MiR-29a-5p In The Differentiation Of Hippocampal Neural Stem Cells Into Neurons Induced By Valproic Acid

ObjectiveThe role of miR-29a-5p in the differentiation of hippocampal neural stem cells into neurons induced by valproate was studied by using microarray and molecular biology techniques.Experiment 1 : Construction of differential expression profiles of mRNAs and miRNAs of rat hippocampal neural stem cells after VPA treatmentMethods(1)Rat hippocampal NSCs were isolated and cultured in vitro.The cells were treated with VPA at concentration of 0.3mM,0.6mM,1mM and 3mM or control vehicle respectively.Immunofluorescence assay was used to detect cell apoptosis.(2)Immunofluorescence assay,western blot and flow cytometry techniques were used to detect the level of histone acetylation and verify the effect of VPA in NSCs proliferation and differentiation.(3)Real-time quantitative PCR was used to detect the dynamic expression of key neuron markers—Synapsin I(Syn1)and Microtubule-associated protein 2(Map2)after VPA treatment,and we collected samples when the expression of these two neuron markers began to increase significantly.(4)Samples were sent to biochip company to construct differential expression profiles of mRNAs and miRNAs.We obtained differentially expressed mRNAs and miRNAs whose differences were more than 1.5 times,and the P values were less than 0.05.(5)Total RNA was extracted from cell samples treated by VPA or vehicle,and the expression of some differential mRNAs and miRNAs was verified by real-time PCR.Results(1)The results showed that the hippocampal NSCs did not show significant apoptosis when they were treated with VPA at a concentration of no more than 1 mM.(2)Immunofluorescence,western blot and flow cytometry showed that VPA could up-regulate the level of histone acetylation,inhibit the proliferation of NSCs,promote the differentiation of NSCs into neurons and inhibit the differentiation of NSCs into astrocytes.(3)Real-time PCR results showed that the expression of neuron markers,Syn1 and Map2,began to increase significantly at 24 h after VPA treatment.(4)Micorarray results showed that there were 2410 mRNAs and 7 miRNAs exhibited significant differential expression.Among them,1660 mRNAs were up-regulated and 750 were down-regulated;6 miRNAs were up-regulated and 1 was down-regulated.(5)The results of real-time PCR showed that the expression of the 12 mRNAs were consistent with microarray results.So did 5 out of the 7 differential miRNAs.Experiment 2: The study on the function of miR-29a-5p in proliferation and differentiation of hippocampal NSCs in ratsMethod(1)The total RNA was extracted from the cerebrum,cerebellum,brainstem,hippocampus,heart,liver and skeletal muscle of SD rats.The tissue expression specificity of miR-29a-5p was detected by real-time quantitative PCR.(2)NSCs,neurons and astrocytes were isolated and cultured in vitro,and total RNA was extracted.Real-time quantitative PCR was used to detect the cell expression specificity of miR-29a-5p.(3)Transfection of NSCs with miR-29a-5p analog(mimic).We set up a concentration gradient of 10 nM,20nM,50 nM,100nM and 200 nM.The transfection efficiency was observed by fluorescence microscope and the overexpression efficiency of miR-29a-5p was detected by real-time quantitative PCR.(4)NSCs were transfected with miR-29a-5p mimic,and the expression of key neuron markers—Map2,Syn1,Neuronal differentiation 1(Neurod1)and Stathmin2(Stmn2)was detected by real-time quantitative PCR.(5)The effect of miR-29a-5p overexpression on the proliferation of NSCs was detected by immunofluorescence,western blot and flow cytometry.(6)The effect of miR-29a-5p overexpression on the differentiation of NSCs was detected by immunofluorescence,western blot.Results(1)The expression of miR-29a-5p showed tissue-specificity and cell-specificity,that it was highly expressed in cerebrum and hippocampus and in NSCs.(2)The miR-29a-5p mimic transfection at the concentration of 20 nM,50nM and 100 nM showed high fluorescence intensity over-expression efficiency.(3)Real-time PCR results showed that the expression of neuron markers—Map2,Syn1,Neurod1,and Stmn2 was significantly up-regulated at 24 h after 20 nM mimic transfection.(4)Immunofluorescence,western blot and flow cytometry results showed that overexpression of miR-29a-5p could inhibit the proliferation of NSCs and promote the differentiation of NSCs into neurons.Conclusions1.VPA could up-regulate the level of histone acetylation,inhibit the proliferation of NSCs,promote the differentiation of NSCs into neurons and inhibit the differentiation of NSCs into astrocytes.2.During the differentiation of hippocampal NSCs induced by VPA,the expression of 12 mRNAs were consistant with microarray results.Expression of 5 out of 7 differentail miRNAs were consistent with microarray results.3.Over-expression of miR-29a-5p could inhibit the proliferation of hippocampal NSCs and promote the differentiation of hippocampal NSCs into neurons.