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Long non-coding RNA Malat1 modulates CXCR4 expression to regulate the interaction between induced neural stem cells and microglia following closed head injury

Stem Cell Research & Therapy volume 16, Article number: 31 (2025) Cite this article

Abstract

Background

Closed head injury (CHI) provokes a prominent neuroinflammation that may lead to long-term health consequences. Microglia plays pivotal and complex roles in neuroinflammation-mediated neuronal insult and repair following CHI. We previously reported that induced neural stem cells (iNSCs) can block the effects of CXCL12/CXCR4 signaling on NF-κB activation in activated microglia by CXCR4 overexpression. Here we aim to uncover the mechanism of CXCR4 upregulation in iNSCs.

Methods

We performed bioinformatic analysis to detect the differentially expressed genes in iNSCs after co-cultured with LPS-activated microglia. Subsequently, we predicted the target genes and performed gain- and loss-of-functional studies, dualluciferase reporter, RNA immunoprecipitation, biotin-coupled miRNA pulldown, fluorescence in situ hybridization and cell transplantation assays to further elucidate the mechanism underlying the immunoregulatory effects of iNSCs. Student’s t-test and one-way analysis of variance (ANOVA) with Tukey’s post hoc test were used to determine statistical significance.

Results

Our results indicated that Malat1 could act as a sponge of miR-139-5p to modulate the expression of CXCR4 that exerted significant influence on the immunoregulatory effects of iNSCs on the secretion of CXCL12, TNF-α and IGF-1 by activated microglia. Furthermore, Malat1 inhibition blocked the immunoregulatory effects of iNSC grafts on microglial activation as well as neuroinflammation in the injured cortices of CHI mice. Interestingly, NF-κB activation in iNSCs augmented the immunoregulatory effects of iNSCs on microglial activation by activating the axis of Malat1/miR-139-5p/Cxcr4. Notably, we found that TNF-α secreted by activated microglia could bind to TNFR1 at the surface of iNSCs to trigger NF-κB activation in iNSCs.

Conclusions

In short, our findings reveal a novel role of Malat1 in the immunomodulatory effects of iNSCs on microglial activation, suggesting that transplanted iNSCs may self-perceive the changes of the activated state of microglia and thus make prudential regulation of the neuroinflammation following CHI.

Introduction

Closed head injury (CHI) is a major cause of disability and mortality globally [24]. CHI provokes a prominent neuroinflammatory response, characterized by microglial activation and upregulated pro-inflammatory mediators including cytokines, chemokines and their receptors, which may lead to long-term health consequences [5, 33, 41]. Microglial activation occurred immediately following CHI can play important and complex roles in neuronal insult and repair [2, 20]. For instance, activated microglia can produce abundant CXCL12 to worsen neuroinflammation by enabling immune cell infiltration and TNF-α to incite secondary damage by inducing NF-κB activation via TNF-α/TNFR1 signaling after the initial injury [14, 28]. In contrast, IGF-1 secreted by activated microglia can exert beneficial effects on neuronal survival post-CHI [3, 42]. Therefore, modulation of secretion of these cytokines and chemokines by activated microglia may be useful for the treatment of CHI. Currently available anti-inflammatory agents (including corticosteroid, non-selective and selective COX inhibitors) may reduce secondary damage caused by neuroinflammation, but at the same time inhibit the role of neuroinflammatory response in promoting neuronal repair [17, 26, 31]. Hence, there is an urgent need for the development of novel therapies that may self-perceive the changes of the activated state of microglia and thus make prudential regulation of the neuroinflammation following CHI.

With advances in regenerative medicine, induced neural stem cells (iNSCs), reprogrammed from autologous somatic cells, have been proposed as a novel treatment for CHI [4, 12, 13]. We previously reported that engrafted iNSCs have the potential to regulate secretory products of resident activated microglia by preventing NF-κB activation in the injured cortices of CHI mice [11]. Further research found that CXCR4 overexpressed at the surface of iNSCs can neutralize CXCL12 secreted by activated microglia to block the effects of CXCL12/CXCR4 signaling on NF-κB activation in these microglia such that they fail to exacerbate neuroinflammation-mediated neuronal insult after CHI [11]. However, it remains unclear the mechanism by which CXCR4 is overexpressed at the surface of iNSCs.

Accumulating data have shown that long non-coding RNAs (lncRNAs) may act as key regulators of neuroinflammation by functioning as competing endogenous RNAs (ceRNAs) of microRNAs (miRNAs) to modulate target mRNA expression [18, 25, 37]. For example, lncRNA 4344 may serve as a ceRNA of miR-138-5p to modulate the expression of NLRP3 that plays pivotal roles in regulating microglial activation as well as the levels of neuroinflammation-related factors [10]. LncRNA SNHG14 may work as a sponge for miR-223-3p and miR-145-5p to increase the levels of NLRP3 and PLA2G4A, thereby activating microglia [9, 30]. LncRNA H19, which has been implicated in promoting microglial activation, may regulate the JAK/STAT signaling by competitively binds to let-7b that inhibits microglial activation by targeting STAT3 [15, 16]. LncRNA Malat1 may trigger NF-κB activation and pro-inflammatory mediator production in LPS-treated microglia by sponging miR-199b [45]. Moreover, Malat1 may act as a ceRNA of miR-30b to upregulate the expression of CNR1, which stimulates the activation of PI3K/AKT signaling and promotes neuronal recovery in neurodegenerative disorder [22]. Therefore, lncRNAs and miRNAs contribute to microglial activation, pro-inflammatory mediator production, neuroinflammation-mediated neuronal injury and repair.

Additionally, several scholars reported that lncRNAs and miRNAs may modulate CXCR4 expression. For instance, Malat1 may regulate the expression of CXCR4 in acute myeloid leukemia by interactions with miR-146a [32]. Furthermore, Malat1 may serve as a ceRNA of miR-204 to modulate CXCR4 expression in human hilar cholangiocarcinoma [36]. Moreover, miR-139-5p, which has been described as a neuron-enriched miRNA in the brain, may directly target CXCR4 and negatively regulate its expression in endothelial cells [27]. Hence, understanding the cross-talk between lncRNAs and miRNAs may provide insights into the mechanism of CXCR4 upregulation in iNSCs.

In this study, we investigated the mechanism by which CXCR4 is overexpressed at the surface of iNSCs. Our results indicated Malat1 as the most upregulated lncRNA and identified the Malat1/miR-139-5p/Cxcr4 axis in iNSCs co-cultured with LPS-activated microglia. We determined the effects of Malat1 and miR-139-5p on the overexpression of CXCR4 in iNSCs and explored their function in modulating the immunoregulatory effects of iNSC grafts on microglial activation in the injured cortices of CHI mice. This is the first study, to our knowledge, to show the role of the Malat1/miR-139-5p/Cxcr4 axis in regulating the interaction between iNSCs and microglia following CHI.

Methods

Cell culture

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 number: 2018-066). The work has been reported in line with the ARRIVE guidelines 2.0. C57BL/6 (B6, Charles River Laboratories, Beijing, China, license No. SCXK (Jing) 2016-0006) mouse iNSCs and microglia mono-culture and co-culture experiments were conducted as previously reported [11, 43] and as shown in Additional Fig. 2. Detailed methods are described in Additional file 1.

Fig. 1
figure 1

Bioinformatic analysis of differential mRNA and lncRNA expression in iNSCs after co-cultured with LPS-activated microglia. (A) Hierarchical clustering analysis of differential mRNA expression between the control and co-culture groups (n = 3/group). (B) Volcano plots of differential mRNA expression between the control and co-culture groups (vertical and horizontal lines represent log2 (fold change (FC)) = ± 1 and -log10(false discovery rate (FDR)) = 1.3) (n = 3/group). (C) Hierarchical clustering analysis of differential lncRNA expression between the control and co-culture groups (n = 3/group). (D) Volcano plots of differential lncRNA expression between the control and co-culture groups (vertical and horizontal lines represent log2 (FC) = ± 1 and -log10(FDR) = 1.3) (n = 3/group). (E) The levels of Cxcr4 and Malat1 in iNSCs between the control and co-culture groups (n = 6/group; Student’s t-test, ***P < 0.001). (F, G) The levels of Cxcr4 (F) and Malat1 (G) in iNSCs among the untransfected (iNSCs treated with PBS), control siRNA-transfected (iNSCs treated with control siRNA), and CXCR4-specific siRNA-transfected (iNSCs treated with CXCR4-specific siRNA) co-culture groups (n = 6/group; one-way analysis of variance with Tukey’s post hoc test, ***P < 0.001). (H, I) The levels of Malat1 (H) and Cxcr4 (I) in iNSCs among the untransfected (iNSCs treated with PBS), Scr-transfected (iNSCs treated with scramble control), and shMalat1-transfected (iNSCs treated with shMalat1) co-culture groups (n = 6/group; one-way analysis of variance with Tukey’s post hoc test, ***P < 0.001). (J) The levels of miR-146a, miR-204 and miR-139-5p in iNSCs between the control and co-culture groups (n = 6/group; Student’s t-test, **P < 0.01). (K) The levels of miR-139-5p in iNSCs among the untransfected, control siRNA-transfected and CXCR4-specific siRNA-transfected co-culture groups (n = 6/group; one-way analysis of variance with Tukey’s post hoc test). (L) The levels of miR-139-5p in iNSCs among the untransfected, Scr-transfected and shMalat1-transfected co-culture groups (n = 6/group; one-way analysis of variance with Tukey’s post hoc test, ***P < 0.001)

