Long non-coding RNA H19 regulates neurogenesis of induced neural stem cells in a mouse model of closed head injury

Mou Gao ,Qin Dong ,Zhijun Yang ,Dan ZouYajuan HanZhanfeng Chen,Ruxiang Xu

Abstract Stem cell-based therapies have been proposed as a potential treatment for neural regeneration following closed head injury.We previously reported that induced neural stem cells exert beneficial effects on neural regeneration via cell replacement.However,the neural regeneration efficiency of induced neural stem cells remains limited.In this study,we explored differentially expressed genes and long non-coding RNAs to clarify the mechanism underlying the neurogenesis of induced neural stem cells.We found that H19 was the most downregulated neurogenesis-associated lncRNA in induced neural stem cells compared with induced pluripotent stem cells.Additionally,we demonstrated that H19 levels in induced neural stem cells were markedly lower than those in induced pluripotent stem cells and were substantially higher than those in induced neural stem cell-derived neurons.We predicted the target genes of H19 and discovered that H19 directly interacts with miR-325-3p,which directly interacts with Ctbp2 in induced pluripotent stem cells and induced neural stem cells.Silencing H19 or Ctbp2 impaired induced neural stem cell proliferation,and miR-325-3p suppression restored the effect of H19 inhibition but not the effect of Ctbp2 inhibition.Furthermore,H19 silencing substantially promoted the neural differentiation of induced neural stem cells and did not induce apoptosis of induced neural stem cells.Notably,silencing H19 in induced neural stem cell grafts markedly accelerated the neurological recovery of closed head injury mice.Our results reveal that H19 regulates the neurogenesis of induced neural stem cells.H19 inhibition may promote the neural differentiation of induced neural stem cells,which is closely associated with neurological recovery following closed head injury.

Key Words: closed head injury;Ctbp2;induced neural stem cell;lncRNA H19;miR-325-3p;neurogenesis

Introduction

Neural insult is a leading cause of neurological disorders following closed head injury (CHI) (Sas et al.,2020;Brett et al.,2022;Maas et al.,2022).With advances in regenerative medicine,stem cell-based therapies have been proposed as a potential treatment for neural regeneration (Miao et al.,2020;Liu et al.,2021;Xiong et al.,2021;He et al.,2023).We previously reported that intracerebral-transplanted induced neural stem cells (iNSCs) can differentiate into TUBB3-,Map2-,and neuronal nuclei (NeuN)-positive neural cells to exert beneficial effects on neural regeneration via cell replacement (Yao et al.,2015;Gao et al.,2016).However,the neural regeneration efficiency of iNSCs is limited,especially in CHI-damaged brains (Gao et al.,2018,2021;Sheng et al.,2018).Therefore,clarifying the mechanism underlying the neurogenesis of iNSCs is critical to identify strategies to promote the neural differentiation of iNSCs in CHI-damaged brains (Kalamakis et al.,2019;Denoth-Lippuner et al.,2021;Wei et al.,2022b).

Recent studies have reported that reprogramming technology may influence gene expression in induced pluripotent stem cells (iPSCs) and iNSCs (Stadhouders et al.,2018;Moghadam et al.,2020;Rodriguez-Polo and Behr,2022).As we know,iPSCs and iNSCs are both generated from somatic cells through reprogramming technology,which can be said to have the same origin.In addition,iPSCs and iNSCs both have the potential to differentiate into neural cells,which are expected to achieve neural regeneration after CHI.Therefore,comparison of difference between iPSCs and iNSCs can reveal more about the mechanisms of neurogenesis of iNSCs.To examine the effect of reprogramming technology on gene expression,we previously performed microarray analysis and found that iPSCs clustered closely with embryonic stem cells (ESCs),whereas iNSCs clustered closely with neural stem cells (NSCs) (Gao et al.,2016).Moreover,most of the upregulated genes in iPSCs were downregulated in iNSCs andvice versa(Gao et al.,2016).For instance,the levels of stemness-and proliferation-associated mRNAs,includingOct4andNanog,were significantly higher in iPSCs than in iNSCs;whereas the levels of neurogenesis-associated mRNAs,includingBrn2,Ncan(Mus musculus neurocan),Nkx2-2(NK2 homeobox 2),Tox3(TOX high mobility group box family member 3),Tubb3(class III β-tubulin),Map2(microtubule-associated protein 2) andSyp(synaptophysin),were markedly higher in iNSCs compared with iPSCs.We also discovered that several differentially expressed long non-coding RNAs (lncRNAs) between iPSCs and iNSCs were related to these mRNAs.Accumulating evidence has revealed that lncRNAs function as crucial regulators of neurogenesis by serving as competing endogenous RNAs (sponges) of miRNAs,which are also considered to be indispensable for neural differentiation (Tsagakis et al.,2020;Whipple et al.,2020;Mirzadeh et al.,2021;Wang et al.,2021b).For example,the lncRNAs1604,Meg3,andRik-201may promote neurogenesis by targetingmiR-200c,miR-128-3pandmiR-96,respectively (Bao et al.,2018;Weng et al.,2018;Zhang et al.,2019).The lncRNAH19was shown to contribute to stroke-augmented neurogenesis by affecting chromatin remodeling proteins and miRNA expression (Fan et al.,2020).However,one study reported thatH19may prevent neurogenesis following ischemic stroke via inhibition of the p53/Notch1 pathway,suggesting that the effects ofH19on neurogenesis remain controversial (Wang et al.,2019b).Additionally,H19contributed to hypoxia-,ischemia-and epilepsyinduced neural apoptosis by acting as a competing endogenous RNA to spongemiR-19aorlet-7b(Xiao et al.,2019;Han et al.,2020).Other reports showed thatH19maintains the stemness and proliferation of NSCs and glioblastoma cells,indicating thatH19may play multiple roles in the central nervous system (Momtazmanesh et al.,2021;Nie et al.,2021;Sievers et al.,2021).

