Unveiling the conversion mechanism of glioblastoma from the mesenchymal to the proneural subtype driven by HDAC1/p-SMAD3-TP53I11 axis

doi:10.21203/rs.3.rs-4733991/v1

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Unveiling the conversion mechanism of glioblastoma from the mesenchymal to the proneural subtype driven by HDAC1/p-SMAD3-TP53I11 axis

https://doi.org/10.21203/rs.3.rs-4733991/v1

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Mesenchymal glioblastoma (MES GBM) is characterized by rapid proliferation, extensive invasion, and formidable treatment resistance. Addressing these characteristics in MES cells is crucial for improving patient prognosis. Here, we discovered the MES GBM subtype conversion mechanism driven by HDAC1/p-SMAD3-TP53I11 in this study. First, the impact of HDAC1 inhibitors and bevacizumab on the phenotypic characteristics of MES cells was assessed. Co-immunoprecipitation (Co-IP) and immunofluorescence techniques elucidated the epigenetic mechanism of HDAC1. Chromatin immunoprecipitation sequencing (ChIP-seq) and RNA-seq identified downstream transcribed genes. We found Inhibition or knockdown of HDAC1 transformed MES characteristics into proneural (PN) characteristics, prolonged survival in patient-derived xenograft (PDX) models, and suppressed in vitro cell proliferation and invasion. RG2833 (an HDAC1 inhibitor) was found to enhance histone acetylation, promoting the binding of the transcription factor p-SMAD3 to the genome. Immunoprecipitation experiments revealed an interaction between p-SMAD3 and HDAC1. RNA-seq and ChIP-seq data analysis from MES cell lines before and after RG2833 treatment identified Tumor Protein P53 Inducible Protein 11 (TP53I11) as a downstream gene. The study indicates that by intervening HDAC1/p-SMAD3-TP53I11, HDAC1 can serve as a promising therapeutic target for the treatment of MES GBM.

MES GBM

PN GBM

HDAC1

TP53I11

subtype conversion

HDAC1 is highly expressed in MES GBM cells and silencing it weakens the MES characteristics of GBM.
HDAC1 combined with bevacizumab significantly inhibit the proliferation and invasion of MES GBM cells.
HDAC1/p-SMAD3 complex plays a role in transcriptional regulation through histone acetylation.
Inhibition of HDAC1 enhances the p-SMAD3 binding genome and thus promotes TP53I11 expression.

The standard therapeutic approach for adult glioblastoma (GBM) patients involves maximal safe resection, complemented by radiotherapy, chemotherapy, targeted pharmacotherapy, and tumor-treating fields (TTFields). Despite these interventions, the median survival time for adult patients with GBM remains just 14.7 months [1, 2]. Intratumoral transcriptomic heterogeneity significantly contributes to the refractoriness of GBM. Advances in transcriptome analysis have led to the classification of GBM tumor cells into three distinct subtypes: classical (CL), mesenchymal (MES), and proneural (PN). The PN subtype exhibits a better prognosis and therapeutic responsiveness compared to the MES subtype [3–5]. Similar to epithelial-mesenchymal transition (EMT), proneural-mesenchymal transition (PMT) facilitates invasion, proliferation, and treatment resistance in GBM [6, 7]. Given the heterogeneous nature of tumors wherein almost all GBMs contain three distinct cell types whose proportions determine patient prognosis [3], it is hypothesized that promoting transformation of MES cells into PN cells within tumor tissues may extend overall patient survival.