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RNA preparation, bioinformatic analysis and quantitative reverse transcription-polymerase chain reaction (qRT-PCR)

RNA preparation, bioinformatic analysis and qRT-PCR were performed as previously reported [13, 34]. RNA extracted from cultured iNSCs and microglia was reversely transcribed into cDNA using the QuantScript RT kit (Tiangen Biotech, Beijing, China). The Agilent gene expression arrays were conducted by the CapitalBio Company (Beijing, China). Differentially expressed genes between the control and co-culture groups 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 carried out to study the functions of differentially expressed genes. TargetScan v7.2 (https://www.targetscan.org/mmu_72/), starBase v2.0 (https://starbase.sysu.edu.cn/starbase2/) and miRDB (https://mirdb.org/) were utilized to predict target genes [6, 39]. For verification, we conducted 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). The qRT-PCR conditions were as follows: 95 °C for 5 min denaturation, followed by 45 cycles of 95 °C for 15 s, and 60 °C for 35 s. Expression levels were calculated relative to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using the ΔCt method (2−ΔΔCt). The sequences of the PCR primer pairs utilized in this study are listed in Additional Table 1 [8, 11, 19, 21, 29, 35, 40].

Cell transfection

CXCR4-specific siRNA (Santa Cruz Biotechnology, Santa Cruz, CA, USA), control siRNA (Santa Cruz Biotechnology), Malat1-specific short hairpin RNA (shMalat1, Sangon Biotech, Shanghai, China), scramble control shRNA (Scr, Sangon Biotech), miR-139-5p mimic (Gentaur Ltd, London, UK), miR-NC (Gentaur Ltd), miR-139-5p inhibitor (Gentaur Ltd), NC inhibitor (Gentaur Ltd), p65-specific siRNA (Santa Cruz Biotechnology) and TNFR1-specific siRNA (Santa Cruz Biotechnology) were utilized to transfect iNSCs as previously described [11]. After transfection, gene expression was measured using qRT-PCR.

Fluorescent in situ hybridization (FISH) assay

FISH assay was conducted using the Ribo™ Fluorescent In Situ Hybridization Kit (RiboBio, Guangzhou, China) with fluorescence-labeled probes (Sangon Biotech) to Malat1 (labeled with FITC green fluorescence), miR-139-5p (labeled with Cy3 red fluorescence) and Cxcr4 (labeled with FITC green fluorescence). 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 analyzed via fluorescence microscopy (DM3000, Leica, Wetzlar, Germany) and confocal laser scanning microscopy (TCS SP5 II, Leica). The fluorescence intensity was counted using image analysis software (Image-Pro plus 5.0, Media Cybernetics, Silver Spring, MD, USA).

Dualluciferase reporter assay

PsiCHECK-2 luciferase vectors (Promega, Madison, WI, USA) containing Malat1 or Cxcr4 3′ untranslated region (UTR) with the wild-type (Malat1 or Cxcr4 WT) or mutant (Malat1 or Cxcr4 MUT) miR-139-5p binding site were co-transfected with miR-139-5p mimic or miR-NC into cells with Lipofectamine™ 2000 (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. Luciferase activity was identified using the Dual-Luciferase Reporter Assay System (Promega) at 48 h post-transfection.

RNA immunoprecipitation (RIP) assay

RIP assay was performed using the Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore, Billerica, MA, USA) following the manufacturer’s instructions. 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 measured by qRT-PCR.

Biotin-coupled miRNA pulldown assay

INSCs were transfected with biotin-labeled miR-139-5p (Bio-miR-139-5p WT, GenePharma, Shanghai, China) or the mutated sequence (Bio-miR-139-5p MUT, GenePharma). At 48 h 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 determined by qRT-PCR.

CHI models

CHI models were established as previously reported [13]. In brief, healthy adult (12–14 weeks old) male B6 mice weighing 22–32 g (Charles River Laboratories) were anaesthetized through the intranasal administration of isoflurane (Thermo Fisher Scientific, Cleveland, OH, USA) (induction: 3% isoflurane; maintenance: 1.25% isoflurane), and received fentanyl (0.05 mg/kg body weight per day, intraperitoneal injection, Janssen-Cilag, Berchem, Belgium) 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 measured the severity of CHI mice at 1 h after injury using the neurological severity score (NSS) (Additional Table 2). Mice with an NSS of 6–8 were enrolled in experiments. CHI-induced neurological impairment and fine-motor coordination deficits were analyzed using the NSS and a beam-walk task as previously described [13]. Detailed methods are described in Additional file 2.

Cell transplantation

Transfected iNSCs were digested with accutase (Invitrogen), and washed with PBS. The number of living cells was counted, and the density of the single-cell suspension was adjusted. Subsequently, the cells were maintained on ice. At 12 h after CHI, the mice were anaesthetized as described above and mounted in a stereotaxic apparatus (Stoelting, Wood Dale, IL, USA). Cell suspension or PBS was separately injected into the brain (motor cortex, 5.0 mm anterior to the lambda suture, 1.0 mm lateral to the middle line and 2.0 mm under the dura) via different sterile 25-µL 22 S Hamilton syringes. Each site received 5 µL of cell suspension containing 1 × 106 cells or PBS at a speed of 0.5 µL/min. Approximately 5 min after injection, the syringe was slowly withdrawn.

Animal euthanasia

At 7 days after CHI, the mice were anaesthetized through the intranasal administration of isoflurane (Thermo Fisher Scientific) (induction: 3% isoflurane; maintenance: 1.25% isoflurane), and received fentanyl (0.1 mg/kg body weight, intraperitoneal injection, Janssen-Cilag) as the analgesic agent. Euthanization was performed under cervical dislocation.

Western blot analysis

Western blot analysis was carried out as previously described [13]. Protein was extracted from iNSCs or brain tissues using the RIPA reagent (Sigma-Aldrich) supplemented with protease and phosphatase inhibitors (Fermentas, Burlington, Canada). Protein concentrations were measured using the BCA assay (Thermo Fisher Scientific). Protein samples were separated by SDS-PAGE (35 µg per lane) 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 h at room temperature with HRP-conjugated secondary antibodies. Immunoblots were visualized using the SuperSignal ECL (Pierce, Rockford, IL, USA). The intensities of bands were identified 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. The antibodies used in this study are listed in Additional Table 3.

Enzyme-linked immunosorbent assay (ELISA)

To determine the levels of cytokines, cell culture supernatants were purified by centrifugation for 20 min at 3000 r.p.m and stored at -80 °C. The levels of CXCL12, TNF-α and IGF-1 (R&D Systems, Minneapolis, MN, USA) were measured in duplicate assays using ELISA kits according to the manufacturer’s protocol.

Morphological analysis

Morphological analysis was conducted as previously reported [13]. Slides of iNSCs, microglia and brain tissues were incubated overnight at 4 °C with primary antibodies. After washes in PBS, they were incubated for 2 h at room temperature with secondary antibodies. Nuclei were stained with DAPI Fluoromount-G (SouthernBiotech) and staining was determined via fluorescent microscopy (DM3000, Leica) and confocal laser scanning microscopy (TCS SP5 II, Leica). The fluorescence intensity was counted using image analysis software (Image-Pro plus 5.0, Media Cybernetics). Detailed methods are described in Additional file 3. The antibodies used in this study are listed in Additional Table 3.

Flow cytometry

Flow cytometry was performed as previously reported [13]. Briefly, iNSCs were incubated with primary antibodies for 30 min at 4 °C. After washing with PBS, the cells were incubated for 30 min at room temperature with secondary antibodies. After several washes, the cells were measured on an Accuri C6 Flow Cytometer System (BD Biosciences, San Jose, CA, USA). Isotype control antibodies were used at the same concentrations. The antibodies used in this study are listed in Additional Table 3.

