MicroRNA-128Regulates The Differentiation Of Rat Bone Mesenchymal Stem Cells Into Neuron-like Cells

Background and PurposeBone marrow mesenchymal stem cells (BMSCs) are a source of multipotent stemcells ideally suited for various cell-based therapies. BMSCs have the potential toundergo multilineage differentiation into multiple connective tissue cell types such asosteoblasts, chondrocytes,adipocytes, myoblasts even neurons under appropriate experimental conditions.Oriented differentiation of BMSCs has become a hot research issue and attractedmore and more attention.MicroRNAs are members of a family of non-protein coding, single-strandedRNAs of~23nucleotides in length. These small RNAs destabilize target mRNAs orsuppress their translation by binding to complementary sequences in the3’untranslated regions (3’-UTRs). MicroRNAs control space-time differentialexpressions of different gene of BMSCs genome at the transcriptional level andpost-transcriptional level, and are involved in a series of important processes ofBMSCs differentiation. MicroRNA-128, a brain-enriched miRNA, is highlyexpressed in the central nervous system, especially in the brain and spinal cord.MicroRNA-128has been reported to play an important role in neural stem cells’self-renewal and differentiation. Previous studies indicated that the activation of thecanonical Wnt signaling pathway is related to BMSCs’ directional differentiation.Using microRNA analysis software, we conjectured the possibility that microRNA-128is targeting the3′-UTR of Wnt3a, a key component of the Wntsignaling pathway. To explore the validity of our prediction, we investigated whethermicroRNA-128could affect or induce the neural differentiation of BMSCs and themolecular mechanism through which microRNA-128regulates BMSCsdifferentiation, with a focus on microRNA-128’s modulation of Wnt3a in the Wntsignaling pathway.Part one: Downregulated microRNA-128drive BMSCs toward developing intoneuron-like cellsMethods1BMSCs were isolated from SD rats aged4weeks. The third-generation cells werecollected and detected of cell antigens(CD29，CD34、CD44、CD45、CD105) withflow cytometry for BMSCs identification.2When the cells reached90%confluence, the cells were divided into four groups: aMimic group, an Inhibitor group, an Inducer group and a Blank group. Cells in theMimic group were transfected by microRNA-128mimics via electroporation; cellsin the Inhibitor group were transfected by microRNA-128inhibitors viaelectroporation; lastly, the cells in the Inducer group and Blank group were nottransfected. BMSCs in three experimental groups except Blank group cell wereinduced in the induction medium supplemented with bFGF and EGF. Cells inBlank group were only cultured under common condition.3Relative levels of microRNA-128mRNA in induced BMSCs were determined byreal-time quantitative RT-PCR, where the U6snRNAs served as a control.4The mRNA expressions of nerve cell markers(β3-tubulin，MAP-2，NF-M，NSE，GFAP，Nestin，Vimentin) and Wnt3a were detected by the TaqMan real-timeRT-PCR.5The protein expressions of nerve cell markers(β3-tubulin，MAP-2，NF-M，NSE，GFAP，Nestin，Vimentin) and Wnt3a were detected by Western blot.6The changes in cell morphology and the protein expressions of GFAP, Nestin wereexamined by immunofluorescence. 7The changes in cell morphology and the protein expressions of Nestin wereexamined by immunohistochemistry test8Statistical treatment was performed with SPSS17.0software. Data were expressedas (x±s). One-factor analysis of variance was used in the comparison amongmultiple groups. The least significant difference method (LSD method) was used inthe comparison between two groups. The statistical significance was established atP＜0.05.Results1The third-generation cells appeared positive BMSCs phenotype. Flow cytometry analysis showed that the percentage of CD29(99.26%), CD44(99.14%)and CD105(98.45%) were higher than that of CD34(1.58%)，CD45(1.82%)which were in line with the internationally recognized criteria for BMSCs.2Real-time quantitative RT-PCR result demonstrated that microRNA-128in the Mimicgroup was overexpressed compared with the Blank group and the Inducer group (p＜0.01) and microRNA-128in the Inhibitor group was repressed when compared withthe Blank group and the Inducer group (p＜0.01). There was no significant differencebetween the level of microRNA-128in the Blank group and the Inducer group(p>0.05).3When the BMSCs induced with bFGF and