The Screening And Function Study Of MiRNAs Related With Insulin Resistance In 3T3-L1 Adipocytes

Part one Differential expression of miRNAs in the 3T3-L1 adipocytes and insulin-resistant adipocytesObjective:1.To investigate the differentially expressed miRNAs in 3T3-L1 adipocytes and insulin-resistant adipocytes(IR- adipocytes).2.To screen several possible miRNAs related with insulin resistance (IR) and find their potential target genes,which contributes to further explore the role of miRNAs in IR.Methods:1.The establishment of IR- adipocyte modelIR- adipocyte model was induced with high glucose(25mmol/L) and high insulin(1μmol/L) in combination(high glucose/ insulin) for 24 hours.To identify the sucession of IR- adipocyte model,the glucose consumption and[³H]-glucose uptake were detected.2.The identification of differentially expressed miRNAsMiRNA microarray was used to investigate the differentially expressed miRNAs of up-regulated or down-regulated expressed by more than two folds in 3T3-L1 adipocytes and IR-adipocytes.3.The screening of several miRNAs related with IR and their target genesAccording to the results of miRNA microarray and by means of bio-informatic methods,we assayed some miRNAs of significant change and screened several possible miRNAs related with IR and their potential target genes. Results:1.The establishment of IR- adipocyte modelInsulin-induced glucose consumption and glucose uptake were significantly inhibited by as much as 75%and 161%in 3T3-L1 adipocytes after incubation with high glucose/insulin for 24 h,which demonstrated the establishment of IR- adipocyte model.2.MiRNA microarray analysisMiRNA microarray analysis showed that 50 miRNAs were up-regulated while 29 miRNAs were down-regulated in IR-adipocytes compared to 3T3-L1 adipocytes,and miR-320 was remarkly upregulated, while miR-21 down-regulated.3.Several miRNAs related with IR and their target genesTo further functional study,we selected three miRNAs(miR-320, 298 and miR-21) related with IR by bio-informatic methods,and predicted Pik3r1[phosphoinositide-3-kinase,regulatory subunit 1(p85 alpha)]may be a potential target mRNA of the three miRNAs.Conclusion:1.IR- adipocyte model was established by adding high glucose and high insulin for 24 hours.2.There were differential expression of miRNAs in 3T3-L1 adipocytes and IR- adipocytes. Part Two The effect of miRNAs on insulin resistance in 3T3-L1 adipocytesObjective:1.We designed the specific antisense oligonucleotides against miR-320 and miR-298,and constructed miR-21 recombinant plasmid inculding pre-miR-21(pSilencer 3.1-miR -21).To investigate the effects of the three Lipofectamine^TM2000 mediated antisense-miRNAs-oligonucleotides (AMO,AMO-miR-298 and AMO-miR-320) and pSilencer 3.1-miR-21 on glucose metabolism of 3T3-L1 adipocytes and IR-adipocytes.To clarify whether the three miRNAs could promote glucose uptake and ameliorate IR in normal and IR-adipocytes.2.We selected miRNAs which could ameliorate IR and further explored the mechanism.Methods:1.The effect of miRNAs on glucose consumption in normal and IR- adipocytesWe set up seven groups,the adipocyte group,the lipofectamine group,the AMO-miR-298 group,the AMO-miR-320 group,the scramble control group(control oligo group),the pSilencer 3.1-miR -21 group and the empty plasmid group.After mature adipocytes were transfected in triplicate with different oligo or pasmid using Lipofectamine^TM2000 for 4 -6 hours,then cells were treated with or without DMEM/high glucose and high insulin media cultured for 24 hours.After 24 hours,the glucose concentration in medium were detected by glucoseoxidase method,be subtracted by the glucose concentration of blank apertures in which there were no cells,then we obtained the glucose consumption quantity.2.The effect of miRNAs on[³H]-glucose uptake in normal and IR- adipocytesCells were divided into seven groups and treated as described in the above.After 24 hours,the glucose uptake rates were detected by glucose uptake test.3.The effect of miRNAs on the expression of PI-3K,Akt and GLUT4 and translocation of GLUT4 in IR-adipocytesWe set up seven groups,the adipocyte group,the IR- adipocyte group,the lipofectamine + IR group,the AMO-miR-320+ IR group,the scramble control + IR group,the pSilencer 3.1-miR -21+ IR group and the empty plasmid+ IR group.24 hours after the treatment,the whole cellular protein and outer-membrane protein were