The unstructured N-termini of core histones are characterized by a high abundance of lysine residues, which naturally carry a positive charge and exhibit an affinity for the negatively charged backbone of DNA. Acetylation neutralizes this positive charge, reducing histone-DNA interaction strength and enhancing chromatin accessibility for transcription, which is crucial in epigenetics [8]. Histone deacetylases (HDACs) form a class of co-regulated proteins that function as complexes. typically interact with transcription factors to exert its role in transcriptional regulation. These complexes enable HDACs to bind specific genomic sites, modulating the process of gene expression from DNA to mRNA. HDAC1, a key member, is extensively involved in gene repression through hypoacetylation of local chromatin domains. Epigenetic therapy strategies targeting HDAC1 have been widely reported, and numerous molecular inhibitors against HDAC1 have been developed and assessed in experimental studies across various cancers. Clinical trials have shown therapeutic effects in treating lymphoma, hematological tumors, breast cancer, and gastrointestinal tumors [9, 10]. HDAC1 has been found to promote the malignant progression of glioma and participate in the EMT process [11, 12]. The investigation focused on the molecular heterogeneity of GBM multiforme, revealing HDAC1's involvement in the subtype conversion and intrinsic epigenetic mechanisms of MES and PN subtypes.

Cell lines, cell culture conditions and GBM samples.

Human glioma cells U251, purchased from ATCC (American Type Culture Collection, Manassas, VA, USA), were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Hyclone, Logan, UT, USA). The acquisition methods for the N9 cell line, TBD0207B, and TBD0220L have been described previously [13]. These cells were grown in DMEM/F12 (1:1; Gibco BRL, Rockville, MA) with 10% FBS. All cell cultures were maintained at 37°C in a 5% CO₂ atmosphere. Additionally, data from 78 GBM samples, including information on known mutations, mRNA expression levels, and subtype classification, were obtained from TCGA (Affymetrix U133A).

GBM tissues and patient-derived cerebrospinal fluid exosomes.

After obtaining informed consent from patients and approval from the ethics committee in accordance with the ethical standards of the 2008 Helsinki Declaration, tumor tissue and cerebrospinal fluid samples were randomly selected from four patients with GBM who underwent surgical resection (Table S1). Tumor grades were determined according to the 2021 World Health Organization (WHO) classification of nervous system tumors.

Exosomes were isolated using a micro exosome total RNA extraction kit (Absin, China). Following the completion of washing, digestion, and elution processes, the total RNA solution was obtained through multiple rounds of vigorous shaking, centrifugation, and adsorption.

SiRNA, reagents, plasmids, Lentiviru and transfection

The siHDAC1 and siTP53I11 was synthesized in Ribobio (Guangzhou, China) (Table S2). The open reading frames (ORFs) of HDAC1 and SMAD3 were cloned with a C-terminal Flag into pENTER (Vigenebio, China). We transfected them into cells using Lipofectamine 3000 (Invitrogen, USA) according to the manufacturer’s protocol. Lentiviral shRNA plasmids targeting TP53I11 were constructed from Genechem (Shanghai, China). Before transfection, adherent cells were seeded at a density of 1×10^5/well in 24-well plates 18–24 hours prior. The next day, 2 ml of fresh culture medium containing 6 µg/ml polybrene was added to replace the original culture medium, followed by the addition of an appropriate amount of virus suspension. After 24 hours, fresh culture medium was added to replace the medium containing the virus, and fluorescent expression could be seen 48 hours after transfection.

RNA sequencing and ChIP sequencing

For RNA sequencing, total RNA was isolated from N9 cells treated with either DMSO or RG2833 (Selleck, Shanghai, China) using TRIzol (Takara, Japan) according to the manufacturer’s protocol. Subsequently, cDNA libraries were prepared and sequenced on the Illumina HiSeq4000 platform. The Hisat2 tool was used to align sequencing data to the human reference genome (hg19).

The species was identified as Homo sapiens_Ensembl_GRCm38.104 in ChIP-seq. The sequencing method employed was Illumina PE150, generating 9G of sequencing data. Samples were divided into six groups (control_1, control_2, control_input, RG2833_1, RG2833_2, and RG2833_input). Initially, nuclear DNA was crosslinked with proteins, followed by cell lysis and DNA fragmentation via sonication. The DNA was then immunoprecipitated with anti-p-SMAD3 (#9520, Cell Signaling Technology, USA) to specifically recognize the target protein. Finally, the DNA was purified, a second-generation library was constructed, and sequencing was performed.

Differentially expressed genes were identified using the DESeq2 R package. Gene Ontology (GO) analyses were conducted using DAVID (https://david.ncifcrf.gov/).