Statistical analysis

Statistical analysis was performed with SPSS17.0 statistical software. Data are presented as mean ± standard deviation. Student’s t-test and one-way analysis of variance (ANOVA) with Tukey’s post hoc test were used to determine statistical significance. P < 0.05 indicated statistical significance.

Results

Bioinformatic analysis of differential mRNA and lncRNA expression in iNSCs after co-cultured with LPS-activated microglia

The sequencing data are listed in Additional Table 4. We performed bioinformatic analysis to detect the differentially expressed genes in iNSCs after co-cultured with LPS-activated microglia (Additional Fig. 2A–C). We identified 51,963 genes (including 34,539 mRNAs and 17,424 lncRNAs) in iNSCs, 3,007 genes (including 2,256 mRNAs and 751 lncRNAs) of which upregulated (P < 0.05, log2(FC) > 1) and 2,395 genes (including 1,682 mRNAs and 713 lncRNAs) downregulated (P < 0.05, log2(FC) < -1) after co-cultured with activated microglia (Fig. 1A-D). Furthermore, volcano plots indicated 192 genes (including 184 mRNAs and 8 lncRNAs) of which upregulated (FDR < 0.05, log2(FC) > 1) and 148 genes (including 137 mRNAs and 11 lncRNAs) downregulated (FDR < 0.05, log2(FC) < -1) in iNSCs after co-cultured with activated microglia. Additionally, the FDR and log2(FC) values of Cxcr4 were 0.0307 and 2.1298, respectively. Notably, Malat1 was the most upregulated lncRNA in iNSCs after co-cultured with activated microglia. The FDR and log2(FC) values of Malat1 were 0.0465 and 4.2888, respectively.

Fig. 2
figure 2

Malat1 and Cxcr4 directly interacts with miR-139-5p in iNSCs. (A, B) Representative images of fluorescence in situ hybridization (FISH) assay show the expression and location of miR-139-5p (red), Malat1 (A, green) and Cxcr4 (B, green) in iNSCs between the control and co-culture groups. Nuclei were counterstained with DAPI (blue). Scale bar: 50 μm (5 μm in the magnified images). (C-E) The relative fluorescence intensity values of Malat1 (C), Cxcr4 (D) and miR-139-5p (E) in the cytoplasm and nucleus of iNSCs between the control and co-culture groups (n = 6/group; Student’s t-test, *P < 0.05, ***P < 0.001). (F) Schematic of the putative binding sites between Malat1 and miR-139-5p. The luciferase activities of iNSCs transfected with Malat1 wild-type (Malat1-WT) or Malat1 mutant (Malat1-MUT) reporter vectors and miR-NC or miR-139-5p mimic (n = 3/group; Student’s t-test, **P < 0.01). (G) Schematic of the putative binding sites between Cxcr4 and miR-139-5p. The luciferase activities of iNSCs transfected with Cxcr4 wild-type (Cxcr4-WT) or Cxcr4 mutant (Cxcr4-MUT) reporter vectors and miR-NC or miR-139-5p mimic (n = 3/group; Student’s t-test, **P < 0.01). (H, I) The levels of Malat1 (H), Cxcr4 (I) and miR-139-5p in iNSCs were measured in the substrate of the RNA immunoprecipitation (RIP) assay by qRT-PCR (n = 6/group; one-way analysis of variance with Tukey’s post hoc test, ***P < 0.001)

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GO enrichment analysis revealed that differentially expressed mRNAs were significantly enriched in multicellular organism development (biological process (BP)), protein binding (molecular function (MF)), and neuron projection (cellular component (CC)) (Additional Fig. 2A–C). KEGG pathway analysis showed that differentially expressed mRNAs were mainly enriched in the cell adhesion molecules (Additional Fig. 2D).

To verify the results of the bioinformatic analysis, we performed qRT-PCR to determine the expression of Cxcr4 and Malat1 in iNSCs and found that Cxcr4 and Malat1 levels in iNSCs were significantly higher in the co-culture group than in the control group (Cxcr4: P < 0.0001; Malat1: P < 0.0001) (Fig. 1E).

To explore the relationship between Cxcr4 and Malat1, we transfected iNSCs with CXCR4-specific siRNA or control siRNA prior to the co-culture studies. QRT-PCR revealed that Cxcr4 levels in iNSCs of the CXCR4-specific siRNA-transfected (iNSCs treated with CXCR4-specific siRNA) co-culture group were markedly lower than those in the untransfected (iNSCs treated with PBS) and control siRNA-transfected (iNSCs treated with control siRNA) co-culture groups (CXCR4-specific siRNA-transfected vs. untransfected: P < 0.0001; CXCR4-specific siRNA-transfected vs. control siRNA-transfected: P < 0.0001) (Fig. 1F). Western blot analysis also confirmed CXCR4 inhibition in iNSCs of the CXCR4-specific siRNA-transfected co-culture group (data not shown), suggesting effective knockdown of CXCR4 expression in these iNSCs. There were no significant differences in the Malat1 levels in iNSCs among the three groups (Fig. 1G).

Furthermore, we used shMalat1 or Scr to transfect iNSCs prior to the co-culture studies and observed that Malat1 levels were significantly lower in the shMalat1-transfected (iNSCs treated with shMalat1) co-culture groups in iNSCs compared with the untransfected (iNSCs treated with PBS) and Scr-transfected (iNSCs treated with scramble control) co-culture groups (shMalat1-transfected vs. untransfected: P < 0.0001; shMalat1-transfected vs. Scr-transfected: P < 0.0001), suggesting effective knockdown of Malat1 expression in iNSCs (Fig. 1H). Remarkably, Cxcr4 levels were substantially lower in the shMalat1-transfected co-culture groups in iNSCs compared with the other two groups (shMalat1-transfected vs. untransfected: P = 0.0001; shMalat1-transfected vs. Scr-transfected: P = 0.0001), indicating that Malat1 inhibition reduced the levels of Cxcr4 in iNSCs after co-cultured with activated microglia (Fig. 1I).

We next predicted the target miRNAs of Malat1 such as miR-146a, miR-204 and miR-139-5p and found that miR-139-5p levels in iNSCs were markedly lower in the co-culture group than in the control group (P = 0.0009) (Fig. 1J). Then, we investigated miR-139-5p levels in iNSCs treated with CXCR4-specific siRNA and observed that there were no significant differences in the miR-139-5p levels in iNSCs among the untransfected, control siRNA-transfected and CXCR4-specific siRNA-transfected co-culture groups (Fig. 1K). However, miR-139-5p levels were substantially higher in the shMalat1-transfected co-culture groups in iNSCs compared with the untransfected and Scr-transfected co-culture groups (shMalat1-transfected vs. untransfected: P < 0.0001; shMalat1-transfected vs. Scr-transfected: P < 0.0001), indicating that Malat1 inhibition increased the levels of miR-139-5p in iNSCs after co-cultured with activated microglia (Fig. 1L).

Together, these data showed that Malat1 was the most upregulated lncRNA in iNSCs after co-cultured with activated microglia. Additionally, Malat1 inhibition decreased Cxcr4 and increased miR-139-5p levels in these iNSCs.

Malat1 and Cxcr4 directly interacts with mir-139-5p in iNSCs

To explore the relationship among Malat1, Cxcr4 and miR-139-5p, we used FISH assay to detect their subcellular localization and observed the partial co-localization of Malat1 and miR-139-5p as well as Cxcr4 and miR-139-5p within iNSCs (Fig. 2A and B). The relative fluorescence intensity (RFI) values of Malat1 and Cxcr4 in iNSCs were significantly higher in the co-culture group than in the control group (Malat1: P = 0.0001; Cxcr4: P = 0.0004) (Additional Fig. 3A and B). Furthermore, the RFI values of Malat1 and Cxcr4 in the nuclear and cytoplasmic fraction of iNSCs were markedly higher in the co-culture group than in the control group (Malat1(nuclear): P = 0.0005; Malat1(cytoplasm): P < 0.0001; Cxcr4(nuclear): P = 0.0219; Cxcr4(cytoplasm): P < 0.0001) (Fig. 2C and D). In contrast, the RFI values of miR-139-5p in the cytoplasmic fraction of iNSCs were substantially lower in the co-culture group than in the control group (P < 0.0001) (Fig. 2E). These data showed that the elevated levels of Malat1 and Cxcr4 were closely correlated with the decreased levels of miR-139-5p in iNSCs after co-cultured with activated microglia.

