Soft Substrates Promote Direct Reprogramming Of Human Fibroblasts Into Neurons Through The MiR-615-3p/ITGB4 Signaling Axis

The research results that the combination of small molecules(SMs)can directly reprogram fibroblasts(FBs)into induced neurons(iNs)bypassing the intermediate stage have greatly promoted the progress of regenerative medicine research.However,based on the existing research results,the gap between the efficiency,quality and quantity of neurons obtained by in vitro reprogramming of FBs and subsequent basic research and practical applications is still an urgent scientific and technical problem to be solved in the field.The maintenance of tissue cell structure and function in the body,the regulation of proliferation and differentiation depend on the microenvironment in which the cells live.Although the effects of physical signals and miRNA on the direct reprogramming of FBs into iNs have been reported successively,the molecular mechanisms of their regulation of neuronal reprogramming have not been systematically understood,and physical factors do not cover all the characteristics of extracellular matrix(ECM).At present,in vitro studies for neural induction scientists mainly use rigid polystyrene tissue culture plate(TCP)(～GPa),and TCP is also very different from ECM in vivo only in terms of its stiffness.It is known that the stiffness of brain tissue is soft in the range of 100Pa-3kPa,although the effect of this stiffness on neural direct reprogramming has not been fully analyzed,softer ECM stiffness can induce a variety of stem cells to differentiate into nerves.and it has been proved that a variety of key physical signals such as Hippo,cytoskeleton and integrin(integrin beta 1,integrin beta 4(ITGB4),etc.)are involved.However,there are no reports related to the connection between mechanical signals and miRNA includes miR-615-3p,which is involved in nerve differentiation.Therefore,on the basis of direct neuronal reprogramming by SMs combination,it is of great scientific significance and application value to explore the effects and mechanisms of miRNA and mechanical signals on neuronal reprogramming.In this study:(1)human foreskin fibroblasts(hFFs)were used as starting cells,neurons with electrophysiological characteristics were used as target cells,and type I collagen was use to prepare soft substrates.The elastic modulus range of soft substrates was detected by nanoindenter.Morphological statistics,quantitative reverse transcription PCR(qRT-PCR),immunofluorescence staining and patch clamp,etc.were used to explore the effects of soft substrates on neural direct reprogramming of SMs combination;(2)Transcriptome sequencing was performed on hFFs on TCP(ThFFs),hFFs on soft substrates(SShFFs),iNs on TCP(TiNs)and iNs on soft substrates(SSiNs)in the reprogramming process,the neural development-related signals and mechanical signals among them by differential expressed(DE)GO and KEGG analysis,mechanical signaling pathways were verified by transmission electron microscopy and immunofluorescence techniques,and competing endogenous RNA(ceRNA)network analysis was performed on whole transcriptome sequencing data to analyze the regulatory mechanism of miRNA-targeted mRNA mechanical signaling that promote neuronal reprogramming by soft substrates,and locking miRNA-mRNA research targets in combination with mechanical signaling pathways;(3)The role and mechanism of the locked miR-615-3p/ITGB4 signaling axis in the process of reprogramming were explored by dual luciferase reporter,cell transfection,qRT-PCR and Western Blot experiments,to propose new ideas and theoretical foundations for regenerative medicine based on nerve repair.1.Soft substrates promote reprogramming of hFFs into glutamatergic neurons with electrophysiological functionsPrimary hFFs were successfully isolated and cultured,they express VIMENTIN and do not express E-CADHERIN,and they are mesenchymal cells without multipotent differentiation,meeting the requirements of neurons reprogramming starting cells;The elastic modulus of the soft substrates prepared with type Ⅰ collagen was in the range of 450-850 Pa and aggregated at about 600Pa,which was consistent with the stiffness range of brain tissue and neural differentiation,and it would not be completely degraded during the reprogramming process;After observation and statistics,it was found that the soft substrates significantly increased the number of neuromorphic cells and neurites.It was also found that the soft substrates promoted the occurrence of mesenchymal-epithelial transition(MET)during direct reprogramming;In the exploration of the neuronal reprogramming stage,we found that TiNs could not proliferate on day 4,while SSiNs could.Therefore,it is necessary to determine the presence of neural stem cells(NSCs)at this point in the subsequent process.SSiNs were in a non-proliferative phase on days 7 and 10;Soft substrates also inhibited the expression of FBs markers VIMENTIN,CTGF,COL1A1 and DKK3,and increased the expression of neural markers such as neuronal class Ⅲ β-tubulin(TUJ1),ASCL1,microtubule-associated protein 2,TAU,NEUN,SYN1,Nav1.3 and Kv4.3.Importantly,the soft substrates increased the expression of the presynaptic protein SYN1,the postsynaptic protein PSD95,and the type I vesicular glutamate transporter(vGLUT1),especially at the neurites,with a significant increase in the number of positive cells.This suggested that soft substrates were beneficial to the formation of iNs synaptic network and expression of vGLUT1.More importantly,soft