In this study,we explored the mechanisms that regulate neurogenesis in iNSCs.Our findings revealedH19as a differentially expressed lncRNA and identified theH19/miR 325 3p/Ctbp2axis in iNSCs.We investigated the effects ofH19on the neurogenesis of iNSCs and determined its function in enhancing the neural regeneration efficiency of iNSCs to restore neural function following CHI.To the best of our knowledge,this is the first study to reveal the role of theH19in the proliferation and neural differentiation of iNSCs.

Methods

Cell culture,differentiation and magnetic activated cell sorter cytokine secretion assays

Cell culture (including mouse embryonic fibroblasts,iPSCs,and iNSCs),cell differentiation and magnetic activated cell sorter cytokine secretion assays were performed as previously reported (Yao et al.,2015;Gao et al.,2016,2018) and as shown inAdditional Figure 1.Detailed methods are provided inAdditional file 1.

Figure 1｜ Bioinformatics analysis of differential mRNA and lncRNA expression between iPSCs and iNSCs.

RNA preparation,microarray analysis and quantitative reverse transcription-polymerase chain reaction

RNA preparation,microarray analysis and quantitative reverse transcriptionpolymerase chain reaction (qRT-PCR) assays were conducted as previously reported (Gao et al.,2016;Sinitcyn et al.,2021).Briefly,RNA extracted from cultured iPSCs,iNSCs and iNSC-derived neurons was reversely transcribed into cDNA.The Agilent gene expression arrays (including mRNA and lncRNA) were performed by the CapitalBio Company (Beijing,China).Differentially expressed genes between iPSCs and iNSCs were selected after fold change (FC) and false discovery rate (FDR) analysis.Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were conducted to study the functions of differentially expressed genes.The gene coexpression network (including the lncRNA-mRNA network) was built by Cytoscape software v3.9.1.(Cytoscape Consortium,San Diego,CA,USA).For verification,we performed qRT-PCR using the SYBR-Green Master Mix (TaKaRa Biotech,Dalian,China) and a ViiA7 Real-Time PCR System (Applied Biosystems,Foster City,CA,USA).Detailed methods are provided inAdditional file 2.The sequences of the PCR primer pairs used in this study are listed inAdditional Table 1and were previously reported (Yao et al.,2015;Gao et al.,2016;Viereck et al.,2020;Sekiya et al.,2021;Wang et al.,2021a).

Cell transfection

H19-specific short hairpin RNA (shH19,Sangon Biotech,Shanghai,China),scramble control shRNA (scr,Sangon Biotech),miR-325-3pmimic (Gentaur Ltd,London,UK),miR-NC(Gentaur Ltd),miR-325-3pinhibitor (Gentaur Ltd),NC inhibitor (Gentaur Ltd),CtBP2-specific siRNA (Santa Cruz Biotechnology,Santa Cruz,CA,USA) and control siRNA (Santa Cruz Biotechnology) were used to transfect iPSCs and iNSCs as previously described (Gao et al.,2017).After transfection,gene expression was assessed using qRT-PCR.

Dual luciferase reporter assay

TargetScan v7.2 (https://www.targetscan.org/mmu_72/),starBase v2.0 (https://starbase.sysu.edu.cn/starbase2/) and miRDB (https://mirdb.org/) were used to predict target genes and binding sites (Chen et al.,2019;Wei et al.,2022a).PsiCHECK-2 luciferase vectors (Promega,Madison,WI,USA) containingH19orCtbp23′ untranslated region (UTR) with the wild-type (H19or Ctbp2 WT) or mutant (H19or Ctbp2 MUT)miR-325-3pbinding site were cotransfected withmiR-325-3pmimic ormiR-NCinto cells with LipofectamineTM2000 (Invitrogen,Carlsbad,CA,USA) following the manufacturer’s instructions.Luciferase activity was determined using the Dual-Luciferase Reporter Assay System (Promega) at 48 hours post-transfection.

RIP assay

RIP assay was conducted using the Magna RIPTMRNA-Binding Protein Immunoprecipitation Kit (Millipore,Billerica,MA,USA) following the manufacturer’s instructions.iPSCs and iNSCs were lysed and incubated with RIP buffer containing magnetic beads conjugated with anti-Argonaute2 (anti-Ago2,Abcam,Cambridge,MA,USA) or anti-IgG (Abcam) antibody.RNA isolated from the immunoprecipitated pellets was detected by RT-qPCR.

Biotin-coupled miRNA pull down assay

iPSCs and iNSCs were transfected with biotin-labeledmiR-325-3p(Bio-miR-325-3pWT,GenePharma,Shanghai,China) or the mutated sequence (BiomiR-325-3pMUT,GenePharma).At 48 hours post-transfection,cells were lysed with lysis buffer,and then incubated with streptavidin magnetic beads (Invitrogen) to pull-down the biotin-coupled RNA complex.RNA isolated from magnetic beads was quantified by qRT-PCR.

FISH assay

FISH assay was performed using the RiboTMFluorescentIn SituHybridization Kit (RiboBio,Guangzhou,China) with fluorescence-labeled probes (Sangon Biotech) toH19(labeled with Cy3 red fluorescence),miR-325-3p(labeled with FITC green fluorescence) andCtbp2(labeled with Cy3 red fluorescence).iPSCs and iNSCs were grown on round coverslips coated with poly-l-lysine (Sigma-Aldrich,St.Louis,MO,USA) and hybridized with FISH probes following the manufacturer’s instructions.After hybridization,the nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) Fluoromount-G (SouthernBiotech,Birmingham,AL,USA).Stained cells were examined via fluorescence microscopy (DM3000,Leica,Wetzlar,Germany) and confocal laser scanning microscopy (TCS SP5 II,Leica).

Fractionation of nuclear and cytoplasmic RNA

Nuclear and cytoplasmic RNA were isolated using the PARISTMKit (Invitrogen) following the manufacturer’s instructions.RNA isolated from the nuclear and cytoplasmic fractions of iPSCs and iNSCs was assessed using qRT-PCR.For normalization,U6 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA were used as endogenous controls for the nucleus and cytoplasm,respectively.