Fig. 3
figure 3

The interplay among Malat1, miR-139-5p and Cxcr4 in regulating the interaction between iNSCs and LPS-activated microglia. (A) Schematic of the putative binding sites between miR-139-5p and Malat1. The levels of Malat1 in iNSCs pulled down by miR-139-5p (n = 6/group; one-way analysis of variance with Tukey’s post hoc test, ***P < 0.001). (B) Schematic of the putative binding sites between miR-139-5p and Cxcr4. The levels of Cxcr4 in iNSCs pulled down by miR-139-5p (n = 6/group; one-way analysis of variance with Tukey’s post hoc test, ***P < 0.001). (C) The levels of CXCR4 in iNSCs among the control, co-culture (Scr) (iNSCs treated with scramble control), co-culture (shMalat1) (iNSCs treated with shMalat1), co-culture (shMalat1 + NC inhibitor) (iNSCs treated with shMalat1 and NC inhibitor), co-culture (shMalat1 + miR-139-5p inhibitor) (iNSCs treated with shMalat1 and miR-139-5p inhibitor), co-culture (shMalat1 + miR-139-5p inhibitor + control siRNA) (iNSCs treated with shMalat1, miR-139-5p inhibitor and control siRNA), and co-culture (shMalat1 + miR-139-5p inhibitor + CXCR4 siRNA) (iNSCs treated with shMalat1, miR-139-5p inhibitor and CXCR4-specific siRNA) groups (full-length blots are presented in Additional Fig. 14). (D) The levels of CXCR4 in iNSCs among the seven groups (n = 6/group; one-way analysis of variance with Tukey’s post hoc test, ***P < 0.001). (E, F) The levels of CXCL12 (E) and TNF-α (F) in cell culture supernatants measured by ELISA among the seven groups (n = 6/group; one-way analysis of variance with Tukey’s post hoc test, ***P < 0.001)

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We then used miR-139-5p mimic or miR-NC to transfect iNSCs (Additional Fig. 4). There were no significant differences in the miR-139-5p levels between the untransfected (iNSCs treated with PBS) and miR-NC-transfected (iNSCs treated with miR-NC) groups in iNSCs. In contrast, miR-139-5p levels in iNSCs were markedly higher in the miR-139-5p mimic-transfected (iNSCs treated with miR-139-5p mimic) group than in the other two groups (miR-139-5p mimic-transfected vs. untransfected: P < 0.0001; miR-139-5p mimic-transfected vs. miR-NC-transfected: P < 0.0001), suggesting effective overexpression of miR-139-5p in iNSCs. Subsequently, we predicted the possible binding sites of Malat1 and miR-139-5p as well as Cxcr4 3′ untranslated region (UTR) and miR-139-5p and constructed reporter vectors for dualluciferase reporter assays (Fig. 2F and G). Luciferase activity was significantly reduced after co-transfection of the Malat1-WT or Cxcr4-WT reporter vector and miR-139-5p mimic in iNSCs (Malat1-WT: P = 0.0011; Cxcr4-WT: P = 0.0018) and was unchanged when the Malat1-MUT or Cxcr4-MUT reporter was used.

Fig. 4
figure 4

Malat1 inhibition attenuates the immunoregulatory effects of iNSC grafts on microglial activation in the injured cortices of CHI mice. (A, B) Representative staining for CXCL12+ (green, A), TNF-α+ (green, B) and Iba1+ (red) cells depicting the distribution of CXCL12+/Iba1+ and TNF-α+/Iba1+ microglia in the injured cortices among the sham, PBS (CHI mice receiving PBS), iNSC (Scr) (CHI mice receiving iNSCs pretreated with scramble control), and iNSC (shMalat1) (CHI mice receiving iNSCs pretreated with shMalat1) groups on Day 7 post-CHI. Nuclei were counterstained with DAPI (blue). Scale bar: 50 μm (5 μm in the magnified images). (C, D) The relative fluorescence intensity of CXCL12 (C) and TNF-α (D) in the injured cortices among the PBS, iNSC (Scr), and iNSC (shMalat1) groups (n = 6/group; one-way analysis of variance with Tukey’s post hoc test, **P < 0.01, ***P < 0.001). (E) The levels of CXCL12, TNF-α, IGF-1, p-p65, p65, p-IκBα, IκBα and GAPDH in the injured cortices among the three groups (full-length blots are presented in Additional Fig. 15). (F-J) The levels of CXCL12 (F), TNF-α (G), IGF-1 (H), p-p65/p65 (I) and p-IκBα/IκBα (J) in the injured cortices among the three groups (n = 6/group; one-way analysis of variance with Tukey’s post hoc test, *P < 0.05, **P < 0.01, ***P < 0.001)

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Additionally, we performed RIP assays and found that Malat1 and miR-139-5p as well as Cxcr4 and miR-139-5p were specifically enriched in Ago2 pellets of iNSC extracts relative to the IgG control group (Malat1: P < 0.0001; Cxcr4: P < 0.0001; miR-139-5p: P < 0.0001) (Fig. 2H and I). Biotin-coupled miRNA pulldown assay also revealed that the incubation of Bio-miR-139-5p WT led to a dramatic increase in Malat1 or Cxcr4 enrichment in iNSCs compared with the Bio-miR-139-5p MUT group (Malat1: P < 0.0001; Cxcr4: P < 0.0001) (Fig. 3A and B). Together, these data indicated that Malat1 and Cxcr4 could directly interact with miR-139-5p in iNSCs.

The interplay among Malat1, mir-139-5p and Cxcr4 in regulating the interaction between iNSCs and LPS-activated microglia

We first performed gain- and loss-of-functional studies to identify the role of miR-139-5p and discovered that there were no significant differences in the Malat1 levels in iNSCs among the miR-NC-transfected (iNSCs treated with miR-NC), miR-139-5p mimic-transfected (iNSCs treated with miR-139-5p mimic), NC inhibitor-transfected (iNSCs treated with NC inhibitor) and miR-139-5p inhibitor-transfected (iNSCs treated with miR-139-5p inhibitor) co-culture groups (Additional Fig. 5A). In contrast, Cxcr4 levels in iNSCs of the miR-139-5p mimic-transfected co-culture group were markedly lower than those in the miR-NC-transfected co-culture group (miR-139-5p mimic-transfected vs. miR-NC-transfected: P = 0.0002), while Cxcr4 levels of the miR-139-5p inhibitor-transfected co-culture group were substantially higher than those in the NC inhibitor-transfected co-culture group (miR-139-5p inhibitor-transfected vs. NC inhibitor-transfected: P < 0.0001) (Additional Fig. 5B). Furthermore, western blot analysis revealed that CXCR4 levels were significantly downregulated in iNSCs treated with miR-139-5p mimic (miR-139-5p mimic-transfected vs. miR-NC-transfected: P < 0.0001) (Additional Fig. 5C and D). However, CXCR4 levels were markedly upregulated in iNSCs treated with miR-139-5p inhibitor (miR-139-5p inhibitor-transfected vs. NC inhibitor-transfected: P < 0.0001). These data further suggested that miR-139-5p could regulate CXCR4 expression in iNSCs co-cultured with activated microglia.

Fig. 5
figure 5

Malat1 inhibition attenuates the immunoregulatory effects of iNSC grafts on the levels of CXCL12 and TNF-α in the injured cortices of CHI mice. (A) Representative images showed the distribution of GFP-labeled grafts (green) and CXCL12+ (red) cells in the injured cortices between the iNSC (Scr) (CHI mice receiving iNSCs pretreated with scramble control) and iNSC (shMalat1) (CHI mice receiving iNSCs pretreated with shMalat1) groups on Day 7 post-CHI. Nuclei were counterstained with DAPI (blue). Scale bar: 50 μm (5 μm in the magnified images). (B, C) Histograms indicated the numbers of GFP+ (B) and GFP+/CXCL12+ (C) cells in the injured cortices between the two groups (n = 6/group; Student’s t-test, ***P < 0.001). (D) The relative fluorescence intensity of CXCL12 in the injured cortices between the two groups (n = 6/group; Student’s t-test, ***P < 0.001). (E) Representative images showed the distribution of GFP-labeled grafts (green) and TNF-α+ (red) cells in the injured cortices between the two groups on Day 7 post-CHI. Nuclei were counterstained with DAPI (blue). Scale bar: 50 μm (5 μm in the magnified images). (F, G) Histograms indicated the numbers of GFP+ (F) and GFP+/TNF-α+ (G) cells in the injured cortices between the two groups (n = 6/group; Student’s t-test). (H) The relative fluorescence intensity of TNF-α in the injured cortices between the two groups (n = 6/group; Student’s t-test, **P < 0.01)

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Additionally, ELISA showed that miR-139-5p upregulation restored the levels of CXCL12 (miR-139-5p mimic-transfected vs. miR-NC-transfected: P < 0.0001), TNF-α (miR-139-5p mimic-transfected vs. miR-NC-transfected: P = 0.0001) and IGF-1 (miR-139-5p mimic-transfected vs. miR-NC-transfected: P < 0.0001) in the supernatant of the co-culture (miR-139-5p mimic) (iNSCs treated with miR-139-5p mimic) group (Additional Fig. 6A-C). In contrast, miR-139-5p inhibition substantially decreased the levels of CXCL12 (miR-139-5p inhibitor-transfected vs. NC inhibitor-transfected: P = 0.0073) and TNF-α (miR-139-5p inhibitor-transfected vs. NC inhibitor-transfected: P = 0.0076) and increased the levels of IGF-1 (miR-139-5p inhibitor-transfected vs. NC inhibitor-transfected: P = 0.0001) in the supernatant of the co-culture (miR-139-5p inhibitor) (iNSCs treated with miR-139-5p inhibitor) group.

