摘要
第一部分miRNAs在3T3-L1脂肪细胞和胰岛素抵抗脂肪细胞中的差异表达
目的:
1.探讨正常3T3-L1脂肪细胞和胰岛素抵抗(insulin resistance,IR)脂肪细胞中微小RNA(miRNAs)的表达是否存在差异。
2.筛选几个可能与IR相关的miRNAs,寻找其靶基因,以进一步研究miRNAs在IR中的作用。
方法:
1.IR脂肪细胞模型的建立
采用高糖/高胰岛素处理3T3-L1脂肪细胞24小时,通过葡萄糖消耗实验和[~3H]葡萄糖摄取实验判断模型是否建立。
2.miRNAs的差异表达
运用miRNAs芯片技术检测3T3-L1脂肪细胞和IR脂肪细胞中差异表达的miRNAs。
3.IR相关的几个miRNAs及其靶基因的筛选
根据基因芯片结果,借助生物信息学方法,对显著变化的某些miRNAs进行分析,筛选几个可能与IR相关的miRNAs及其调控的靶基因。
结果:
1.IR脂肪细胞模型的建立
高糖/高胰岛素分别使3T3-L1脂肪细胞葡萄糖消耗量和[~3H]葡萄糖摄取率下降75%和161%,与脂肪细胞组相比差异有显著性,结果表明IR脂肪细胞模型成立。
2.miRNAs芯片分析
miRNAs芯片结果显示:miRNAs在IR脂肪细胞中比在脂肪细胞中表达水平升高2倍以上者有50个,表达水平降低至1/2以下者有29个;且miR-320上调最明显,miR-21下调最明显。
3.IR相关的几个miRNAs及其靶基因
应用生物信息学方法,我们选取了3个可能与IR相关的miRNAs(miR-320,298和miR-21)进行下一步的功能实验,Pik3r1可能是他们的靶基因。
结论:
1.高糖和高胰岛素可诱导3T3-L1脂肪细胞产生IR。
2.脂肪细胞和IR脂肪细胞中存在miRNAs的差异表达。
第二部分miRNAs对3T3-L1脂肪细胞胰岛素抵抗的调节作用及其分子机制
目的:
1.针对miR-320和miR-298设计相应的反义寡核苷酸(AMO-miR-320,AMO-miR-298),同时构建含miR-21前体的重组质粒(pSilencer 3.1-miR-21)。以脂质体为载体介导,观察AMO-miR-320,AMO-miR-298及pSilencer 3.1-miR-21对3T3-L1脂肪细胞和IR脂肪细胞糖代谢的影响。了解这3个miRNAs在正常和病理状况下有无促进葡萄糖摄取和改善IR的作用。
2.对能改善脂肪细胞IR的miRNAs,进一步探讨其作用机制。
方法:
1.miRNAs对正常及IR脂肪细胞葡萄糖消耗的影响
实验分7组:脂肪细胞组、脂质体组、AMO-miR-298组、AMO-miR-320组、miRNA无关对照组(control oligo组)、pSilencer3.1-miR-21组和空质粒组。脂肪细胞转染4-6h小时后,用含0.2%BSA的正常糖/正常胰岛素或高糖/高胰岛素培养液培养24小时,以葡萄糖氧化酶法检测每孔培养液中葡萄糖的含量,与未接种细胞的空白孔的糖含量均值相减,计算葡萄糖的消耗量。
2.miRNAs对正常及IR脂肪细胞葡萄糖摄取的影响
实验分组及细胞处理同上,24小时后,通过葡萄糖摄取实验检测各组脂肪细胞对葡萄糖摄取的影响。
3.miRNAs对IR脂肪细胞PI-3K、Akt、GLUT4的表达和GLUT4转位的影响
实验分7组:脂肪细胞组、IR脂肪细胞组(IR组)、AMO-miR-320+IR组、脂质体+IR组、miRNA无关对照+IR组、pSilencer 3.1-miR-21+IR组和空质粒+IR组。实验结束时,通过western-blot检测各组细胞(P1-3K,Akt和GLUT4)的蛋白表达及细胞外膜蛋白中GLUT4蛋白的含量;荧光定量PCR检测P1-3K,Akt和GLUT4基因表达水平。
结果:
1.miRNAs对正常3T3-L1脂肪细胞葡萄糖消耗和摄取的影响
基础状态下,3种miRNAs对3T3-L1脂肪细胞葡萄糖消耗和摄取无显著性影响;在100nmol/L胰岛素存在的条件下,各组脂肪细胞对葡萄糖的消耗和摄取都显著增加,分别是基础状态下的2.2-2.3倍和4.2-4.4倍,但组间无显著性差异。
2.miRNAs对IR脂肪细胞葡萄糖消耗和摄取的影响
基础状态下,IR组和脂肪细胞组葡萄糖消耗量和摄取率无显著性差异。在100nmol/L胰岛素存在下,与脂肪细胞组相比,IR组葡萄糖消耗量和摄取率显著下降;与IR组比较,AMO-miR-320+IR组和pSilencer 3.1-miR-21+IR组葡萄糖消耗量分别增加了18.5%和14.9%(P＜0.05),葡萄糖摄取率分别增加了57.3%和46.2%(P＜0.05);其他各组与IR组相比,葡萄糖消耗量和摄取率无显著性差异。
3.miRNAs对IR脂肪细胞PI-3K、Akt、GLUT4的表达和GLUT4转位的影响