Cell Counting Kit-8 assay

Cell proliferation ability was evaluated by Cell Counting Kit-8 (CCK-8) assay (Beyotime,Shanghai,China) following the manufacturer’s instructions.Transfected iNSCs were cultured in 96-well plates and assayed at 0,24,48 and 72 hours.A microplate reader (Thermo Fisher Scientific,Cleveland,OH,USA) was used to measure the optical density at 450 nm (OD450),which reflected the cell proliferation ability.

The 5-ethynyl-20-deoxyuridine incorporation assay

The 5-ethynyl-20-deoxyuridine (EdU) incorporation assay (RiboBio) was used to determine the proliferation ability of transfected cells following the manufacturer’s instructions.Transfected iNSCs grown on poly-l-lysinecoated round coverslips were incubated with EdU.The nuclei were stained with DAPI Fluoromount-G (SouthernBiotech),and staining was examined by fluorescence microscopy (DM3000,Leica).

Western blot analysis

Western blot analysis was conducted as previously described (Gao et al.,2017).In brief,protein was extracted from transfected iNSCs.Protein concentrations were determined using the BCA assay (Thermo Fisher Scientific).Protein samples were separated by SDS-PAGE and transferred to PVDF membranes (Millipore).The blots were incubated with primary antibodies at 4°C overnight.After several washes,the blots were incubated for 1 hour at room temperature (RT) with HRP-conjugated secondary antibodies.Immunoblots were visualized using the SuperSignal ECL (Pierce,Rockford,IL,USA).The intensities of bands were determined using the Image Lab Version 4.0 software (Bio-Rad Laboratories,Hercules,CA,USA).The results were expressed relative to the control and normalized to GAPDH.Detailed methods are provided inAdditional file 3.The antibodies used in this study are listed inAdditional Table 2.

Morphological analysis

Morphological analysis was performed as previously reported (Gao et al.,2017).Briefly,slides of transfected iNSCs and brain tissues were incubated overnight at 4°C with primary antibodies.After washes in PBS,they were incubated for 1-2 hours at RT with secondary antibodies.Nuclei were stained with DAPI Fluoromount-G (SouthernBiotech) and staining was detected via fluorescent microscopy (DM3000,Leica) and confocal laser scanning microscopy (TCS SP5 II,Leica).The number of positive cells was manually counted at 20× magnification and adjusted using image analysis software (Image-Pro plus 5.0,Media Cybernetics,Silver Spring,MD,USA).The relative levels of positive cells were calculated as follows: (number of positive cells/total number of cells)×100%.Detailed methods are provided inAdditional file 4.The antibodies used in this study are listed inAdditional Table 2.

Terminal deoxynucleotidyl transferase dUTP nick end labeling staining

Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining in transfected iNSCs was performed as previously described (Gao et al.,2018).In brief,TUNEL staining was performed using the In Situ Cell Death Detection Kit with TMR red (Roche,Mannheim,Germany).The nuclei were counterstained with DAPI Fluoromount-G (SouthernBiotech).The relative levels of TUNEL-positive cells were calculated as follows: (the number of TUNEL-positive cells/the total number of DAPI-positive cells) ×100%.Detailed methods are provided inAdditional file 5.

Flow cytometry

Flow cytometry was conducted as previously reported (Gao et al.,2018).Briefly,transfected iNSCs were incubated with primary antibodies for 30 minutes at 4°C.After washing with PBS,the cells were incubated for 30 minutes at RT with secondary antibodies.After several washes,the cells were analyzed on an Accuri C6 Flow Cytometer System (BD Biosciences,San Jose,CA,USA).Detailed methods are provided inAdditional file 6.The antibodies used in this study are listed inAdditional Table 2.

CHI models and cell transplantation

CHI models were established as previously reported (Gao et al.,2017).In brief,healthy adult (12-14 weeks old) male C57BL/6 mice weighing 2432 g (Charles River Laboratories,Beijing,China,license No.SCXK (Jing) 2016-0006) were anaesthetized through intranasal administration of isoflurane (Fisher Scientific);the mice received fentanyl as the analgesic agent.After shaving and cleaning the skin,the parietal bone was exposed by a midline scalp incision.A free-falling rod with a blunt tip of 3.0 mm diameter was dropped onto the skull (2.0 mm anterior to the lambda suture and 2.0 mm lateral to the middle line) at a falling height of 3.0 cm.The scalp wound was sutured and treated with povidone-iodine solution.Sham-operated mice underwent the same procedures (anesthesia,analgesia and scalp incision),but not head trauma.Two blinded,trained investigators evaluated the animals at 1 hour post-CHI using the neurological severity score (NSS) (Additional Table 3).Mice with an NSS of 6-8 were used in experiments.

CHI-induced neurological impairment and fine-motor coordination deficits were evaluated using the NSS and a beam-walk task as previously described (Gao et al.,2017).At 12 hours after CHI,cell transplantation was performed,as previously described (Gao et al.,2017).Detailed methods are provided inAdditional file 7.

All experimental procedures were in compliance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health and approved by the Committee on the Ethics of Animal Experiments of the Chinese PLA General Hospital (approval No.2018-066,approval date: January 21,2019).

Statistical analysis

Statistical analysis was performed with SPSS17.0 statistical software.Data are presented as mean ± standard deviation.Student’st-test,one-way analysis of variance (ANOVA) and two-way ANOVA with Tukey’spost hoctest were used to determine statistical significance.P<0.05 indicated statistical significance.

Results

Bioinformatic analysis of differential mRNA and lncRNA expression between iPSCs and iNSCs

To examine the differentially expressed genes between iPSCs and iNSCs in neurogenesis,we performed microarray analysis.The results identified 51,963 genes in iPSCs and iNSCs,including 6836 upregulated (P<0.05,log2(FC) >1) and 7531 downregulated (P<0.05,log2(FC) <-1) genes in iNSCs compared with iPSCs.A total of 32,649 mRNAs were identified,including 4449 upregulated (P<0.05,log2(FC) >1) and 5020 downregulated (P<0.05,log2(FC) <-1) mRNAs in iNSCs compared with iPSCs (Figure 1AandB).GO enrichment analysis revealed that differentially expressed mRNAs were significantly enriched in protein binding (molecular function (MF)),regulation of transcription,DNA-dependent (biological process (BP)) and integral to membrane (cellular component (CC)) (Additional Figure 2A-C).KEGG pathway analysis showed that differentially expressed mRNAs were mainly enriched in the MAPK signaling pathway and cytokine-cytokine receptor interaction (Additional Figure 2D).