Fig. 6
figure 6

NF-κB activation regulates Malat1, miR-139-5p and Cxcr4 expression in iNSCs. (A) Representative images show the expression and location of p-p65 (red) in iNSCs between the control and co-culture groups. Nuclei were counterstained with DAPI (blue). Scale bar: 50 μm (5 μm in the magnified images). (B) The relative fluorescence intensity values of p-p65 in the nucleus of iNSCs between the two groups (n = 6/group; Student’s t-test, ***P < 0.001). (C) The levels of p-p65, p65, p-IκBα, IκBα and GAPDH in iNSCs between the two groups (full-length blots are presented in Additional Fig. 16). (D, E) The levels of p-p65/p65 (D) and p-IκBα/IκBα (E) in iNSCs between the two groups (n = 6/group; Student’s t-test, ***P < 0.001). (F-I) The levels of p65 (F), Malat1 (G), miR-139-5p (H) and Cxcr4 (I) in iNSCs among the untransfected, control siRNA-transfected (iNSCs treated with control siRNA), and p65-specific siRNA-transfected (iNSCs treated with p65-specific siRNA) co-culture groups (n = 6/group; one-way analysis of variance with Tukey’s post hoc test, ***P < 0.001). (J) The levels of p-p65, p65, CXCR4 and GAPDH in iNSCs among the three groups (full-length blots are presented in Additional Fig. 17). (K-M) The levels of p65 (K), p-p65/p65 (L) and CXCR4 (M) in iNSCs among the three groups (n = 6/group; one-way analysis of variance with Tukey’s post hoc test, ***P < 0.001). (N-P) The levels of CXCL12 (N), TNF-α (O) and IGF-1 (P) in cell culture supernatants measured by ELISA among the three groups (n = 6/group; one-way analysis of variance with Tukey’s post hoc test, *P < 0.05, **P < 0.01, ***P < 0.001)

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To determine the mechanism underlying the changes of these cytokines, we performed morphological analysis and observed that miR-139-5p downregulation in iNSCs significantly decreased the levels of CXCL12 (co-culture (miR-139-5p inhibitor) vs. control: P < 0.0001) and TNF-α (co-culture (miR-139-5p inhibitor) vs. control: P < 0.0001) in activated microglia of the co-culture (miR-139-5p inhibitor) group (Additional Fig. 7). In contrast, miR-139-5p upregulation in iNSCs restored the levels of CXCL12 (miR-139-5p mimic vs. miR-139-5p inhibitor: P < 0.0001) and TNF-α (miR-139-5p mimic vs. miR-139-5p inhibitor: P = 0.0077) in activated microglia of the co-culture (miR-139-5p mimic) group. Additionally, the regulation of miR-139-5p in iNSCs also modulated IGF-1 expression in activated microglia (data not shown). These results revealed that miR-139-5p exerted significant influence on the immunoregulatory effects of iNSCs on the secretion of CXCL12, TNF-α and IGF-1 by activated microglia.

Fig. 7
figure 7

TNFR1 inhibition reduces the levels of Malat1 and CXCR4 expression as well as NF-κB activation in iNSCs. (A-D) The levels of Tnfr1 (A), Malat1 (B), miR-139-5p (C) and Cxcr4 (D) in iNSCs among the untransfected, control siRNA-transfected (iNSCs treated with control siRNA), and TNFR1-specific siRNA-transfected (iNSCs treated with TNFR1-specific siRNA) co-culture groups (n = 6/group; one-way analysis of variance with Tukey’s post hoc test, *P < 0.05, **P < 0.01, ***P < 0.001). (E) The levels of TNFR1, p-p65, p65, p-IκBα, IκBα, CXCR4 and GAPDH in iNSCs among the three groups (full-length blots are presented in Additional Fig. 18). (F-I) The levels of TNFR1 (F), p-p65/p65 (G), p-IκBα/IκBα (H) and CXCR4 (I) in iNSCs among the three groups (n = 6/group; one-way analysis of variance with Tukey’s post hoc test, **P < 0.01, ***P < 0.001). (J-M) Representative flow cytometric analysis of TNFR1 (J) and CXCR4 (L) expression in iNSCs among the untransfected (red line), control siRNA-transfected (green line), and TNFR1-specific siRNA-transfected (blue line) co-culture groups. Isotype antibodies were used as controls (black line). Histograms showing the median fluorescence intensity (MFI) values of TNFR1 (K) and CXCR4 (M) expression in iNSCs among the three groups (n = 3/group; one-way analysis of variance with Tukey’s post hoc test, ***P < 0.001)

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To clarify the interplay among Malat1, miR-139-5p and Cxcr4, we used shMalat1, miR-139-5p inhibitor or CXCR4-specific siRNA to transfect iNSCs and observed that Malat1 levels showed no significant changes in iNSCs with co-transfection with miR-139-5p inhibitor and CXCR4-specific siRNA (Additional Fig. 8A and B). In contrast, Cxcr4 levels markedly decreased after transfection with shMalat1 and CXCR4-specific siRNA, and the miR-139-5p inhibitor restored the effect of shMalat1 but not CXCR4-specific siRNA (P < 0.0001). These data indicated that Malat1 could serve as a sponge of miR-139-5p to regulate Cxcr4 expression in iNSCs co-cultured with activated microglia.

We then performed western blot analysis and discovered that Malat1 inhibition decreased the levels of CXCR4 in iNSCs after co-cultured with activated microglia (shMalat1 vs. Scr: P < 0.0001) (Fig. 3C and D). Notably, miR-139-5p inhibitor reversed the effect of Malat1 inhibition (miR-139-5p inhibitor vs. NC inhibitor: P < 0.0001), while CXCR4 inhibition with co-transfection with shMalat1 and miR-139-5p inhibitor substantially reduced CXCR4 expression in iNSCs co-cultured with activated microglia (CXCR4 siRNA vs. control siRNA: P < 0.0001). These data showed that Malat1 and miR-139-5p could modulate CXCR4 expression in iNSCs co-cultured with activated microglia. Moreover, miR-139-5p exerted significant influence on the effects of Malat1 on CXCR4 expression in these iNSCs.

Subsequently, we found by ELISA that Malat1 inhibition restored the levels of CXCL12 (shMalat1 vs. Scr: P < 0.0001), TNF-α (shMalat1 vs. Scr: P < 0.0001) and IGF-1 (shMalat1 vs. Scr: P < 0.0001) in the supernatant of the co-culture (shMalat1) (iNSCs treated with shMalat1) group (Fig. 3E and F and Additional Fig. 9). Remarkably, miR-139-5p inhibitor reversed the effect of Malat1 inhibition on the levels of CXCL12 (miR-139-5p inhibitor vs. NC inhibitor: P < 0.0001), TNF-α (miR-139-5p inhibitor vs. NC inhibitor: P < 0.0001) and IGF-1 (miR-139-5p inhibitor vs. NC inhibitor: P < 0.0001) in the supernatant of the co-culture (shMalat1 + miR-139-5p inhibitor) (iNSCs treated with shMalat1 and miR-139-5p inhibitor) group. Moreover, CXCR4 inhibition with co-transfection with shMalat1 and miR-139-5p inhibitor significantly increased the levels of CXCL12 (CXCR4 siRNA vs. control siRNA: P < 0.0001) and TNF-α (CXCR4 siRNA vs. control siRNA: P < 0.0001) and decreased the levels of IGF-1 (CXCR4 siRNA vs. control siRNA: P < 0.0001) in the supernatant of the co-culture (shMalat1 + miR-139-5p inhibitor + CXCR4 siRNA) (iNSCs treated with shMalat1, miR-139-5p inhibitor and CXCR4-specific siRNA) group. These data implied that the interplay among Malat1, miR-139-5p and Cxcr4 could regulate the levels of CXCL12, TNF-α and IGF-1 in the co-culture supernatant.