substrates decreased the resting membrane potential of iNs,increased the maximum Na inward current,and promoted the occurrence of fast Na+ inward current and action potential;By judging the expression of NSCs markers NESTIN and PAX6 during reprogramming and the ability of spherulation,no dedifferentiated NSCs intermediates that could form spheroids were found.These results suggested that the soft substrates of 450-850Pa promoted direct reprogramming of human FBs into elec trophy siological glutamatergic neurons.2.miRNA and mechanical signaling mechanism of soft substrates in neuronal reprogrammingDE analysis was performed between SShFF and ThFFs,between SSiNs and TiNs,and between the two groups of common DE RNAs,it was found that soft substrates promoted neural development,manifested in the development of neural bulge,neuronal cell bodies,axons,synapses,glutamate metabolism,axon guidance,etc.,which further illustrated that the soft substrates promoted the generation of glutamatergic neurons,at the same time,some mechanical cues that soft substrates promoted neuronal reprogramming were found,such as focal adhesions,stress fibers,microtubules binding,actin filaments binding,integrin binding,Hippo signaling pathway,regulation of actin cytoskeleton and mesenchymal epithelial transition,etc.Analysis of significantly enriched Hippo and cytoskeletal signaling pathways alone also confirmed that multiple DE transcripts were enriched in both pathways;The transmission electron microscope observation of each group showed that the microfilaments decreased and changed from gathering around the nucleus to dispersed in the cytoplasm.The nuclear localization statistics of Yes-associated protein(YAP)showed that the soft substrates promoted the nuclear export of YAP during neuronal reprogramming.After ceRNA network analysis,the mRNA was intersected with genes in the cytoskeleton and Hippo signaling pathway,and ITGB4 and its upstream miR-615-3p were found,it was confirmed that the expression trends of the two were opposite on soft substrates by qRT-PCR.These results suggested that soft substrates might promoted neuronal reprogramming by affecting the cytoskeleton and Hippo signaling pathway,while miR-615-3p and ITGB4 may serve as neuronal reprogramming targets for continued research.3.Soft substrates promote neuronal reprogramming by inhibiting miR-615-3p targeting ITGB4miR-615-3p and ITGB4 have sequence complementary binding sites.The double luciferase report suggested that miR-615-3p and ITGB4 might interact structurally,and after overexpression or inhibition of miR-615-3p,the expression of ITGB4 at RNA and protein levels could be reduced or increased respectively.Therefore,we explored whether miR-615-3p inhibition could improve the expression of neural markers and whether miR-615-3p overexpression could reverse the neuronal reprogramming effect of soft substrates.The results showed that miR-615-3p inhibition could increase the expression of TUJ1 and ASCL1 in ThFFs and the expression of TUJ1 and TAU in TiNs,whereas miR-615-3p overexpression could decrease the expression of TAU and ASCL1 in SSiNs,suggested that the soft substrates could promote neuronal reprogramming by inhibiting miR-6153p.After successful construction of hFFs stably transfected cell lines overexpressing and knocking down ITGB4,they were subjected to neuronal reprogramming.It was found that ITGB4 overexpression increased the expression of TUJ1 and ASCL1 in ThFFs and the expression of TUJ1 and TAU in TiNs,whereas its inhibition reduced the expression of TUJ1,TAU,and ASCL1 in SSiNs.These results suggested that soft substrate could promote neuronal reprogramming through overexpression of ITGB4.More importantly,miR-615-3p was further overexpressed on the basis of overexpression of ITGB4,and miR-615-3p was further inhibited on the basis of knockdown of ITGB4.It was found that miR-615-3p could restore the influence of ITGB4 on the expression of neural markers in neural reprogramming,suggesting that there was a targeting relationship between miR-615-3p and ITGB4,and that the soft substrates were likely to promote neuronal reprogramming by inhibiting of miR-615-3p targeting ITGB4.In summary,we demonstrated for the first time that the 450-850Pa soft substrates significantly improved the generation efficiency and quality of iNs without the need for co-culture with glial cells during reprogramming,promoted the MET process and generated more glutamatergic neurons with electrophysiological function in less time.Analysis of sequencing data suggested that the soft substrates might promote reprogramming of glutamatergic neurons by affecting mechanical signals such as actin cytoskeleton,Hippo signaling pathway,and integrins,while the combined analysis of competing endogenous RNA networks and mechanical signals provided new targets for neuronal reprogramming,namely ITGB4 and miR-615-3p.On this basis,we had demonstrated for the first time that miR-615-3p was likely to have a targeting relationship with ITGB4,and the soft substrates might promote neuronal reprogramming by inhibiting miR-615-3p targeting ITGB4,thereby affecting the cytoskeleton.The improvement of the efficiency and quality of neuronal reprogramming will provide a sufficient number of functional neurons for the follow-up basic and clinical research of regenerative medicine,and provide new insights for further revealing the mechanism by which the soft substrates improve the reprogramming efficiency.The design of medical biomaterials provides new ideas for nerve regeneration.