Figure 2｜ Expression of H19 and its target miRNAs in iPSCs,iNSCs,and iNSC-derived neurons.

A total of 17,424 lncRNAs were identified,including 2217 upregulated (P<0.05,log2(FC) >1) and 2352 downregulated (P<0.05,log2(FC) <-1) lncRNAs in iNSCs compared with iPSCs (Figure 1CandD).We analyzed the foldchange levels of neurogenesis-associated lncRNAs which were identified by literature search and KEGG pathway analysis and found thatH19was the most downregulated lncRNA in iNSCs compared with iPSCs (Figure 1E).We selected 12 neurogenesis-associated lncRNAs (includingH19) with significant fold-change levels to construct a lncRNA-mRNA coexpression network (Figure 1F).We also conducted GO and KEGG pathway enrichment analyses of theH19coexpressed mRNAs (Figure 1G-J).GO enrichment analysis indicated thatH19coexpressed mRNAs were markedly enriched in protein binding (MF),multicellular organism development (BP) and membrane (CC) (Figure 1G-I).KEGG pathway analysis indicated thatH19coexpressed mRNAs were mainly enriched in pathways in cancer (Figure 1J).To visualize the relationship betweenH19and coexpressed mRNAs,we constructed anH19-mRNA coexpression network,which revealed thatH19was connected with 49 mRNAs,including 20 upregulated and 29 downregulated mRNAs in iNSCs compared with iPSCs (Figure 1K).H19levels were positively correlated with the levels of stemness-and proliferation-associated mRNAs in iNSCs but were negatively correlated with the levels of neurogenesis-associated mRNAs.

Together,these data indicated thatH19was the most downregulated neurogenesis-associated lncRNA in iNSCs compared with iPSCs.

Expression of H19 and its target miRNAs in iPSCs,iNSCs and iNSC-derived neurons

To verify the results of the microarray analysis,we examined the expression ofH19in iPSCs and iNSCs by qRT-PCR and confirmed thatH19levels in iNSCs were substantially lower than those in iPSCs (P=0.0002;Figure 2A).Furthermore,H19levels in iNSC-derived neurons were significantly lower than those in iNSCs (P=0.0001;Figure 2B).We next predicted the target miRNAs ofH19and examined their expressions in iNSCs and iPSCs.miR-21a-5plevels in iNSCs were significantly lower than those in iPSCs (P=0.0072),whereas miR-325-3p levels in iNSCs were significantly higher than those in iPSCs (P=0.0056) (Figure 2C).miR-21a-5plevels showed no significant differences between iNSCs and iNSC-derived neurons,whereasmiR-325-3plevels in iNSCs were markedly lower than those in iNSC-derived neurons (P=0.0106;Figure 2D).Therefore,miR-325-3pwas selected as a candidate target miRNA ofH19for further investigation.

To clarify the relationship betweenH19andmiR-325-3p,we used shH19or scr to transfect iPSCs and iNSCs (Figure 2E-GandAdditional Figure 3A-C).There were no significant differences inH19ormiR-325-3plevels between the untransfected and scr-transfected groups in iPSCs,iNSCs and iNSCderived neurons.H19levels were substantially lower in the shH19-transfected groups in iPSCs (shH19-transfectedvs.untransfected groups:P<0.0001;shH19-transfectedvs.scr-transfected groups:P<0.0001),iNSCs (shH19-transfected vs.untransfected groups:P<0.0001;shH19-transfectedvs.scrtransfected groups:P=0.0003) and iNSC-derived neurons (shH19-transfectedvs.untransfected groups:P=0.0002;shH19-transfectedvs.scr-transfected groups:P=0.0001) compared with the other two groups,indicating effective knockdown.Notably,miR-325-3p levels were significantly higher in the shH19-transfected groups in iPSCs (shH19-transfectedvs.untransfected groups:P=0.0003;shH19-transfectedvs.scr-transfected groups:P=0.0001),iNSCs (shH19-transfectedvs.untransfected groups:P=0.0006;shH19-transfectedvs.scr-transfected groups:P=0.0032) and iNSC-derived neurons (shH19-transfectedvs.untransfected groups:P=0.0031;shH19-transfectedvs.scr-transfected groups:P=0.0007) compared with the other two groups,suggesting thatH19inhibition increased the levels ofmiR-325-3p.

Figure 3｜ H19 directly interacts with miR-325-3p in iPSCs and iNSCs.

We then usedmiR-325-3pmimic or miR-NC to transfect iNSCs and iPSCs and performed qRT-PCR (Figure 2HandAdditional Figure 4A).There were no significant differences in themiR-325-3plevels between the untransfected and miR-NC transfected groups in iNSCs and iPSCs.miR-325-3plevels in iNSCs (miR-325-3pmimic-transfectedvs.untransfected groups:P<0.0001;miR-325-3pmimic-transfectedvs.miR-NC transfected groups:P<0.0001) and iPSCs (miR-325-3pmimic-transfectedvs.untransfected groups:P<0.0001;miR-325-3pmimic-transfectedvs.miR-NC transfected groups:P<0.0001) were substantially higher in themiR-325-3pmimic-transfected group than in the other two groups.To confirm thatH19is a target ofmiR-325-3pin iNSCs and iPSCs,we predicted the possible binding sites ofH19andmiR-325-3pand constructed reporter vectors for dual luciferase reporter assays (Figure 2IandJandAdditional Figure 4AandB).Luciferase activity was markedly reduced after cotransfection of theH19-WT reporter vector andmiR-325-3pmimic in iNSCs (P=0.0015) and iPSCs (P=0.0038) but was unchanged when theH19-MUT reporter was used.These findings implied that miR-325-3p is a target miRNA ofH19.