extracted.Then,the quantity of P1-3K,Akt and GLUT4 in the whole cellular protein and the quantity of GLUT4 in the outer - membrane protein were detected by western-blot.In addition,total RNAs were extracted and mRNA expression levels of PI-3K,Akt and GLUT4 were examined by quantitative real-time RT-PCR(qPCR).Results:1.The effect of miRNAs on glucose consumption and uptake in normal 3T3-L1 adipocytesThe three miRNAs had no significant effect on glucose consumption and uptake in basal state.However,insulin induced glucose consumption and uptake significantly increased by 2.2-2.3 folds and 4.2-4.4 folds respectively in 3T3-L1 adipocytes in all groups.2.The effect of miRNAs on glucose consumption and uptake in IR-adipocytesGlucose consumption and uptake tests showed that there was no significant difference in basal state between IR group and adipocyte group.In the presence of 100nmol/L insulin,Compared with adipocyte group,glucose consumption and uptake rate displayed a remarkable reduction in IR group.Compared with IR group,glucose consumption of AMO-miR-320+ IR group and pSilencer 3.1-miR-21 + IR group increased 18.5%and 14.9%respectively(P＜0.05),and the glucose uptake rate increased 57.3%and 46.2%respectively(P＜0.05).There was no significant difference in the other groups compared to IR group.3.The effect of miRNAs on the expression of PI-3K,Akt and GLUT4 and translocation of GLUT4 in IR- adipocytesWestern-blot assay showed that the level of PI-3K p85 protein expression and phosphorylation of Akt exhibited a significant 42.7% and 54.8%decrement in IR group compared with adipocyte group.The decrement of PI-3K p85 protein expression was fully recovered by the treatment of AMO-miR-320 and miR-21 in IR-adipocytes.While AMO-miR-320 and miR-21 significantly increased the level of phosphorylation of Ak by 28.5%and 33.9%in IR-adipocytes, respectively.GLUT4 protein level of the IR group presented a significant 51.1%reduction compared with adipocyte group,this decrement was significantly recovered(29.6%recovery) by the treatment of AMO-miR-320.However,GLUT4 protein level slightly increased in the pSilencer 3.1-miR-21+ IR group.GLUT4 translocation assay showed the translocation of GLUT4 was significantly suppressed in the IR group,only 33.7%as the adipocyte group.The mount of GLUT4 translocation in IR group was acted as the base value(100%),and the relative quantity of GLUT4 translocation was caculated.AMO-miR-320 and miR-21 significantly increased the translocation of GLUT4 by 168.7%and 149.4%in IR-adipocytes.QPCR assay showed the mRNA expression levels of PI3K,Akt and GLUT4 remarkably increased by 8.57,16 and 18.4-fold in the adipocyte group compared with IR group,respectively.Compared with IR group, the Akt and GLUT4 mRNA expression levels significantly increased by 4.29 and 12.1-fold;but PI3K mRNA expression had no significant change in the AMO-miR-320+ IR group.MiR-21 increased significantly the mRNA expression levels of PI3K,Akt and GLUT4 of the IR group by 4.92,6.06 and 6.96-fold,respectively.All the above-mentioned indexes had no significant difference in the other groups as comparing to IR group.Conclusion:1.AMO-miR-320 and miR-21 could promote glucose metabolism and improve insulin resistance in IR-adipocytes.2.AMO-miR-320 and miR-21 might improve IR by targeting PI-3K p85 and PI-3K p85 upstream genes,respectively;increasing the level of Akt phosphorylation,and promoting the translocation of GLUT4 in IR-adipocyes. Part Three Differential expression of miRNAs during adipocyte differentiationObjective:1.To investigate the differentially expressed miRNAs in the procession of adipocyte differentiation of 3T3-L1 preadipocytes and mesechymal stem cells(MSC).2.To explore the effects of miR-320,miR-21 and miR-375 on 3T3-L1 preadipocyte differentiation.We selected miRNAs which could affect adipocyte differentiation and further clarify the mechanism.Methods:1.The identification of differentially expressed miRNAs during adipocyte differentiation3T3-L1 preadipocytes and MSC were cultured and induced to differentiate,the extent of adipocyte differentiation were assessed by Oil Red O staining and reverse trancription PCR(RT-PCR) methods. MiRNA microarray was used to investigate the differentially expressed miRNAs in 3T3-L1 adipocytes and adipocytes,MSC and MSC derived adipocytes.2.The screening of several miRNAs related with adipocyte differentiation and their target genesAccording