MRI Detection Of The Neural Differentiation Of Stem Cells Base On A Microrna-124-Responsive Reporter Gene

Background and objective Stem cells have attracted much attention in the field of regenerative medicine because of their multi-directional differentiation potential.Stem cells can be induced to differentiate into neurons,thereby repair or replace damaged cells and tissues,which provides a new strategy for the treatment of neurological diseases.However,in order to achieve clinical application,it is necessary to use non-invasive imaging methods to effectively monitor and evaluate the occurrence of stem cell differentiation.At present,there are three imaging methods for stem cell monitoring:optical imaging,nuclear medicine imaging and magnetic resonance imaging(MRI).MRI is widely used due to its non-radiation,strong tissue penetration and good soft tissue contrast.Molecular MR imaging often relies on reporter genes.MRI reporter genes can be divided into reporter genes encoding enzymes,reporter genes encoding cell membrane receptors and endogenous reporter genes.Ferritin gene is an endogenous reporter gene which is one of the most mature and widely used MRI reporter genes.In the previous studies,we have used the FTH1 reporter gene for MRI tracing studies on neural differentiation of stem cells.Although the reporter gene-based MRI has been verified to be feasible for longitudinal monitoring the process of stem cell differentiation,the cells before and after neural differentiation cannot be effectively distinguished,that means occurrence of stem cell differentiation cannot be detected.In recent years,more and more evidence showed that micro RNAs(miRNAs)are closely involved in the regulation of neural differentiation of stem cells.The miRNA-124(miR-124)is one of the most abundant miRNAs in neural cells.The expression of miR-124 changes significantly in the neural differentiation of stem cells.Considering the differential expression of miR-124 before and after neural differentiation of stem cells,it is possible to realize the dynamic monitoring of stem cell differentiation through constructing a miR-124 responsive MRI reporting gene system.Recent studies have found that the miR-124 can target and specifically bind to the 3'untranslated sequences(3'UTR)of the ionotropic glutamate receptor AMPA2(GRIA2)and inhibit the expression of GRIA2.Inspired by this,we assume that the 3'UTR of FTH1 could be replaced by the3'UTR of GRIA2(GRIA2 3'UTR),which could be specifically bound with miR-124,thus control the expression of FTH1 reporter gene.Then,the intracellular miR-124 could be detected through the change of reporter gene expression,thereby determining the events of stem cell differentiation.Based on the above background and assumptions,the present study intended to use the GRIA2 3'UTR to modify the FTH1 reporter gene and construct a miR-124-responsive FTH1 gene reporting system(FTH1-GRIA2 3'UTR).The gene reporting system was transduced into mouse P19 stem cells.The transgenic P19 stem cells were induced to differentiate into neurons,and the effect of neural differentiation on reporter gene expression was observed before and after differentiation.It was expected that the expression of miR-124 will increase after the neural differentiation of stem cells and specifically target to the GRIA2 3'UTR of FTH1 gene,thus inhibiting the FTH1 gene expression.It is possible to visualize the dynamic changes of miR-124 in cells by MRI through the changes of intracellular reporter gene expression before and after neural differentiation,so as to effectively monitor stem cell differentiation events.This strategy could also provide a new non-invasive MRI method for researching the targeting relationship between the miRNA and their possible target gene.Methods(1)Dual-luciferase reporter gene assay The experiment was to use Target Scan to predict the binding site between miR-124 and GRIA2 3'UTR.To validate the miR-124 for targeting the GRIA2,4 experimental groups was designed as follows:GRIA2 3'UTR vector plasmid+miR-124 vector plasmid;GRIA2 3'UTR empty plasmid(GRIA2 3'UTR-NC)+miR-124 vector plasmid;GRIA23'UTR plasmid+miR empty plasmid(miR-NC);GRIA2 3'UTR empty plasmid(GRIA2 3'UTR-NC)+miR-NC.The positive reference plasmids were divided into two groups: TRAF6 3'UTR vector plasmid+miR-146 b vector plasmid,TRAF6 3'UTR vector plasmid+miR-NC.These were used to verify whether the whole transfection detection system was effective.(2)Detection of FTH1 expression by western blot Western blot was used to detect whether FTH1 was inhibited after FTH1-GRIA2 3'UTR combined with miR-124 in 293 T cells.The experiment was divided into two groups: FTH1-GRIA2 3'UTR vector plasmid+miR-124 vector plasmid and FTH1-GRIA2 3'UTR plasmid+miR-NC plasmid.The FTH1 expression level in 2 groups was detected.Each experiment was repeated three times.(3)Construction of lentiviral vector The vector plasmid GV367(Ubi-MCS-SV40-EGFP-IRES-puromycin)was digested by Age I/Nhe Ienzyme,which wasdesigned by Shanghai Genechem Co.,Ltd.The synthetic FTH1 sequence and the 3'UTR of GRIA2 were connected with the vector and transformed to obtain the recombinant lentiviral vector plasmid GV367-FTH1-3'UTR.The monoclonal clones were sequenced and identified.After the successful construction,293 T cells were infected to produce the lentivirus.After 48 h,the virus was collected and centrifuged and labeled as CMV-FTH1-GRIA2 3'UTR.The virus was packaged and stored at-80°C.The CMV-FTH1 was constructed as a positive control and CMV-NC as a negative control.