EGF are compared with the Blank group,the mRNA-relative quantitation of NSE, Nestin, GFAP, NF-M, MAP-2, Vimentinand β3-tubulin were found to be significantly higher in all3experimental groups(p<0.05). In the Mimic group, nerve cell markers mRNA levels were significantlyreduced compared with the Inducer group (p<0.05). In the Inhibitor group, nervecell markers mRNA levels were significantly elevated compared with the Inducergroup (p<0.05). Furthermore, identical experiments were performed, whichprovided similar results to Wnt3a.4Western blot image showed that cells in Blank group didn’t express7nerve cellmarkers, while cells in other3groups induced by bFGF and EGF express7nerve cell marker much higher compared to cells in Blank group. The protein expressionsof nerve cell markers were significantly increased in the Inhibitor group, butsignificantly deceased in the Mimic group compared to the Inducer group. Here wefound microRNA-128have an impact on the7nerve cell markers in the inducedBMSCs. The expression of the7nerve cell markers was suppressed by themicroRNA-128mimic but elevated by the microRNA-128inhibitor.5For both GFAP and Nestin, the fluorescence intensity of the Blank group and theInducer group were negative and positive, respectively. Particularly, the intensity offluorescence in the Mimic group provided the strongest inhibitory effect while theintensity of fluorescence in the Inhibitor group provided the strongest enhancementeffect.6The immunochemical staining result of Nestin in the Blank group, the Inducergroup, the Mimic group and the Inhibitor group were respectively negative,positive, weak positive and strong positive. Most cells in the Inhibitor group shrunkand became round-like or cone-like with long processus forming networks whichwere the typical structures of nerve cells.Part two: The molecular mechanism through which microRNA-128regulatesBMSCs differentiation into neurons-like cellsMethods1The relationship between the Wnt3a–3′-UTR and its targeted miRNAs waspredicted through bioinformatics analysis by miRanda， miRDB， RNA22，TargetScan and miRanda that showed that the3′-UTR of Wnt3a might be targetedby microRNA-128.2To construct a luciferase reporter vector,the wild type3’UTR of Wnt3a wasamplified using genomic DNA from the rat as templates. The corresponding mutantconstructs were created by overlap PCR. BMSCs were cotransfected withmicroRNA-128mimics/microRNA-128inhibitors and luciferase reporter plasmid(wild type/mutant type).At48h after transfection, cells were harvested andluciferase activity was measured. 3To determine whether Wnt3a is involved in the microRNA-128-induced thedifferentiation, we performed rescue assays. We created the vectors containingWnt3a gene (without3′-UTR region).4To determine whether Wnt3a acts as a critical mediator of microRNA-128’s role inBMSCs differentiation, we performed rescue assay targeted knockdown of Wnt3aexpression using siRNA.Results1The relationship between the Wnt3a–3′-UTR and its targeted miRNAs waspredicted through bioinformatics analysis by TargetScan that showed that the3′-UTR of Wnt3a might be targeted by microRNA-128and contains a complementsite for the8mer seed region.2The reporter vectors consisting of the luciferase coding sequence followed by the3′UTR of Wnt3a (wild type/mutant type)were verified by sequencing.Cotransfection experiments showed that microRNA-128decreased the luciferaseactivity of wild type（P<0.05）, but this was not observed on mutant type.3Overexpression of microRNA-128and Wnt3a (without3′-UTR region) nullified theinhibitory effect of microRNA-128on the expression of Wnt3a compared with cellstransfected with microRNA-128and the empty expression vector groups. Thesedata demonstrated that forced expression of Wnt3a significantly rescuedmicroRNA-128induced inhibiting effect.4The reduction of Wnt3a expression level by siRNA led to a marked decrease in thedifferentiation of BMSC into neurons, similar to those induced by microRNA-128Mimic.Conclusions1The results showed that downregulated microRNA-128could promote thedifferentiation of BMSC toward neuron-like cells by bFGF and EGF, whileupregulated microRNA-128may suppress mesenchymal stem cells toward aneuronal fate.2MicroRNA-128can bind to the3′-UTR of Wnt3a mRNA to suppress Wnt3a expression. By binding to a specific site in the3’-UTR of Wnt3a in BMSCs,downregulated microRNA-128may lead to the neural differentiation ofmesenchymal stem cells.