to the results of miRNA microarray,we selected the miRNA of significant change and two miRNAs(miR-320 and miR-21) which had been proved to regulate insulin resistance.Subsequently,we predicted their potential target genes by means of bio-informatic methods.3.The effect of miR-21 and miR-375 on 3T3-L1 preadipocyte differentiationWe constructed miR-21 and miR-375 recombinant plasmid inculding pre-miR-21 and pre-miR-375(pSilencer 3.1-miR-21 and pSilencer 3.1-miR-375).The pSilencer 3.1-miR-21,pSilencer 3.1-miR -375 and empty plasmid were transfected into 3T3-L1 preadipocyte with Lipofectamine^TM2000,and the positive clones were screened by G418. Subsequently,cells were induced to differentiate,nine days later, differentiation was assessed by Oil Red O staining.4.The effect of AMO-miR-320 on 3T3-L1 preadipocyte differentiationWe set up four groups,the 3T3-L1 control group,the 3T3-L1 adipocyte group,the AMO-miR-320 group and the scramble control group(control oligo group).3T3-L1 preadipocytes were transfected prior to induction of differentiation with AMO-miR-320 oligo or scramble control oligo using Lipofectamine^TM2000.Three days after ASO transfection,when the cells reached confluence,cells were induced to differentiate.At the end of experiment,the extent of adipocyte differentiation were assessed by Oil Red O staining.5.MiR-375 promote 3T3-L1 preadipocyte differentiation and its possible mechanismsTo further observe whether miR-375 promote 3T3-L1 preadipocyte differentiation and its possible mechanisms.3T3-L1 preadipocytes of normal and stable expressed miR-375 were induced to differentiate.The mRNA expression of aP2 and PPARγ2 were examined by RT-PCR methods at the different time(the 0,3,5,7,9 days of differentiation);the content of triglyceride(TG) were detected by high performance liquid chromatography(HPLC);the protein expression levels of extracellular signal-regulated protein kinase(ERK1/2),aP2 and PPARγ2 were detected respectively by western -blot. Results:1.MiRNA microarray analysisAt the end of experiment,cytoplasmic accumulation of lipid droplets was observed and confirmed in about 90%of the cells by positive reddish color in Oil Red O staining.The mRNA expression levels of peroxisome proliferator activated receptorγ2(PPARγ2) and adipocyte fatty acid binding protein(aP2) were remarkably increased.These results confirmed 3T3-L1 pre-adipocytes and MSC differentiated into mature adipocytes. MiRNA microarray showed that 26 miRNAs were up- regulated and 2 miRNAs were down-regulated in 3T3-L1 adipocytes compared with 3T3-L1 preadipocytes,while 20 miRNAs were up- regulated and 55 miRNAs were down-regulated in MSC derived adipocytes compared with MSC.2.Several miRNAs related with adipocyte differentiation and their target genesWe selected three miRNAs(miR-320,21 and miR-375) related with adipocyte differentiation,and predicted MAPK1(ERK2) may be a potential target mRNA of the three miRNAs by bio-informatic methods.3.The effect of miRNAs on 3T3-L1 preadipocyte differentiationOil Red O staining test showed that miR-375 increased significantly the value of OD,while miR-21 and AMO-miR-320 had no significant effect on the value of OD in 3T3-L1 adipocytes.These results indicated that miR-375 might promote 3 T3-L1 preadipocyte differentiation.4.miR-375 promote 3T3-L1 preadipocyte differentiation and its possible mechanismsRT-PCR assay showed the mRNA expression levels of PPARγ2 and aP2 gradually increased during 3T3-L1 preadipocyte differentiation. However,at the beginning of the 5 day,PPARγ2 and aP2 mRNA expression remarkably increased in the miR-375 group compared with 3T3-L1 preadipocyte group.At the 9 day,the content of triglyceride(TG) also significantly increased(12.1±0.46 vs 10.8±0.34 mg/mg protein, p＜0.01) in the stable expressed miR-375 adipocyte compared with normal 3T3-L1 adipocyte.Western-blot assay showed that miR-375 significantly decreased the levels of ERK1 and ERK2 protein expression by 27.4%and 28.2%,increased PPARγ2 and aP2 by 49.6%and 52.8%in 3T3-L1 adipocytes,respectively(P＜0.01) Empty plasmid had no significant effect on the ERK1/2,aP2 and PPARγ2 protein expression in 3T3-L1 adipocytes.Conclusion:1.There were differentially expressed miRNAs during 3T3-L1 preadipocytes and MSC differentiation.2.miR-375 can promote 3T3-L1 preadipocyte differentiation by inhibiting ERK1/2 protein expression and increasing PPARγ2 and aP2 protein expression.