(4)Establishment of stable strains by lentiviral transfection of P19 cells and puromycin screening When the confluence of P19 cells reached about 30% in the orifice plate,the virus CMV-FTH1-GRIA2 3'UTR of experimental group,CMV-FTH1 of positive control group and CMV-NC of negative group were respectively added into the culture medium.After 48 h,green fluorescence protein expression was observed under a fluorescence microscope.The stable strain was screened by puromycin and named P19-FTH1-GRIA2 3'UTR,P19-FTH1 and P19-NC.(5)Induction and identification of the neural differentiation of P19 cells Taking 8 d as a cycle,the cells of P19-FTH1-GRIA2 3'UTR and P19-FTH1 were suspended in 10 ? M at RA medium for 4 d.Then cells were resuspended and centrifuged,and maintained in acomplete medium without at RA on polylysine-coated petri dish for next 4 d.Under a microscope,the cell morphology changes on the 0th,4th,and 8th day.The cells on the 8th day were chosen for immunofluorescence staining.PCR was performed to detect the expression changes of the intracellular miR-124 on the 0,2,4,6 and 8th day.The above three methods were used to determine whether Neurons-FTH1-GRIA2 3'UTR,Neurons-FTH1 cells were successfully differentiated.(6)FTH1 expression and iron collection before and after neural differentiation The intracellular iron content was detected by Prussian blue staining and iron content reagent Kit.The FTH1 expression was detected by Western blot.The T2 WI signal and R2 change was observed on MRI images in vitro,before and after neuronal differentiation.Results(1)Dual-luciferase reporter assay Target Scan analysis showed that miR-124 had binding sites with GRAI2 3'UTR region.The results of dual-luciferase reporter assay showed that the luciferase activity of GRIA2 3'UTR + miR-124 group was significantly lower than that of GRIA2 3'UTR-NC + miR-124 group,and the difference was statistically significant(P<0.05).In addition,the luciferase activity in the TRAF6 3'UTR + miR-146 b group was significantly lower than that in the TRAF6 3'UTR + miR-NC group,and the difference was statistically significant(P<0.05),indicating that there was no abnormality in the transfection detection system. (2)Western blotdetect the expression of FTH1 in 293 T Western blot analysis showed that the expression of FTH1 in FTH1-GRIA2 3'UTR + miR-124 group was lower than that in FTH1-GRIA2 3'UTR + miR-NC group(P<0.05).(3)Identification and virus packaging of FTH1-GRIA2 3'UTR lentiviralvector After vector digestion,target gene fragment acquisition,recombinant vector construction(PCR identification)and sequencing,The CMV-FTH1-GRIA2 3'UTR and CMV-FTH1 lentivirus vector were successfully constructed,and the virus titer was 1.5×109 TU/m L.(4)Obtainment of stable cell strains After 72 h of lentivirus transfection,green fluorescence expression was observed under the microscope.After 7 d of screening with 2?g/m L puromycin,the cell concentration was reduced to half,and then screened for 3 days.We successfully obtained P19-FTH1-GRIA2 3'UTR,P19-FTH1 and P19-NC stable strain cells.(5)Neural differentiation of P19 cells Under the microscope,the cells of P19-FTH1-GRIA2 3'UTR,P19-FTH1 were observed to be adherent on 0th day,and some cells were clone-like.The cells gradually grew into clusters and formed spherical embryoids with clear boundaries on 4th day after at RA induction.The cell morphology began to transform to neurons,and synapse formation was visible in the next 4 days in complete medium.Immunofluorescence detected obvious expression of TUJ1 in the induced cells.PCR showed that the expression of miR-124 gradually increased with differentiation days.Neurons-FTH1-GRIA2 3'UTR and Neurons-FTH1 cells were successfully differentiated.(6)The intracellular FTH1 expression and iron uptake effect before and after neural differentiation Prussian blue staining showed a large amount of intracellular iron particles in P19-FTH1-GRIA2 3'UTR group,P19-FTH1 group and Neurons-FTH1 group.Less iron particles accumulated in cells of the Neurons-FTH1-GRIA2 3'UTR group,and no obvious iron particles accumulated in cells of the P19-NC group.The results of iron reagent kit showed that the intracellular iron content in Neurons-FTH1-GRIA2 3'UTR group was significantly lower than that in P19-FTH1-GRIA2 3'UTR group,and the difference was statistically significant(P<0.05).Western blot analysis showed obvious FTH1 expression in the P19-FTH1-GRIA2 3'UTR,P19-FTH1,and Neurons-FTH1 groups,while only a small amount in the Neurons-FTH1-GRIA2 3'UTR group,and hardly in the P19-NC group,and the difference was statistically significant(P<0.05).MRI showed that the T2 WI signal of the Neurons-FTH1-GRIA2 3'UTR cells was significantly higher than that of P19-FTH1-GRIA2 3'UTR cells.The R2 of Neurons-FTH1-GRIA2 3'UTR cells on T2 WI was significantly lower than that of P19-FTH1-GRIA2 3'UTR cells,and the difference was statistically significant(P<0.05).Conclusion Based on the characteristics of miR-124 targeting GRIA2 3'UTR,the miR-124 responsive gene reporting system FTH1-GRIA2 3'UTR was successfully constructed through gene technology.After neural differentiation of P19 stem cells,the intracellular miR-124 expression was significantly increased.The miR-124 specifically targeted to GRIA23'UTR of FTH1 gene reporting system,thereby inhibiting the FTH1 gene expression,which leaded to intracellular iron accumulation and allowed for MRI detection.This study confirmed the feasibility of the miR-124-responsive reporter gene system for MRI displaying the intracellular miR-124 activity and provided a new imaging strategy for detecting neural differentiation of stem cells.This strategy may also be expanded to noninvasively study the targeting relationship between the interested miRNAs and their possible target genes.