Figure 4｜ Expression of H19 coexpressed mRNAs in iPSCs,iNSCs,and iNSC-derived neurons.

H19 directly interacts with miR-325-3p in iPSCs and iNSCs

To examine the potential interaction ofmiR-325-3pandH19in cells,we performed FISH assay to determine the subcellular localization ofH19andmiR-325-3pin iPSCs and iNSCs.The results demonstrated colocalization ofH19andmiR-325-3pin iPSCs and iNSCs (Figure 3A).The relative fluorescence intensity (RFI) values ofH19in iPSCs were substantially higher than those in iNSCs (P<0.0001;Figure 3B).In contrast,the RFI values ofmiR-325-3pin iPSCs were markedly lower than those in iNSCs (P<0.0001;Figure 3C).The RFI values ofH19andmiR-325-3pin the cytoplasmic fraction of iPSCs (H19:P<0.0001;miR-325-3p:P<0.0001) and iNSCs (H19:P<0.0001;miR-325-3p:P<0.0001) were significantly higher than those in the nuclear fraction (Figure 3DandE).To confirm the results of the FISH assay,we performed nuclear and cytoplasmic fractionation assays and observed thatH19levels were substantially higher in the cytoplasmic fraction of iPSCs (P<0.0001) and iNSCs (P<0.0001) than the nuclear fractions,suggesting thatH19was mainly localized in the cytoplasmic fraction;similar results were observed formiR-325-3p(Additional Figure 5AandB).

Figure 5｜Ctbp2 directly interacts with miR-325-3p in iPSCs and iNSCs.

To further elucidate the interaction betweenH19andmiR-325-3p,we used RIP assays and discovered thatH19andmiR-325-3pwere specifically enriched in Ago2 pellets of iPSC and iNSC extracts relative to the IgG control group (Figure 3FandG).Biotin-coupled miRNA pull down assay revealed that the incubation of Bio-miR-325-3pWT led to a dramatic increase inH19enrichment in iPSCs and iNSCs compared with the Bio-miR-325-3pMUT group (Figure 3H-J).

Together,these data indicated thatH19directly interacts withmiR-325-3pin the cytoplasm of iPSCs and iNSCs.

Expression of H19 coexpressed mRNAs in iPSCs,iNSCs and iNSC-derived neurons

To explore the potential mechanism ofH19in iNSCs,we performed KEGG pathway analysis of theH19coexpressed mRNAs and found that theH19coexpressed mRNAs were involved in many pathways,including pathways in cancer,GABAergic synapse and cell adhesion molecules (Figure 4A).We summarized the putative target mRNAs ofH19andmiR-325-3p(a total of 32,649 mRNAs were identified for analysis,and the putative target mRNAs forH19andmiR-325-3pwere predicted and overlapping mRNAs (Gabbr1,Slit2,Trpc3,Ppp3ca,Pdgfra,Ctbp2,Gli1andPax8) were selected) and discovered thatCtbp2levels (Ctbp2was just found in the KEGG Pathways in cancer term and was not pursued among any other candidates) in iNSCs were markedly lower than those in iPSCs (P=0.0002;Figure 4B).Additionally,Ctbp2levels were significantly lower in iNSC-derived neurons than in iNSCs (P=0.0006;Figure 4C).To study the relationship betweenH19andCtbp2,we downregulatedH19in cells as described above (Figure 4D-F).There were no significant differences inCtbp2levels between the untransfected and scr-transfected groups in iPSCs,iNSCs or iNSC-derived neurons.However,Ctbp2levels in iPSCs (shH19-transfectedvs.untransfected groups:P=0.0005;shH19-transfectedvs.scrtransfected groups:P=0.0001),iNSCs (shH19-transfected vs.untransfected groups:P=0.0001;shH19-transfectedvs.scr-transfected groups:P<0.0001) and iNSC-derived neurons (shH19-transfectedvs.untransfected groups:P<0.0001;shH19-transfectedvs.scr-transfected groups:P=0.0013) were substantially lower in the shH19-transfected group than in the other two groups,suggesting thatH19inhibition reduced the levels ofCtbp2.

We next performed qRT-PCR to detect the levels ofCtbp2in iPSCs and iNSCs transfected withmiR-325-3pmimic or miR-NC (Figure 4GandH).There were no significant differences inCtbp2levels in iPSCs or iNSCs between the untransfected and miR-NC transfected groups.However,Ctbp2levels in iPSCs (miR-325-3pmimic-transfectedvs.untransfected groups:P=0.0049;miR-325-3pmimic-transfectedvs.miR-NCtransfected groups:P=0.0191) and iNSCs (miR-325-3pmimic-transfected vs.untransfected groups:P=0.0012;miR-325-3pmimic-transfectedvs.miR-NCtransfected groups:P=0.0005) were markedly lower in themiR-325-3pmimic-transfected group than in the other two groups,indicating thatmiR-325-3poverexpression decreased the levels ofCtbp2.

To confirm thatCtbp2is a target ofmiR-325-3pin iNSCs and iPSCs,we predicted the possible binding sites in theCtbp2mRNA 3′ untranslated region (UTR) andmiR-325-3pto construct reporter vectors and performed dual luciferase reporter assays (Figure 4IandJandAdditional Figure 6).The luciferase activity was substantially reduced after cotransfection of the Ctbp2-WT reporter andmiR-325-3pmimic in iNSCs (P=0.0024) and iPSCs (P=0.0048),and no significant changes were observed upon transfection with theCtbp2-MUT construct.These data suggested that Ctbp2 is a target mRNA ofH19andmiR-325-3p.

Figure 6｜H19 functions as a sponge of miR-325-3p,which targeted Ctbp2,influencing proliferation of induced neural stem cells (iNSCs).