Western-blot显示:与脂肪细胞组相比,IR组PI3K p85蛋白表达和Akt丝氨酸磷酸化水平分别下降了42.7%和54.8%;AMO-miR-320和miR-21可完全恢复IR脂肪细胞PI3K p85表达;同时使IR脂肪细胞Akt丝氨酸磷酸化水平分别提高28.5%和33.9%。此外,IR组较脂肪细胞组GLUT4蛋白表达显著下降51.1%,AMO-miR-320可使IR脂肪细胞GLUT4蛋白水平提高29.6%;pSilencer 3.1-miR-21+IR组GLUT4蛋白表达水平轻微提高,但与IR组相比无显著性差异。葡萄糖转位分析表明:IR脂肪细胞GLUT4蛋白转位显著降低,仅为正常脂肪细胞的33.7%。我们以IR组GLUT4蛋白转位量作为基值,各组与之相比计算GLUT4蛋白转位的相对定量。AMO-miR-320和miR-21分别使IR脂肪细胞GLUT4蛋白转位增加168.7%和149.4%。荧光定量PCR结果表明:脂肪细胞组PI3K、Akt和GLUT4 mRNA的表达分别是IR组的8.57,16和18.4倍;AMO-miR-320可使IR组Akt和GLUT4 mRNA表达升高4.29和12.1倍,但不影响PI3K mRNA表达水平;miR-21可使IR组PI3K、Akt和GLUT4 mRNA的表达分别提高4.92,6.06和6.96倍。其余各组与IR组相比,上述指标无显著性差异。
结论:
1.AMO-miR-320和miR-21可促进IR脂肪细胞糖代谢、改善IR。
2.AMO-miR-320和miR-21可能是通过作用PI3K P85和PI3KP85上游基因,提高Akt丝氨酸磷酸化水平,促进GLUT4的转位来发挥改善IR的作用。
第三部分脂肪细胞分化过程中miRNAs差异表达及功能研究
目的:
1.探讨3T3-L1前脂肪细胞和间质干细胞(MSC)分化成脂肪细胞过程中差异表达的miRNAs。
2.观察miR-320、miR-21和miR-375对3T3-L1脂肪细胞分化的影响;对能影响脂肪细胞分化的miRNAs,进一步探讨其作用机制。
方法:
1.脂肪细胞分化过程中miRNAs的差异表达
分别将3T3-L1前脂肪细胞和分离培养的MSC诱导分化,通过油红O染色检测细胞内脂质蓄积情况、RT-PCR方法检测过氧化物酶体增殖物激活受体γ2(PPARγ2)和脂肪性脂肪酸结合蛋白(aP2)的表达,判断3T3-L1前脂肪细胞和MSC分化情况。运用miRNAs芯片技术检测3T3-L1前脂肪细胞和3T3-L1脂肪细胞;MSC和MSC-脂肪细胞中差异表达的miRNAs。
2.脂肪细胞分化相关的miRNAs的筛选及其靶基因的预测
根据基因芯片结果,借助生物信息学方法,对3T3-L1前脂肪细胞分化过程中显著变化的1个miRNA及前面已经证明对脂肪细胞IR有作用的2个miRNAs(miR-320和miR-21)进行靶基因预测。
3.miR-21和miR-375对3T3-L1前脂肪细胞分化的影响
构建含miR-21和miR-375前体的重组质粒(pSilencer 3.1-miR-21和pSilencer 3.1-miR-375),通过脂质体转染法将重组质粒稳定转染3T3-L1细胞。随后将正常和稳定转染的3T3-L1前脂肪细胞诱导分化,实验结束时进行油红O染色,判断脂肪细胞的分化情况。
4.AMO-miR-320对3T3-L1前脂肪细胞分化的影响
实验共分4组:分别为3T3-L1对照组、3T3-L1脂肪细胞组、AMO-miR-320组和miRNAs无关对照组。3T3-L1前脂肪细胞常规培养,在诱导分化前分别将AMO-miR-320和无关对照序列转入细胞。转染3天后当细胞生长融合时开始诱导,实验结束时进行油红O染色,判断脂肪细胞的分化情况。
5.miR-375促进3T3-L1前脂肪细胞的分化及可能机制
为进一步观察miR-375是否促进3T3-L1前脂肪细胞的分化及其可能机制,我们将3T3-L1前脂肪细胞(对照组)和pSilencer3.1-miR-375稳定转染3T3-L1前脂肪细胞(miR-375组)诱导分化,通过RT-PCR实验检测在分化的不同时间(0,3,5,7,9天)aP2和PPARγ2基因表达;高效液相色谱检测脂肪细胞内甘油三酯含量;Western-blot检测分析各实验组ERK1/2、PPARγ2和aP2蛋白的表达。
结果:
1.miRNAs芯片分析
诱导分化结束时,两种脂肪细胞内均含许多脂滴,90%以上的细胞能被油红O染色;且PPARγ2和aP2的基因表达显著增加,这些结果表明3T3-L1前脂肪细胞和MSC分化成成熟的脂肪细胞。芯片结果显示:3T3-L1前脂肪细胞分化成脂肪细胞上调的miRNAs有26个,下调的miRNAs有2个;小鼠MSC分化成脂肪细胞上调的miRNAs有20个,下调的miRNAs有55个。
2.脂肪细胞分化相关的miRNAs及其靶基因
应用生物信息学方法,我们选择了3个可能与脂肪细胞分化相关的miRNAs(miR-320,21和miR-375),且MAPK1(ERK2)可能是这3个miRNAs的靶基因。
3.miRNAs对3T3-L1前脂肪细胞分化的影响
油红O染色结果表明:miR-375显著增加脂肪细胞的OD值,而miR-21和AMO-miR-320对脂肪细胞的OD值无显著影响,提示miR-375能促进3T3-L1前脂肪细胞分化。
4.miR-375促进3T3-L1前脂肪细胞的分化及可能机制
RT-PCR结果表明:对照组和miR375组在分化过程中PPARγ2和aP2表达逐日增加;但miR375组从分化的第5天起PPARγ2和aP2表达显著高于对照组。高效液相色谱分析发现:miR375使脂肪细胞内甘油三酯含量显著增加(12.1±0.46 vs 10.8±0.34 mg/mg prot,p＜0.01)。Western-blot结果显示:miR-375使3T3-L1脂肪细胞ERK1和ERK2蛋白分别下降了27.4%和28.2%,PPARγ2和aP2蛋白表达分别提高了49.6%和52.8%(P＜0.01);空质粒对上述指标无显著影响。
结论:
1.3T3-L1前脂肪细胞和MSC分化成脂肪细胞过程中存在差异表达的miRNAs。
2.miR-375可促进3T3-L1前脂肪细胞分化,其机制可能是通过抑制ERK1/2蛋白的表达、提高PPARγ2和aP2蛋白的表达。
Part one Differential expression of miRNAs in the 3T3-L1 adipocytes and insulin-resistant adipocytes
Objective:
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 model
IR- 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 miRNAs
MiRNA 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 genes
According 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 model
Insulin-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 analysis
MiRNA 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 genes
To 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 adipocytes
Objective:
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~(TM)2000 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- adipocytes
We 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~(TM)2000 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- adipocytes
Cells 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-adipocytes
We 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 adipocytes
The 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-adipocytes
Glucose 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- adipocytes
Western-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 differentiation
Objective:
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 differentiation
3T3-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 genes
According 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 differentiation
We 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~(TM)2000,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 differentiation
We 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~(TM)2000.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 mechanisms
To 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 analysis
At 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 genes
We 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 differentiation
Oil 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 mechanisms
RT-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.
引文
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