To study the mechanism underlying the changes of these cytokines, we performed morphological analysis and observed that Malat1 inhibition in iNSCs restored the levels of CXCL12 (shMalat1 vs. Scr: P < 0.0001) and TNF-α (shMalat1 vs. Scr: P = 0.0077) in activated microglia of the co-culture (shMalat1) (iNSCs treated with shMalat1) group (Additional Fig. 10). Additionally, Malat1 inhibition in iNSCs also restored the levels of IGF-1 in activated microglia of the co-culture (shMalat1) group (data not shown). These results indicated that Malat1 exerted significant influence on the immunoregulatory effects of iNSCs on the secretion of CXCL12, TNF-α and IGF-1 by activated microglia.

Together, these findings suggested that Malat1 could act as a sponge of miR-139-5p to modulate CXCR4 expression in iNSCs co-cultured with activated microglia and the Malat1/miR-139-5p/Cxcr4 axis could play important roles in regulating the interaction between iNSCs and microglia.

Malat1 inhibition attenuates the immunoregulatory effects of iNSC grafts on microglial activation and neuroinflammation in the injured cortices of CHI mice

To determine the influence of Malat1 inhibition in iNSC grafts on microglial activation in the injured cortices of CHI mice, we first used double-labelling experiments and found that CXCL12+/Iba1+ and TNF-α+/Iba1+ microglia were evident in the injured cortices of the PBS (CHI mice receiving PBS) group on Day 7 after CHI (Fig. 4A and B). No CXCL12+/Iba1+ or TNF-α+/Iba1+ microglia were observed in the sham group. Quantitatively, the levels of CXCL12+/Iba1+ (P < 0.0001) and TNF-α+/Iba1+ (P = 0.0001) microglia were markedly lower in the iNSC (Scr) (CHI mice receiving iNSCs pretreated with scramble control) group than in the PBS group (Fig. 4C and D). However, Malat1 inhibition in iNSC grafts restored the levels of CXCL12+/Iba1+ (shMalat1 vs. Scr: P = 0.0001) and TNF-α+/Iba1+ (shMalat1 vs. Scr: P = 0.0074) microglia of the iNSC (shMalat1) (CHI mice receiving iNSCs pretreated with shMalat1) group.

Next, we performed western blot analysis and found that in contrast to the PBS group, iNSC grafts substantially decreased the levels of CXCL12 (P = 0.0002), TNF-α (P = 0.0001), phospho-p65/p65 (P = 0.0042) and phospho-IκBα/IκBα (P = 0.0045) and increased the levels of IGF-1 (P = 0.0001) in the injured cortices of CHI mice of the iNSC (Scr) group (Fig. 4E-J). Remarkably, Malat1 inhibition in iNSC grafts restored the levels of CXCL12 (shMalat1 vs. Scr: P = 0.0041), TNF-α (shMalat1 vs. Scr: P = 0.0010), IGF-1 (shMalat1 vs. Scr: P = 0.0016), phospho-p65/p65 (shMalat1 vs. Scr: P = 0.0266) and phospho-IκBα/IκBα (shMalat1 vs. Scr: P = 0.0351) in the injured cortices of CHI mice of the iNSC (shMalat1) group.

Furthermore, we found that GFP-labeled grafts migrated into the injured cortices of CHI mice (Fig. 5A and E). Quantitatively, the levels of GFP-labeled grafts had no significant differences between the iNSC (Scr) and iNSC (shMalat1) groups (Fig. 5B and F). Remarkably, we observed the partial co-localization of GFP and CXCL12 and determined that the levels of GFP+/CXCL12+ grafts were markedly higher in the iNSC (Scr) group than in the iNSC (shMalat1) group (P < 0.0001) (Fig. 5C), no matter that the RFI values of CXCL12 in the injured cortices were significantly lower in the iNSC (Scr) group than in the iNSC (shMalat1) group (P = 0.0001) (Fig. 5D). Additionally, we also observed the partial co-localization of GFP and TNF-α and detected that the levels of GFP+/TNF-α+ grafts had no significant differences between the two groups (Fig. 5G). However, the RFI values of TNF-α in the injured cortices were significantly lower in the iNSC (Scr) group than in the iNSC (shMalat1) group (P = 0.0016) (Fig. 5H).

Together, these results demonstrated that Malat1 inhibition blocked the immunoregulatory effects of iNSC grafts on microglial activation and neuroinflammation in the injured cortices of CHI mice. Moreover, Malat1 inhibition reduced the levels of CXCL12 co-localized to iNSC grafts, while failed to regulate the levels of TNF-α co-localized to iNSC grafts.

NF-κB activation regulates Malat1, mir-139-5p and Cxcr4 expression in iNSCs

To clarify the mechanism underlying the upregulation of Malat1 in iNSCs after co-cultured with LPS-activated microglia, we detected several related signaling pathways and observed that p65 levels in iNSCs were markedly higher in the co-culture group than in the control group (data not shown). To determine the role of NF-κB p65 in the upregulation of Malat1, we performed morphological analysis to identify the subcellular localization of phospho-p65 within iNSCs and discovered that the RFI values of phospho-p65 in the nuclear fraction of iNSCs were significantly higher in the co-culture group than in the control group, suggesting that there was a significant enhancement of phosphorylation and nuclear translocation of p65 in iNSCs after co-cultured with activated microglia (P < 0.0001) (Fig. 6A and B). Moreover, western blot analysis indicated that the levels of phospho-p65/p65 (P < 0.0001) and phospho-IκBα/IκBα (P < 0.0001) in iNSCs were substantially higher in the co-culture group than in the control group (Fig. 6C-E). These data indicated NF-κB activation in iNSCs after co-cultured with activated microglia.

Subsequently, we transfected iNSCs with p65-specific siRNA or control siRNA prior to the co-culture studies. QRT-PCR showed that p65 levels in iNSCs of the p65-specific siRNA-transfected (iNSCs treated with p65-specific siRNA) co-culture group were markedly lower than those in the untransfected (iNSCs treated with PBS) and control siRNA-transfected (iNSCs treated with control siRNA) co-culture groups (p65-specific siRNA-transfected vs. untransfected: P < 0.0001; p65-specific siRNA-transfected vs. control siRNA-transfected: P < 0.0001) (Fig. 6F). Western blot analysis also confirmed p65 inhibition in iNSCs of the p65-specific siRNA-transfected co-culture group (data not shown), suggesting effective knockdown of p65 expression in these iNSCs. Notably, p65 inhibition significantly decreased the levels of Malat1 (p65-specific siRNA-transfected vs. untransfected: P < 0.0001; p65-specific siRNA-transfected vs. control siRNA-transfected: P < 0.0001) and Cxcr4 (p65-specific siRNA-transfected vs. untransfected: P < 0.0001; p65-specific siRNA-transfected vs. control siRNA-transfected: P < 0.0001) and increased the levels of miR-139-5p (p65-specific siRNA-transfected vs. untransfected: P < 0.0001; p65-specific siRNA-transfected vs. control siRNA-transfected: P = 0.0003) in iNSCs of the p65-specific siRNA-transfected co-culture group, suggesting that p65 could regulate the Malat1/miR-139-5p/Cxcr4 axis in iNSCs co-cultured with activated microglia (Fig. 6G-I).

We then performed western blot analysis and demonstrated that p65 inhibition substantially decreased the levels of p65 (p65-specific siRNA-transfected vs. untransfected: P < 0.0001; p65-specific siRNA-transfected vs. control siRNA-transfected: P < 0.0001), phospho-p65/p65 (p65-specific siRNA-transfected vs. untransfected: P < 0.0001; p65-specific siRNA-transfected vs. control siRNA-transfected: P < 0.0001) and CXCR4 (p65-specific siRNA-transfected vs. untransfected: P < 0.0001; p65-specific siRNA-transfected vs. control siRNA-transfected: P < 0.0001) in iNSCs of the p65-specific siRNA-transfected co-culture group, indicating that p65 could regulate CXCR4 expression in iNSCs co-cultured with activated microglia (Fig. 6J-M).

Additionally, ELISA showed that p65 inhibition markedly increased the levels of CXCL12 (p65-specific siRNA-transfected vs. untransfected: P = 0.0006; p65-specific siRNA-transfected vs. control siRNA-transfected: P = 0.0001) and TNF-α (p65-specific siRNA-transfected vs. untransfected: P = 0.0350; p65-specific siRNA-transfected vs. control siRNA-transfected: P = 0.0013) and decreased the levels of IGF-1 (p65-specific siRNA-transfected vs. untransfected: P = 0.0018; p65-specific siRNA-transfected vs. control siRNA-transfected: P = 0.0263) in the supernatant of the co-culture (p65 siRNA) (iNSCs treated with p65-specific siRNA) group (Fig. 6N-P).

