| Background:Spinal Muscular Atrophy syndrome(SMA)is a rare autosomal genetic disease caused by the mutation of SMN1 gene,which leads to the degeneration of motor neurons and muscle atrophy.Downregulation of SMN protein can cause damage to neuronal axons,decrease of neuromuscular junctions,and obstruction of signal transduction.The PCDH gene cluster is essential in neural development and has functions such as axon formation,dendrite self-avoidance and neuron survival.The selective expression of PCDH subgene can be used as a surface marker for the unique structure and function of neuron cells.However,the function of PCDH gene cluster in motor neuron development and the regulatory mechanism of selective expression remain unclear,and the selective expression pattern and regulatory mechanism of PCDH gene cluster in degenerated motor neuron cells and atrophic muscle cells during the pathogenesis of SMA disease remain to be clarified.Objective:By establishing a neural differentiation model of SMA disease in vitro,the selective expression of PCDH subgene in normal and SMA progenitor cells and the mechanism of regulating the selective abnormal expression of PCDH subgene were studied.Method:1.The pluripotency of SMA-iPSCs was verified by immunofluorescence staining,flow cytometry,RT-qPCR and other experiments.Teratoma formation test and EB formation test were used to verify the differentiation ability in vitro and in vivo.The in vitro differentiation system of iPSCs into neural progenitor cells was established by SMADI nerve induction culture.The neural progenitor cell markers were verified by immunofluorescence staining and RT-qPCR.2.The differential genes of SMA disease group and normal group during neural differentiation in vitro were investigated by RNA-seq sequencing and verified by RT-qPCR.Then small interfering RNA transfection was used to knock down differential genes to explore the changes of other differential genes.3.The ChIP-qPCR experiment was used for immunoco-precipitation of DNA fragments in the differential gene enrichment region,and RT-qPCR was used to quantify the DNA expression level to further verify the regulatory relationship between the differential genes.Result:1.To verify the pluripotency and in vitro differentiation of SMA-iPSCs induced reprogramming in vitro.The differentiation system of iPSCs into neural progenitor cells in normal and SMA patients was successfully established in vitro,and the expressions of neural progenitor cell markers PAX6,SOX1 and NESTIN were upregulated.2.Transcriptomic sequencing found that the selective expression of the progenitor gene PCDHα6 was abnormally elevated during the differentiation of neural progenitor cells with SMA disease in vitro,which may be one of the main causes of the abnormal function of nerve cells.3.The abnormal up-regulation of PCDHα6 was mainly due to the up-regulation of the expression of RAD21,an important subunit of chromatin ring.After transfection with siRNA knockdown of RAD21,the expression of PCDHα6 was down-regulated.The results indicated that the abnormal increase of RAD21 in SMA neural progenitor cells led to the abnormally high expression of PCDHα6.4.In terms of mechanism,we found that during the differentiation of SMA neural progenitors,RAD21 significantly increased its enrichment in the PCDHα6 promoter and its enhancer HS5-1a region,indicating that RAD21 regulates the expression level of PCDHα6 promoter and enhancer by regulating the activity of PCDHα6 promoter and enhancer.Conclusion:In the process of iPSCs differentiation into neural progenitor cells,the expression of RAD21 of the important subunit of the chromatin ring(Cohesin complex)increases abnormally,and the enrichment of promoters and enhancers of PCDHα6 subgene is up-regulated,which pulls the distal enhancer towards the PCDHα6 promoter and enhances its expression activity.The expression of PCDHα6 in SMA neural progenitor cells was abnormally upregulated,which may be the cause of the dysfunction of SMA neural progenitor cells. |
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