CircHIPK2 Regulates Neural Stem Cell Differentiation And Its Role In The Treatment Of Ischemic Stroke

Part I circHIPK2 is involved in the differentiation of NSCsAims: Neural stem cells(NSCs)can differentiate into neurons,astrocytes,and oligodendrocytes.NSCs differentiated neurons can replace neurons lost due to injury,aging and neurodegenerative diseases.Circ RNAs(circular RNAs)are abundantly expressed in the central nervous system(CNS),but the regulatory mechanism of circRNAs involved in the differentiation of NSCs is still unknown.Our previous studies indicated that circHIPK2 is involved in neuroinflammatory regulation.Therefore,this chapter aims to explore the regulatory effect of circHIPK2 in the differentiation of NSCs and exploreits molecular mechanism.Methods: Real-time quantitative PCR(q PCR)was used to detect the expression of circHIPK2 during the differentiation of NSCs.NSCs were transducted with lentiviral circHIPK2 si RNA(si-circHIPK2-NSCs)and Bromodeoxyuridine(Brd U)staining was used to detect the proliferation of NSCs.Western blot was used to detect glial fibrillary acidic protein(GFAP)and ?-III Tubulin(TUJ1)expression.Immunofluorescence staining was used to detect the expression of GFAP and TUJ1 in NSCs differentiated cells.Results: 1)During the differentiation of NSCs,the expression of circHIPK2 decreased in a time-dependent manner..2)Silencing of circHIPK2 significantly promoted the differentiation of NSCs into neurons,but did not affect the differentiation of NSCs into astrocytes.3)Overexpression of circHIPK2 in NSCs significantly reduced the number of differentiated neurons,but did not affect the differentiation of NSCs into astrocytes.4)Lentivirus did not affect the differentiation of NSCs.Conclusion: This section of the study found that during the differentiation of NSCs,the expression of circHIPK2 gradually decreased.Silencing of circHIPK2 promoted the differentiation of NSCs into neurons,but did not affect their differentiation into astrocytes.It is shown that silencing of circHIPK2 promoted the differentiation of NSCs into neurons.Part II Tracing of SPIO-labeled NSCs in vivoAims: In the application of stem cell therapy,monitoring the biological distribution and fate of injected cells is crucial.Superparamagnetic iron oxide nanoparticles(SPIOs)can successfully label many types of mammalian cells.Magnetic resonance imaging(MRI)and near-infrared fluorescence imaging(NIF)dual-mode were used to detect the movement and distribution of transplanted SPIO-labeled NSCs in vivo.In this section,the authenticity of tracing of SPIO-labeled si-circHIPK2-NSCs in transient middle cerebral artery occlusion(t MCAO)mice will be explored to provide a theoretical basis for their therapeutic effects.Methods: Cell counting kit-8(CCK-8),Prussian blue staining,MRI,inductively coupled plasma mass spectrometry(ICP-MS)and Brd U experiments were used to detect the characteristics of SPIO.After t MCAO model mice were established,SPIO-labeled NSCs was microinjected to the lateral ventricle.The migration and distribution of SPIO-labeled NSCs in the brain was monitored by MRI and NIF.Immunofluorescence staining was performed to confirm the authenticity of SPIO labeled NSCs in vivo.Results: 1)The uptake of SPIOs by NSCs was concentration-dependent.2)High concentrations of SPIOs had better magnetic properties than low concentrations of SPIOs.3)The labeling concentration 200 ?g/m L of SPIOs had no significant effect on the proliferation and differentiation of NSCs.4)A large number of iron signals were detected by MRI both at the injection site and the infarcted brain side in si-circCon-NSC group and si-circHIPK2-NSC group,indicating that SPIO-labeled NSCs could migrate to the infarcted brain area.5)NIF studies showed that there was no significant difference between the si-circCon-NSC group and si-circHIPK2-NSC group in fluorescence intensity and distribution in vivo.A large number of GFP-positive NSCs existed at the injection site and the infarcted cerebral cortex,and the injected NSCs gradually migrated from the injection site to the lesioned tissue.6)The authenticity of SPIO-labeled NSCs was confirmed by immunofluorescence staining in vivo.Conclusion: This section investigates the characteristics of SPIOs.The uptake of SPIOs by NSCs was concentration-dependent.The labeling concentration 200 ?g/m L of SPIOs had no significant effect on the proliferation and differentiation of NSCs.Dual-mode imaging enables real-time tracking of NSCs in vivo,providing a visual basis for evaluating the therapeutic effect of si-circHIPK2-NSCs.Part ? The mechanism of circHIPK2 regulates NSCs on ischemic stroke treatmentAims: Ischemic stroke can lead to the loss of large numbers of nerve cells and neurological deficit.Stem cell therapy can enhance neural plasticity and promote neural function recovery after ischemic stroke.Our previous studies have confirmed that circHIPK2 regulates the differentiation of NSCs and the distribution of transplanted NSCs can be monitored in t MCAO model mice in vivo in real time.This section will further explore the therapeutic effect of transplanted si-circHIPK2-NSCs on ischemic stroke,and explore its molecular mechanism in the pathological process of ischemic stroke,in order to provide a certain theoretical basis for treatment of ischemic stroke.Methods: MRI was used to detect the effect of NSC transplantation on the infarct volume of t MCAO mice.Modified neurological severity score,cylinder test and adhesive removal test were used to evaluate the functional recovery of mice after t MCAO.Immunofluorescence staining and western blot were used to detect the expression of synaptophysin and PSD-95 and TUJ1.Golgi staining was used to detect dendritic spine density in cortical neurons of t MCAO model mice.The m RNA expression profiles of si-circCon-NSCs and si-circHIPK2-NSCs were obtained by RNA sequencing.Results: 1)Transplantation of si-circHIPK2-NSCs promoted neurological recovery after stroke,but did not influence the volume of cerebral infarction.2)Compared with other organs,circHIPK2 was highly expressed in the brain.The expression level of circHIPK2 in the infarcted cortex of t MCAO mice did not change significantly at different time points.3)Silencing of circHIPK2 promoted the differentiation and neuroplasticity of differentiated neurons in vitro.4)Silencing of circHIPK2 triggered NSC-differentiated neurons and facilitates neuroplasticity in t MCAO mice.5)circHIPK2 regulated the differentiation of NSCs through downstream Spermine oxidase(Smox).Conclusion: The findings in this section shows that si-circHIPK2-NSCs promotes neurological recovery in t MCAO mice.It is verified that silencing of circHIPK2 can enhance the number of differentiated neurons and promote the neuroplasticity of t MCAO mice.Silencing of circHIPK2 in NSCs can regulate the differentiation of NSCs through the downstream Smox.The aim of this study is to provide preclinical experimental basis for si-circHIPK2-NSCs in promoting the recovery of brain injury after stroke.