To determine the influence of p65 inhibition in iNSC grafts on neuroinflammation in the injured cortices of CHI mice, we performed western blot analysis and found that in contrast to the PBS group, iNSC grafts substantially decreased the levels of CXCL12 (P < 0.0001), TNF-α (P < 0.0001), phospho-p65/p65 (P < 0.0001) and phospho-IκBα/IκBα (P < 0.0001) and increased the levels of IGF-1 (P < 0.0001) in the injured cortices of CHI mice of the iNSC (control siRNA) group (Additional Fig. 11). Remarkably, p65 inhibition in iNSC grafts restored the levels of CXCL12 (p65 vs. control: P = 0.0002), TNF-α (p65 vs. control: P = 0.0028), IGF-1 (p65 vs. control: P < 0.0001), phospho-p65/p65 (p65 vs. control: P = 0.0013) and phospho-IκBα/IκBα (p65 vs. control: P < 0.0001) in the injured cortices of CHI mice of the iNSC (p65 siRNA) group.

Together, these data indicated that NF-κB inhibition exerted significant effects on the Malat1/miR-139-5p/Cxcr4 axis to attenuate the immunoregulatory effects of iNSCs on microglial activation. Moreover, NF-κB inhibition could block the immunoregulatory effects of iNSC grafts on neuroinflammation in the injured cortices of CHI mice.

TNFR1 inhibition reduces the levels of Malat1 and CXCR4 expression as well as NF-κB activation in iNSCs

Considering that the non-contact interaction between iNSCs and LPS-activated microglia was mainly mediated by cytokines, chemokines and their receptors, such as CXCL12/CXCR4 as we reported previously [11], and NF-κB activation was closely correlated to TNF-α and its receptors, we performed qRT-PCR and found that Tnfr1 levels in iNSCs were significantly higher in the co-culture group than in the control group (data not shown). To study the role of TNFR1 in regulating the Malat1/miR-139-5p/Cxcr4 axis in iNSCs, we transfected iNSCs with TNFR1-specific siRNA or control siRNA prior to the co-culture studies. QRT-PCR showed that Tnfr1 levels in iNSCs of the TNFR1-specific siRNA-transfected (iNSCs treated with TNFR1-specific siRNA) co-culture group were markedly lower than those in the untransfected (iNSCs treated with PBS) and control siRNA-transfected (iNSCs treated with control siRNA) co-culture groups (TNFR1-specific siRNA-transfected vs. untransfected: P < 0.0001; TNFR1-specific siRNA-transfected vs. control siRNA-transfected: P < 0.0001) (Fig. 7A). Western blot analysis also demonstrated TNFR1 inhibition in iNSCs of the TNFR1-specific siRNA-transfected co-culture group (data not shown), suggesting effective knockdown of TNFR1 expression in these iNSCs. Notably, TNFR1 inhibition significantly decreased the levels of Malat1 (TNFR1-specific siRNA-transfected vs. untransfected: P = 0.0037; TNFR1-specific siRNA-transfected vs. control siRNA-transfected: P = 0.0342) and Cxcr4 (TNFR1-specific siRNA-transfected vs. untransfected: P = 0.0002; TNFR1-specific siRNA-transfected vs. control siRNA-transfected: P = 0.0006) and increased the levels of miR-139-5p (TNFR1-specific siRNA-transfected vs. untransfected: P = 0.0012; TNFR1-specific siRNA-transfected vs. control siRNA-transfected: P = 0.0003) in iNSCs of the TNFR1-specific siRNA-transfected co-culture group, suggesting that TNFR1 could regulate the Malat1/miR-139-5p/Cxcr4 axis in iNSCs co-cultured with activated microglia (Fig. 7B-D).

We then performed western blot analysis and demonstrated that TNFR1 inhibition substantially decreased the levels of TNFR1 (TNFR1-specific siRNA-transfected vs. untransfected: P < 0.0001; TNFR1-specific siRNA-transfected vs. control siRNA-transfected: P < 0.0001), phospho-p65/p65 (TNFR1-specific siRNA-transfected vs. untransfected: P < 0.0001; TNFR1-specific siRNA-transfected vs. control siRNA-transfected: P < 0.0001), phospho-IκBα/IκBα (TNFR1-specific siRNA-transfected vs. untransfected: P < 0.0001; TNFR1-specific siRNA-transfected vs. control siRNA-transfected: P < 0.0001) and CXCR4 (TNFR1-specific siRNA-transfected vs. untransfected: P = 0.0005; TNFR1-specific siRNA-transfected vs. control siRNA-transfected: P = 0.0018) in iNSCs of the TNFR1-specific siRNA-transfected co-culture group, indicating that TNFR1 could regulate NF-κB activation and CXCR4 expression in iNSCs co-cultured with activated microglia (Fig. 7E-I).

Notably, flow cytometry analysis revealed that TNFR1 inhibition markedly decreased the levels of TNFR1 (TNFR1-specific siRNA-transfected vs. untransfected: P = 0.0013; TNFR1-specific siRNA-transfected vs. control siRNA-transfected: P = 0.0034) and CXCR4 (TNFR1-specific siRNA-transfected vs. untransfected: P = 0.0010; TNFR1-specific siRNA-transfected vs. control siRNA-transfected: P = 0.0025) in iNSCs of the TNFR1-specific siRNA-transfected co-culture group, suggesting that TNFR1 could regulate the expression of CXCR4 at the surface of iNSCs co-cultured with activated microglia (Fig. 7J-M).

To determine the influence of TNFR1 inhibition in iNSC grafts on neuroinflammation in the injured cortices of CHI mice, we also performed western blot analysis and found that in contrast to the PBS group, iNSC grafts substantially decreased the levels of CXCL12 (P < 0.0001), TNF-α (P < 0.0001), phospho-p65/p65 (P < 0.0001) and phospho-IκBα/IκBα (P < 0.0001) and increased the levels of IGF-1 (P < 0.0001) in the injured cortices of CHI mice of the iNSC (control siRNA) group (Additional Fig. 12). Remarkably, TNFR1 inhibition in iNSC grafts restored the levels of CXCL12 (TNFR1 vs. control: P < 0.0001), TNF-α (TNFR1 vs. control: P < 0.0001), IGF-1 (TNFR1 vs. control: P < 0.0001), phospho-p65/p65 (TNFR1 vs. control: P < 0.0001) and phospho-IκBα/IκBα (TNFR1 vs. control: P < 0.0001) in the injured cortices of CHI mice of the iNSC (TNFR1 siRNA) group.

In short, these data indicated that TNFR1 inhibition exerted significant effects on the Malat1/miR-139-5p/Cxcr4 axis to attenuate the immunoregulatory effects of iNSCs on microglial activation (Additional Fig. 13). Moreover, TNFR1 inhibition could block the immunoregulatory effects of iNSC grafts on neuroinflammation in the injured cortices of CHI mice.

Discussion

Microglia can play pivotal and complex roles in neuroinflammation-mediated neuronal insult and repair following CHI [2, 20]. Hence, it is important for the development of novel therapies that may self-perceive the changes of the activated state of microglia and thus make prudential regulation of the neuroinflammation to reduce secondary damage and promote neurological recovery after the initial injury. The transplantation of iNSCs, which may restore neural function via cell replacement and immune modulation, holds promise as a potential treatment for CHI [4, 12, 13]. We previously reported that engrafted iNSCs can regulate secretory products of resident activated microglia by overexpression of CXCR4 to neutralize CXCL12 secreted by microglia and block the effects of CXCL12/CXCR4 signaling on NF-κB activation in these microglia [11]. In this study, we investigated the mechanism of CXCR4 upregulation in iNSCs and found that Malat1 was the most upregulated lncRNA in iNSCs after co-cultured with LPS-activated microglia. Malat1 was reported to be elevated and correlate with microglial activation, neuroinflammation-mediated neuronal injury and repair in various neuropathologic disorders such as brain ischemia/reperfusion injury, spinal cord injury and Parkinson’s disease[1, 38, 45]. Subsequently, we demonstrated the co-expression relationship between Malat1 and Cxcr4 and discovered that Malat1 could act as a sponge of miR-139-5p to modulate CXCR4 expression in iNSCs co-cultured with activated microglia, which was partially consistent with previous researches suggesting that Malat1 could regulate CXCR4 expression by functioning as a ceRNA of miRNAs such as miR-146a and miR-204 and that miR-139-5p could target CXCR4 directly and reduce its expression [27, 32, 36].