Ctbp2 directly interacts with miR-325-3p in iPSCs and iNSCs

To examine the interaction ofmiR-325-3pandCtbp2,we performed FISH assays to detect the subcellular localization ofCtbp2andmiR-325-3pin iPSCs and iNSCs.We found colocalization ofCtbp2andmiR-325-3pin iPSCs and iNSCs (Figure 5A).The RFI values ofCtbp2in iPSCs were markedly higher than those in iNSCs (P<0.0001;Figure 5B),whereas the RFI values ofmiR-325-3pin iPSCs were substantially lower than those in iNSCs (P<0.0001;Figure 5C).Additionally,RFI values ofCtbp2andmiR-325-3pin the cytoplasmic fraction of iPSCs (Ctbp2:P<0.0001;miR-325-3p:P<0.0001) and iNSCs (Ctbp2:P<0.0001;miR-325-3p:P<0.0001) were significantly higher than those in the nuclear fraction (Figure 5DandE).To confirm the results of the FISH assay,we conducted nuclear and cytoplasmic fractionation assays and found thatCtbp2levels in the cytoplasmic fraction of iPSCs (P<0.0001) and iNSCs (P<0.0001) were markedly higher than those in the nuclear fraction,suggesting thatCtbp2,similar tomiR-325-3p,was mainly localized in the cytoplasmic fraction (Additional Figure 7AandB).

Figure 7｜H19 regulates the proliferation and neural differentiation of iNSCs.

To further clarify the interaction betweenCtbp2andmiR-325-3p,we performed RIP assays and found thatCtbp2andmiR-325-3pwere specifically enriched in Ago2 pellets of iPSC and iNSC extracts relative to the IgG control group (Figure 5FandG).Biotin-coupled miRNA pull down assay revealed that the incubation of Bio-miR-325-3pWT led to a dramatic increase inCtbp2enrichment in iPSCs and iNSCs compared with the Bio-miR-325-3pMUT group (Figure 5H-J).These findings indicated thatCtbp2directly interacts withmiR-325-3pin iPSCs and iNSCs.

H19 serves as a sponge of miR-325-3p,which targets Ctbp2,influencing proliferation of iNSCs

To investigate the mechanism underlying the regulation ofH19in the neurogenesis of iNSCs,we performed a rescue study using shH19,miR-325-3pinhibitor or CtBP2-specific siRNA to transfect iNSCs.We found thatH19levels showed no significant changes in cells with cotransfection withmiR-325-3pinhibitor and CtBP2-specific siRNA (Additional Figure 8AandB).In contrast,Ctbp2levels substantially decreased after transfection with shH19and CtBP2-specific siRNA,and themiR-325-3pinhibitor restored the effect of shH19but not CtBP2-specific siRNA (P<0.0001).Together this indicates thatH19serves as a sponge ofmiR-325-3p,which targetsCtbp2.

We conducted CCK8 assays and found thatH19inhibition reduced the proliferation of iNSCs (Figure 6A).Notably,miR-325-3pinhibitor reversed the effect ofH19inhibition (24 hours:P=0.0026;48 hours:P=0.0067;72 hours:P=0.0022),while CtBP2 inhibition with cotransfection with shH19and miR-325-3p inhibitor significantly reduced the proliferation of iNSCs (24 hours:P=0.0189;48 hours:P=0.0034;72 hours:P=0.0015),suggesting thatmiR-325-3pandCtbp2exerted significant influence on the effects ofH19on iNSC proliferation.Furthermore,EdU assays showed that silencingH19reduced the proliferation of iNSCs (P=0.0018) and this reduction was eliminated bymiR-325-3pinhibitor cotransfection (P=0.0086;Figure 6BandC).Moreover,silencing CtBP2 in cells with shH19andmiR-325-3pinhibitor substantially decreased the proliferation of iNSCs (P=0.0013).

We performed qRT-PCR to evaluate neurogenesis-associated mRNAs in iNSCs.H19inhibition significantly reduced the levels of stemness-and proliferationassociated mRNAsSox2(P=0.0022) andNes(P=0.0124),whereas it markedly increased the levels of neurogenesis-associated mRNAsBrn2(P=0.0100),Tubb3(P=0.0031) andSyp(P=0.0078;Figure 6D).Western blot analysis revealed that Sox2 (P=0.0026),Nestin (P=0.0017) and CtBP2 (P=0.0034) levels in iNSCs of the shH19group were substantially lower than those in the control group,while TUBB3 (P=0.0132) and SYP (P=0.0019) levels were significantly higher in the shH19group than in the control group (Figure 6EandF).Immunofluorescence staining showed thatH19inhibition markedly reduced the levels of Sox2+(P=0.0012) and CtBP2+(P=0.0061) iNSCs (Figure 6GandH).TUNEL assays revealed no significant differences in the number of TUNEL+cells between the shH19and control groups,suggesting thatH19inhibition did not lead to cell damage (Figure 6I).

These results implied that theH19can regulate the neurogenesis of iNSCs.

H19 regulates the proliferation and neural differentiation of iNSCs

We further performed double-labeling experiments.We found thatH19inhibition substantially reduced the levels of CtBP2 (P=0.0021) in iNSCs while markedly increasing the levels of TUBB3 (P=0.0001) and SYP (P=0.0002) in iNSCs (Figure 7A-C).Furthermore,flow cytometry analysis demonstrated thatH19inhibition significantly decreased the levels of CtBP2 (P=0.0047) and Nestin (P=0.0034) in iNSCs and substantially increased the levels of NeuN (P=0.0028),TUBB3 (P=0.0012) and SYP (P=0.0005) in iNSCs (Figure 7DandE).Therefore,these results revealed thatH19inhibition promoted the neural differentiation of iNSCs.

We next examined the influence ofH19inhibition on neural differentiation of iNSCsin vivo.We examined the neural differentiation of GFP-labeled iNSCs in the injured cortices of control (CHI mice receiving iNSCs transfected with scr) and shH19(CHI mice receiving iNSCs transfected with shH19) groups.There were no significant differences in the number of GFP+and NeuN+cells in the injured cortices of the two groups.However,the amount of GFP+/NeuN+iNSC-derived neurons was significantly higher in the shH19group than in the control group,suggesting thatH19inhibition promoted the neural differentiation of intracerebral-transplanted iNSCs in the injured cortices of CHI mice (P=0.0013;Figure 7FandG).These data suggested thatH19suppressed the neural differentiation of iNSCsin vivo.

