation,whereas miR-150-3p,miR-135a-5p,miR-1a-3p,miR-23b-5p,miR-133a-3p and miR-491-5p expression were decreased.However,miR-9769-3p,miR-203-5p,miR-7068-3p,miR-1a-1-5p,miR-363-3p,miR-1941-5p,and miR-133a-5p expression showed no significant difference after cold exposure.;2)Treatment of primary brown pre-adipocytes after fully differentiation with CL and Fsk for showed the enhancement of thermogenic program via significant up-regulation of Ucp-1 and Pgc1? m RNA expression;3)we identified miRNAs(miR-455-5p miR-133a-3p,miR-150-3p,miR-135a-5p,miR-23b-5p and miR-491-3p)differentially responsive to ?3-adrenergic stimuli.Conclusion: We found the differential expression of miRNA in brown adipocytes after cold stimulation by miRNA sequencing.We also mimic the regulatory role of ?3-adrenergic-c AMP signaling in miRNAs expression of BAT upon prolonged cold exposure to analyze the expression of cold-responsive miRNAs.Some selected miRNAs have been reported to be induced by cold exposure and play the definite roles in brown adipocytes function,which further supports the results of our cold stimulus chip screening.?3-adrenergic signaling are necessary for the regulation of cold sensitive-miRNAs(miR-23b-5p,miR-491-3p),whereas others(miR-455-5p,miR-133a-3p,miR-150-3p,miR-135a-5p)are only sensitive to the intracellular c AMP signaling.The expression of miRNA-23b-5p is relatively high(CT<30),significant difference,and there are no research reports in adipose tissue and cells.Thus miR-23b-5p becomes a candidate miRNA,which is worthy of further functional study.Part ? MiR-23b-5p regulation of brown fat function and analysisObjective: To further explore the expression of miR-23b-5p under drug stimulation,We over-expressed miR-23b-5p by lentivirus vectors in BAT precursor cells,then adipogenic and thermogenic capacities of BAT were detected.Methods: The expression levels of miR-23b-5p in brown adipocytes and white adipocytes were detected under cold stimulation and drug stimulation models,respectively,and the expression level of miR-23b-5p in brown adipocytes of different age mice was detected.Using lentivirus to express miR-23b-5p,infected with a part of the stromal vascular fraction(SVF)source of brown fat precursor cells in the scapular i BAT of mice,the same cells transfected with empty virus were used as the control group.Insulin,dexamethasone,rosiglitazone,1-methyl-3(MIX),indomethacin and T3 wereused to induce cell differentiation and maturation.Oil red O was used to observe the formation of lipid droplets during the process of adipogenic differentiation.The content of triglyceride in mature adipocytes was detected by enzyme colorimetry.Real-time PCR was used to detect the key genes of adipocyte differentiation(Ppar ?,C/ebp ?,C/ebp ? and Fabp4).Using the mitochondrial DNA copy number and Mitotraker Red staining(m Nd1 and m Cox1)in the differentiated brown adipocytes to evaluate function of mitochondrial.We studied real-time bioenergetic kinetics using the Seahorse extracellular analyser.Using Realtime PCR to test the specific genes of brown fat.Western Blot technology was used to detect the changes in the expression of UCP-1.Results: 1)MiR-23b-5p expression in BAT was significantly down-regulated to 50% after the cold exposure in vivo.Specifically,the responsiveness of miR-23b-5p upon cold stimulation showed no significance in WAT.MiR-23b-5p expression level displayed a remarkable reduction both in BAT and WAT after the ?3-adrenergic agonist(CL-316,243)stimulation in vivo;2)We observed a progressively increased expression of miR-23b-5p from mice with the varying ages;3)Oil Red O staining and triglyceride(TG)content revealed miR-23b-5p over-expression have a weak effect on the lipid accumulation.Consistent with these results,the forced expression of miR-23b-5p did not change the m RNA expression levels of common differentiation markers including Ppar ?,C/ebp ?,C/ebp ? and Fabp4 :4)The mitochondrial DNA copy number(m Nd1 and m Cox1)expression was repressed in miR-23b-5p-expressing adipocytes in basal and thermogenic status,Mitotraker Red staining also confirmed the attenuated abundance of mitochondria after miR-23b-5p over-expression at the basal status as well as the thermogenic propensity by Fsk stimulation;5)The m RNA expression of brown adipocytes marker genes including Ucp-1,Dio2,Cidea and Cyto C was lower in miR-23b-5p-expressing adipocytes.Ucp-1 protein levels assessed by immunofluorescent staining and western blot were significantly declined after miR-23b-5p over-expression.Conclusion: In summary,these observations showed that miR-23b-5p expression was differentially modulated in the activated BAT(cold and ?3-AR stimulation)and aged-associated BAT,suggesting its potential role in regulating BAT function.MiR-23b-5p over-expression have a weak effect on the lipid accumulation.However,lentivirus-mediated miR-23b-5p expression could decrease mitochondrial content,repress oxidative capacity and lead to the decline of brown adipocyte specific genes expression.Part ? Studyon the mechanism of miR-23b-5pregulation of brown fatObjective: To investigate the possible mechanism of miR-23b-5p regulation of brown adipocytes.Methods: Basedon the miRNA target prediction analysis(www.targetscan.org),target genes of miR-23b-5p were selected.The potential target genes were verified by Realtime PCR technology and luciferase reporter gene detection.Using Realtime PCR and Western Blot to test such genes(AMPK?1,AMPK?2,AMPK?2,HSL,Atgl and GLUT4)involving in the respiratory chain/oxidative phosphorylation system(Ox Phos),lipolysis metabolism and fatty acid ?-oxidation(FAO).Results: 1)We further detected these potential targets expression by q PCR in miR-23b-5p over-expressing adipocytes and results showed a significant decrease of Ern1.Ern1 expression was increased by CL and Fsk stimulation.However,luciferase reporter assays showed that miR-23b-5p over-expression did not change the activity of the reporter construct harboring a wild-type(WT1 and WT2)or mutant(MUT1 and MUT2)3'UTRs;2)QPCR results showed a decreased expression of AMP-activated protein kinase alpha 1 catalytic subunit(AMPK?1),AMP-activated protein kinase alpha 2 catalytic subunit(AMPK?2),protein kinase AMP-activated non-catalytic subunit beta 1(AMPK?2),hormone sensitive lipase(Hsl),patatin-like phospholipase domain containing 2(Atgl)and Glut4,all of which were involved in lipolysis;3)Characteristic mitochondrial FAO enzymes in BAT compared to WAT including Acyl-Coenzyme A(acyl-Co A)synthetase short-chain family member 1(Acss1),Acyl-Co A synthetase-1(Acsl1),Acyl-Co A synthetase-5(Acsl5),medium chain acyl-Co A dehydrogenase(Acadm),short chain acyl-Co A dehydrogenase(Acads),long-chain acyl-Co A dehydrogenase(Acadl)and 3-oxoacyl-Co A thiolase(Acaa2)revealed a notable decrease :4)Genes associated with Ox Phos system were similar between NC and miR-23b-5p over-expression groups.;5)Western blot detection further revealed miR-23b-5p-transduced cells showed the reduced protein expression(AMPK?,HSL,Atgl,GLUT4,Acsl1,Acadm,Acss1 and Acads)involved in lipolysis and fatty acid ?-oxidation pathwaysConclusion: Over-expression of miR-23b-5p inhibited the thermogenic program through lipolysis metabolism and fatty acid ?-oxidation pathways.