Effect Of Let-7g On Proliferation, Migration And Neural Differentiation Of Bone Marrow Mesenchymal Stem Cells In Vitro

Background and objectiveIn recent years, bone marrow mesenchymal stem cells(MSCs) transplantation for treatment of neurological diseases were given high hopes, but the pathological changes of brain structure and function in patients may directly affect the survival and differentiation of MSCs. MicroRNA(miRNAs) are a class of endogenous non-coding RNA with length of about 20 to 25 nucleotides, which involve in the regulation of proliferation, differentiation, signal transduction and apoptosis of MSCs. As a member of miRNAs, let-7g can regulate the function of many genes, involving in the regulation of cell proliferation, differentiation, apoptosis, invasion, migration and so on. Research about effect of let-7g on biological activity of MSCs has not been seen. The lentiviral vectors of let-7g-up and let-7g-down were constructed. And then transfecting into MSCs in vitro using these lentiviral vectors, resulting in that let-7g was high or low expression within the cells. To investingate the effect and possible mechanism of let-7g on proliferation, migration, and nerve differentiation of MSCs. In order to find a new way to improve the curative effect of MSCs transplantation. Materials and methods1. MSCs were isolated from SD rats aged 6-8 weeks and weighed 100-120 g by the method of whole bone marrow cultureing, and expanded in vitro for more than six generations.2. The lentiviral vectors of let-7g-up and let-7g-down were constructed and transfected into rat MSCs in vitro. Optimal multiplicity of infection was screened. The cells were divided into four groups: non-transfected group(transfected nothing), negative control group(transfected with empty lentivirus + EGFP), transfected enhancement group(transfected with LV-rno-Let-7g-up + EGFP), transfected inhibition group(transfected with LV-rno-Let-7g-5p-inhibition + EGFP). The miRNA exprission of let-7g in each group were detedted by RT-PCR.3. Immunocytochemical method and western blot were used to detect the effects of different expression level of let-7g on the expression of high mobility group-AT-hook 2(HMGA2). In vitro, the proliferation and migration ability of MSCs were assessed by MTT assay, transwell migration assay and wound-healing assay respectively.4. MSCs were treated with fasudil as an inducer for triggering them to differentiate into neurons.5. The expression of neuron-specific enolase(NSE), microtubule-associated protein 2(MAP-2) and Notch-1 in each inducde groups were measured by immunocytochemistry method and western blot assay. The mRNA expression of microtubule-associated protein 2 and Notch-1 were measured by RT-PCR. To investigate the role and possible mechanisms of let-7g and Notch signaling pathway in MSCs differentiating into neurons.6. The potential target genes of let-7g were predicted by bioinformation softwares. Luciferase reporter assay was performed to detect whether the Notch-1 was regulated by let-7g.7. The PKH26-labelled MSCs were transplanted into ventricles of SD rats. The distribution of MSCs in rat brain was observed under fluorescence microscope at 2 weeks after transplantation. Results1. Under the inverted fluorescence microscope we found lentiviral vector can transfected MSCs efficiently. There was no significant difference of EGFP expression between transfectioned groups. RT-PCR results demonstrated that the expression level of let-7g in the transfected enhancement group were significantly higher than that in the negative control group(P<0.05), while in the transfected Inhibitor group, the expression level of let-7g was lower than that in the negative control group(P<0.05). There was no significant difference between the expression level of let-7g in the non-transfected group and the negative control group(P>0.05).2. The proliferation and migration capacity and the expression level of HMGA2 in the transfected enhancement group were significant lower than those in the negative contral group(P<0.05), while in the transfected inhibition group, they were higher than those in the negative contral group(P<0.05).3. Fasudil induced MSCs to differentiate into neurons. The induction efficiency and expression levels of NSE 、 MAP-2 and MAP-2 mRNA in the transfected enhancement group were significant higher than those in the negative contral group(P<0.05), while in the transfected inhibition group, they were lower than those in the negative contral group(P<0.05). Comparing with the negative contral group, expression levels of Notch-1 and Notch-1 mRNA in the transfected enhancement group were higher, while those in the transfected inhibition group were lower(P<0.05).4. Notch-1 was predicted as a target of let-7g by TargetScan and miRBase database. Luciferase activity of cells transfected with Notch-1 3’UTR+ rno-let-7g-5p was significantly reduced than corresponding control group(P<0.05).5. In brain, PKH26-labelled cells were mainly distributed in the lateral ventricle, some migrated. Conclusions1. Let-7g could inhibit proliferation and migration of MSCs by downregulation of HMGA2.2. Let-7g could promote MSCs to differentiate into neurons. Notch-1 was one of the target genes of let-7g. Let-7g could promote MSCs neuronal differentiation by inhibition of the notch signaling pathway.3. MSCs transplanted into the lateral ventricle of rat survived well, and some migrated.