To explore the effect of engrafted iNSCs transfected with shH19on the neurological recovery of CHI mice,we used the NSS and performed evaluations at 1,3 and 7 days after CHI (Additional Figure 9A).Two-way ANOVA indicated that time (P<0.0001),treatment (P<0.0001) and the interaction between time and treatment (P=0.0220) exerted significant effects on NSS.Simple main effects analysis showed that the NSS was lower in the control group than in the PBS (CHI mice receiving PBS) group and was lower in the shH19group than in the control group (controlvs.PBS group:P<0.0001;controlvs.shH19group:P<0.0001).Moreover,NSS was lower on day 3 post-CHI than on day 1 post-CHI and was higher on day 3 post-CHI than on day 7 post-CHI among the three groups (day 1vs.day 3:P<0.0001;day 3vs.day 7: P <0.0001).

We also performed a beam-walk task to assess CHI-induced fine-motor coordination deficits at 1,3 and 7 days after CHI (Additional Figure 9B).Twoway ANOVA revealed that time (P<0.0001),treatment (P<0.0001) and the interaction between time and treatment (P<0.0001) exerted obvious effects on fine-motor coordination.Simple main effects analysis implied that the numbers of foot faults were lower in the control group than in the PBS group and lower in the shH19group than in the control group (controlvs.PBS group:P<0.0001;controlvs.shH19group:P<0.0001).Furthermore,the numbers of foot faults were lower on day 3 post-CHI than on day 1 post-CHI and was higher on day 3 post-CHI than on day 7 post-CHI among the three groups (day 1vs.day 3:P<0.0001;day 3vs.day 7:P<0.0001).Therefore,thein vitroandin vivostudies demonstrated that theH19could regulate the neurogenesis of iNSCs,which was closely associated with neurological recovery after CHI.

Discussion

The transplantation of iNSCs,which have the capacity to generate neurons to restore neural function,holds promise as a treatment for neurological disorders following CHI (Yao et al.,2015;Gao et al.,2016,2017,2018).In this study,we explored the mechanism underlying the neurogenesis of iNSCs and found thatH19was the most downregulated neurogenesis-associated lncRNA in iNSCs compared with iPSCs.Recent studies have reported thatH19plays important and differential roles in neural regeneration after brain injury (Wang et al.,2019b;Fan et al.,2020).To study the function ofH19in the neurogenesis of iNSCs,we conducted bioinformatic analysis and found thatH19levels were positively correlated with the levels of stemness-and proliferation-associated mRNAs,includingOct4andNanog,and negatively correlated with the levels of neurogenesis-associated mRNAs,includingBrn2,Ncan,Nkx2-2,Tox3,Tubb3,Map2,andSyp.Furthermore,H19levels in iNSCs were markedly lower than those in iPSCs but were substantially higher than those in iNSC-derived neurons,suggesting that the function ofH19is closely related to neurogenesis.Furthermore,H19coexpressed mRNAs were mainly enriched in pathways in cancer,which is in agreement with previous research in the central nervous system showing thatH19exerts multiple effects on cell growth and apoptosis (Momtazmanesh et al.,2021;Nie et al.,2021;Sievers et al.,2021).Thus,we speculated thatH19may participate in the regulation of iNSC proliferation and differentiation.

To verify this hypothesis,we predicted the target genes ofH19and discovered a significantly negative relationship betweenmiR-325-3pandH19in iPSCs,iNSCs and iNSC-derived neurons,which is partially consistent with previous studies indicating thatmiR-325-3prepresses genes related to ESC proliferation and inhibits cell growth and metastasis in human glioma and thatmiR-325-3plevels in glioma tissues were significantly lower than those in adjacent normal brain tissues (Xiong and Su,2021;Carvelli et al.,2022).Mechanistic studies using dual luciferase reporter,RIP,biotin-coupled miRNA pull down and FISH assays demonstrated thatH19directly interacts withmiR-325-3pin iPSCs and iNSCs.Additionally,we summarized the putative target mRNAs ofH19andmiR-325-3pand found a substantially positive relationship betweenCtbp2andH19in iPSCs,iNSCs and iNSC-derived neurons,which was partially in line with a previous study suggesting thatCtbp2levels were downregulated during ESC differentiation (Kwak et al.,2018;Arthur et al.,2019;Karaca et al.,2020).CtBP2 is an evolutionarily conserved transcriptional corepressor to repress neurogenesis and is essential for embryonic development (Wang et al.,2019a;Moser et al.,2020).Mechanistic studies demonstrated thatCtbp2directly interacts withmiR-325-3pin iPSCs and iNSCs.Furthermore,H19inhibition markedly increased the levels ofmiR-325-3pbut reduced the levels ofCtbp2.Moreover,miR-325-3psuppression blocked the effect ofH19inhibition,but not the effect ofCtbp2inhibition,onCtbp2expression.Therefore,we concluded thatH19serves as a sponge ofmiR-325-3p,which targetsCtbp2.

We found that silencingH19orCtbp2impaired iNSC proliferation,andmiR-325-3psuppression restored the effect induced byH19inhibition but not the effect ofCtbp2inhibition.H19inhibition promoted the neural differentiation of iNSCs without causing cell damage.Given the effects of silencingH19on the proliferation and differentiation of iNSCsin vitro,we investigated the role ofH19inhibition in iNSC grafts on neurological recovery in mouse models of CHI.In accordance with thein vitroexperimental results,silencingH19enhanced the neural differentiation of intracerebral-transplanted iNSCs in the injured cortices of CHI mice.Notably,H19inhibition in iNSC grafts also markedly accelerated the neurological recovery of CHI mice.These results indicated thatH19silencing in iNSC grafts led to the acceleration of neurological recovery via the promotion of neural differentiation of grafted iNSCs.Thus,these data demonstrate thatH19may regulate the neurogenesis of iNSCs,which is closely associated with neurological recovery after CHI.