In particular, we performed gain- and loss-of-functional studies to identify the role of Malat1 and miR-139-5p and revealed that Malat1 could serve as a sponge of miR-139-5p to modulate the expression of CXCR4 that exerted significant influence on the immunoregulatory effects of iNSCs on microglial activation and secretion of CXCL12, TNF-α and IGF-1. Similar to previous studies that CXCR4 was negatively regulated by miR-139-5p in endothelial cells, our findings indicated that miR-139-5p could directly target CXCR4 and suppress its expression in iNSCs [27]. In addition, we first reported that Malat1 could act as an upstream regulator of the miR-139-5p/Cxcr4 axis in regulating the interplay between iNSCs and microglia. Based on the novel observation that Malat1 may modulate the cross-talk between iNSCs and microglia via miR-139-5p/Cxcr4 axis, we detected the influence of Malat1 inhibition in iNSC grafts on microglial activation in the injured cortices of CHI mice. In accordance with the in vitro functional results, Malat1 inhibition blocked the immunoregulatory effects of iNSC grafts on microglial activation as well as neuroinflammation leading to the elevated levels of pro-inflammatory mediators (including TNF-α and CXCL12) and the activation of NF-κB while the decreased levels of IGF-1 in the injured cortices of CHI mice. Moreover, Malat1 inhibition reduced the levels of CXCL12 co-localized to iNSC grafts, suggesting that Malat1 could regulate CXCR4 expression in iNSC grafts. Therefore, this is the first study, to our knowledge, to show the role of the Malat1/miR-139-5p/Cxcr4 axis in regulating the interaction between iNSCs and microglia following CHI.

Additionally, we noted that several lncRNAs were reported to modulate target gene expression via interacting with NF-κB signaling and NF-κB p65 was found to directly bind to the promoter of Malat1 and promote its transcription [7, 23, 44]. Here we determined a significant enhancement of phosphorylation and nuclear translocation of p65 that could work as an upstream regulator of the Malat1/miR-139-5p/Cxcr4 axis in regulating the cross-talk between iNSCs and microglia. Interestingly, in contrast to previous findings that iNSCs attenuated neuroinflammation by inhibiting NF-κB signaling in activated microglia as well as the injured cortices of CHI mice, our data implied that NF-κB activation in iNSCs augmented the immunoregulatory effects of iNSCs on microglial activation by activating the axis of Malat1/miR-139-5p/Cxcr4. In contrast, NF-κB inhibition blocked the immunoregulatory effects of iNSC grafts on neuroinflammation in the injured cortices of CHI mice.

To clarify the mechanism underlying the activation of NF-κB signaling in iNSCs, we further explored the functions of cytokines, chemokines and their receptors and uncovered that TNF-α secreted by activated microglia could bind to TNFR1 at the surface of iNSCs to trigger NF-κB activation in iNSCs. Moreover, the role of TNF-α/TNFR1 signaling in regulating the expression of CXCR4 at the surface of iNSCs was confirmed by the knockdown of TNFR1. Hence, TNF-α/TNFR1 signaling had positive effects on NF-κB activation and CXCR4 expression in iNSCs. Remarkably, TNFR1 inhibition also could block the immunoregulatory effects of iNSC grafts on neuroinflammation in the injured cortices of CHI mice. As described above, NF-κB activation increased the levels of Malat1, which could act as a sponge of miR-139-5p to modulate CXCR4 expression in iNSCs. Moreover, CXCR4 expressed at the surface of iNSCs could neutralize CXCL12 secreted by activated microglia to block the effects of CXCL12/CXCR4 signaling on NF-κB activation in these microglia as we previously reported [11]. Therefore, the interplay among TNF-α/TNFR1, NF-κB, Malat1/miR-139-5p/Cxcr4 and CXCL12/CXCR4 signaling pathways may constitute a system to regulate the interaction between iNSCs and microglia.

This study had several limitations. For example, some scholars reported that Malat1 could modulate NF-κB DNA-binding activity to reduce the production of pro-inflammatory mediators (including NF-κB-dependent TNF-α) by directly binding to NF-κB p65 in the nucleus of LPS-activated macrophages [44]. Whether Malat1 interacts with NF-κB and functions as a feedback regulator of NF-κB signaling in iNSCs co-cultured with LPS-activated microglia remains unclear. Furthermore, the influence of the interaction among TNF-α/TNFR1, NF-κB, Malat1/miR-139-5p/Cxcr4 and CXCL12/CXCR4 signaling pathways on the biological behavior of iNSCs such as neurogenesis and neurotrophy as well as the neurological recovery of CHI mice is not fully elucidated.

Further studies exploring the interaction between Malat1 and NF-κB signaling in iNSCs are expected to clarify the mechanism underlying the immunomodulatory effects of iNSCs on microglial activation and neuroinflammation following CHI. Moreover, the development of advanced methods may help identify the effect of the interaction among TNF-α/TNFR1, NF-κB, Malat1/miR-139-5p/Cxcr4 and CXCL12/CXCR4 signaling pathways on the biological behavior of iNSCs and the neurological recovery of CHI mice.

Conclusions

Overall, our results show that Malat1 may work as a ceRNA of miR-139-5p, which directly targets Cxcr4 and modulate CXCR4 expression in iNSCs. In addition, the axis of Malat1/miR-139-5p/Cxcr4 may exert significant influence on the immunoregulatory effects of iNSCs on the secretion of CXCL12, TNF-α and IGF-1 by activated microglia. Furthermore, our findings reveal a novel role of Malat1 in the immunomodulatory effects of iNSCs on microglial activation and neuroinflammation following CHI. Lastly, the interplay among TNF-α/TNFR1, NF-κB, Malat1/miR-139-5p/Cxcr4 and CXCL12/CXCR4 signaling pathways may constitute a system to regulate the cross-talk between iNSCs and microglia, suggesting that transplanted iNSCs may self-perceive the changes of the activated state of microglia and thus make prudential regulation of the neuroinflammation after CHI. Hence, iNSC-based therapy holds promise in the treatment of CHI, having played these important roles with its peculiarity.

Data availability

All relevant data are within the paper and its Additional files.

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Acknowledgements

We sincerely thank Dr. Hui Yao, Yan Zhang, Cuiying Wu, Kai Sun and Ning Liu (Chinese PLA General Hospital) for helpful advice and technical support. The authors declare that they have not used Artificial Intelligence in this study.

Funding

This work was supported by grants from the National Natural Science Foundation of China, Nos. 82271397 (to MG), 82001293 (to MG), 82171355 (to RX), 81971295 (to RX) and 81671189 (to RX) and the Medical Science Research Project Plan of the Hebei Provincial Health Commission, Nos. 20250191 (to MG) and 20251064 (to ZY).

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  1. Qin Dong, Pengyu Chen, Wenqiao Qiu, Zhijun Yang and Yanteng Li contributed equally to this work.

Authors and Affiliations

  1. Department of Neurology, Fu Xing Hospital, Capital Medical University, Beijing, 100038, China

    Qin Dong

  2. College of Traditional Chinese Medicine, Fujian University of Traditional Chinese Medicine, Fuzhou, Fujian Province, 350122, China

    Pengyu Chen

  3. Department of Neurosurgery, Sichuan Provincial People’s Hospital, School of Medicine, Sichuan Academy of Medical Sciences, University of Electronic Science and Technology of China, Chengdu, Sichuan Province, 610072, China

    Wenqiao Qiu, Zhijun Yang, Yuhui Zhou, Lili Guo, Dan Zou & Ruxiang Xu

  4. Hebei Leren Biotechnology Co., LTD, Shijiazhuang, Hebei Province, 050000, China

    Zhijun Yang

  5. Department of Neurosurgery, The First Medical Centre, Chinese PLA General Hospital, Beijing, 100853, China

    Yanteng Li & Mou Gao

  6. Chuanxing Middle School, Xichang, Sichuan Province, 615000, China

    Yuhui Zhou

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Contributions

QD conducted cell transplantation experiments and performed bioinformatic and statistical analysis. PC performed cell transfection assay. WQ and ZY performed western blot analysis. YL performed dualluciferase reporter assay. YZ performed morphological analysis. LG and DZ performed flow cytometry. RX designed and revised the manuscript. MG conducted cell culture and transplantation experiments and wrote the manuscript. All the authors read and approved the manuscript.

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(1) Title of the approved project: Effect and mechanism of lincRNA on the safety of induced neural stem cell transplantation; (2) Name of the institutional approval committee: Research Ethics Committee at Chinese PLA General Hospital; (3) The approval number: 2018-066; (4) The approval date: January 21, 2019.

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Dong, Q., Chen, P., Qiu, W. et al. Long non-coding RNA Malat1 modulates CXCR4 expression to regulate the interaction between induced neural stem cells and microglia following closed head injury. Stem Cell Res Ther 16, 31 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13287-024-04116-1

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Stem Cell Research & Therapy

ISSN: 1757-6512