H19has emerged as an important regulator in the pathology of brain injury with functions in the promotion of NSC proliferation,regulation of axon growth,prevention of neurogenesis,induction of cell apoptosis and modulation of neuroinflammation (Hu et al.,2020;Ghafouri-Fard et al.,2022;Li et al.,2022).Our findings are partially consistent with prior studies,and the differences in the function ofH19may be from the multiple mechanisms involved in neural regeneration in CHI-damaged brains transplanted with iNSCs (Wang et al.,2019b;Fan et al.,2020).For example,one study reported thatH19knockdown reduced the levels of DCX which is used to measure the level of neurogenesis and inhibited NSC proliferation and differentiation (Gan et al.,2022).Therefore,the mechanism of neurogenesis regulated byH19may involve multiple pathways with different functions.

In this study,we revealed the interaction amongH19,miR-325-3pandCtbp2in regulating the proliferation of iNSCs (Figure 7H).We observed an increase in the number of astrocytes and oligodendrocytes derived from iNSCs transfected with shH19or CtBP2-specific siRNA,although this difference was not significant,suggesting thatH19plays complicated roles in regulating the differentiation of iNSCs.Further studies exploring the mechanism underlying the modulation of theH19/miR-325-3p/Ctbp2axis in iNSC differentiation should be conducted to develop an effective therapy for neurological disorders post-CHI.

This study had several limitations.Because of the lack of a more sensitive and specific assessment of CHI-induced neurological impairment,we were unable to accurately evaluate the long-term effects (more than 28 days post-CHI) of the engrafted iNSCs transfected with shH19on the neurological recovery of CHI mice.Furthermore,whether theH19/miR-325-3p/Ctbp2axis promotes the neural differentiation of iPSCs or influences the neurotrophic and immunomodulatory effects of iNSCs remains unclear (Schiebinger et al.,2019).Whether the neurological recovery of CHI mice is associated with the neurotrophic and immunomodulatory effects of engrafted iNSCs transfected with shH19on the mitigation of CHI-induced progressive loss of neurological function is also unknown.

The development of more accurate,sensitive and specific methods may help determine the long-term effects of the neurogenesis,neurotrophy and immunomodulation of engrafted iNSCs transfected with shH19on the neurological recovery of CHI mice.Further studies exploring the interaction between engrafted iNSCs transfected with shH19and microglia are expected to clarify the immunomodulatory effects of engraftment on the mitigation of CHI-induced progressive loss of neurological function.Moreover,considering the downregulatedH19and Ctbp2 during the neural differentiation of iNSCs,subsequent research is essential to explore the factors positively associated with the neural differentiation of iPSCs and iNSCs to further clarify and better understand the mechanism underlying the neurogenesis of iPSCs and iNSCs.

In conclusion,Our findings show thatH19may act as a sponge ofmiR-325-3p,which targetsCtbp2in iPSCs and iNSCs.Furthermore,H19regulates the neurogenesis of iNSCs.H19inhibition may promote the neural differentiation of iNSCs,which is closely associated with neurological recovery following CHI.Our results reveal a novel role ofH19in the neurogenesis of iNSCs,suggesting a potential new approach to promote the neural differentiation of iNSCs by regulating the levels ofH19.

Acknowledgments:We sincerely thank Dr.Hui Yao,Yan Zhang,Cuiying Wu and Ning Liu (Chinese PLA General Hospital) for helpful advice and technical support.

Author contributions:MG conducted cell culture and transplantation experiments and wrote the manuscript.QD performed statistical analysis.ZY performed Western blot analysis.DZ and YH performed flow cytometry.ZC and RX designed and revised the manuscript.All the authors read and approved the manuscript.

Conflicts of interest:All authors declare that they have no conflicts of interest.No conflicts of interest exist between Zhongsai Stem Cell Genetic Engineering Co.,Ltd.and publication of this paper.

Data availability statement:All relevant data are within the paper and its Additional files.

Open access statement:This is an open access journal,and articles are distributed under the terms of the Creative Commons AttributionNonCommercial-ShareAlike 4.0 License,which allows others to remix,tweak,and build upon the work non-commercially,as long as appropriate credit is given and the new creations are licensed under the identical terms.

Additional files:

Additional file 1:Cell culture,differentiation and magnetic activated cell sorter cytokine secretion assays.

Additional file 2:RNA preparation,microarray analysis and qRT-PCR assays.

Additional file 3:Western blot analysis.

Additional file 4:Morphological analysis.

Additional file 5:TUNEL staining.

Additional file 6:Flow cytometry.

Additional file 7:CHI models and cell transplantation.

Additional Table 1:Primer sequences used for qRT-PCR assay.

Additional Table 2:Antibodies were used in this study.

Additional Table 3:Neurological severity score (NSS).

Additional Figure 1:Study flowchart illustrating the experimental procedure.

Additional Figure 2:Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses.

Additional Figuer 3:The levels of H19 in induced pluripotent stem cells (iPSCs),induced neural stem cells (iNSCs),and iNSC-derived neurons.

Additional Figure 4:A dual luciferase reporter assay confirmed that H19 was the target of miR 325 3p in induced pluripotent stem cells (iPSCs).

Additional Figure 5:The subcellular localisation of H19.

Additional Figure 6:The luciferase activities of induced pluripotent stem cells (iPSCs) transfected with Ctbp2-WT or Ctbp2-MUT and miR-NC or miR-325-3p mimic,respectively.

Additional Figure 7:The subcellular localization of Ctbp2.

Additional Figure 8:The levels of H19 and Ctbp2 in induced neural stem cells (iNSCs).

Additional Figure 9:Closed head injury (CHI)-induced neurological impairment and fine-motor coordination deficits were evaluated using a neurological severity score (NSS) and a beam-walk task.

Additional Figure 10:Full-length western blots of Sox2,Nestin,Brn-2,TUBB3,SYP and CtBP2 in induced neural stem cells (iNSCs) between the control (iNSCs treated with scramble control (scr)),and shH19 (iNSCs treated with shH19) groups.