Western blotting analysis and Co-IP

Analysis was performed as described in the Supplementary materials and methods.

Immunohistochemistry, HE staining, and Quantitative real-time PCR

Analysis was performed as described in the Supplementary materials and methods.

Immunofluorescence and confocal imaging

Confocal microscopy was performed as previously described [14]. Confocal images of the cells and correlation analyses of protein localization were acquired using a confocal microscope (FV500) with FluoView software (Olympus).

Cell proliferation and Transwell assay

Analysis was performed as described in the Supplementary materials and methods.

ChIP

Cells were harvested for ChIP using an EZ-ChIP Kit (Millipore) according to the manufacturer’s protocol. Chromatin was extracted, and cross-linked DNA was fragmented into segments of approximately 200 to 1000 base pairs. Protein G agarose was added to the antibody/chromatin complexes, and the mixture was incubated overnight at 4°C. A p-SMAD3 antibody was used to pull down DNA from formaldehyde cross-linked chromatin. The protein G agarose antibody/chromatin complexes were resuspended in a wash buffer and centrifuged to collect the protein/DNA complexes. The protein/DNA cross-links were cleaved to yield free DNA, which was then quantified using qPCR.

Animals

All animal experiments were conducted in accordance with the ethical obligations and animal care guidelines approved by Qingdao University. Based on the previous description [13], patient-derived xenografts (PDX) were constructed from TBD0220L cells. The PDX tissues were implanted into the cranial region of nude mice using skull-guided screws under stereotactic guidance, and the overall survival time of the mice was monitored. Starting from the second day after transplantation, the treatment group received oral administration of RG2833 at a dose of 150 mg/kg every other day until death. The growth of intracranial tumors was monitored using bioluminescence imaging on days 7, 14, and 21. After death, the brains were carefully removed and fixed in 10% formaldehyde.

Stable transfection of sh-TP53I11 and control lentivirus in U251 cells was achieved following the manufacturer’s protocol. These transfected cells were then implanted into the cranial region of nude mice using skull-guided screws. Twenty days later, the mice were euthanized, and the size and weight of the intracranial tumors were measured before being placed in 10% formaldehyde.

Statistical analysis

The figures and figure legends provide detailed statistical parameters, including definitions and precise values of n, the statistical tests used, and the level of statistical significance. The chi-squared test assessed significant differences between two groups. Kaplan–Meier survival plots were used to generate survival curves, with significance determined by the log-rank test. Two-sided Pearson’s correlation tests evaluated correlations between tissues. Statistical significance for functional analyses was determined using Student’s t-test or ANOVA. Statistical analyses were conducted using Prism 8.0 software, with p-values < 0.05 considered statistically significant.

The expression of HDAC1 is significantly elevated in the MES subtype GBM and shows a positive correlation with the expression of MES-specific genes.

To assess the variation in HDAC1 expression across different GBM subtypes, TCGA GBM data were analyzed, revealing significantly elevated mRNA expression in the MES subtype and reduced expression in the PN subtype (Fig. 1A). Previously validated MES subtype GBM cell line TBD0220L/N9 and PN subtype GBM cell line TBD0207B/U251 [13] were used in this study. HDAC1 mRNA expression was notably higher in TBD0220L than in TBD0207B (Fig. 1B) and in N9 compared to U251 (Fig. 1C), with similar results for HDAC1 protein expression (Fig. 1D). Characteristic genes for each subtype were evaluated using Verhaak RG classification data (Table S5). Cerebrospinal fluid specimens from four patients with GBM (Table S1) were analyzed to evaluate the correlation between HDAC1 mRNA and subtype marker gene. HDAC1 expression positively correlated with the MES subtype marker gene MMP7 and negatively with the PN subtype marker gene HOXD3 (Fig. 1E). Immunofluorescence detection confirmed a positive correlation between HDAC1 and MMP7 expression (Fig. 1F). To inhibit HDAC1, siHDAC1 was designed to prevent protein synthesis, and an HDAC1 inhibitor (RG2833) was used to block its biological function (Supplementary Fig. 1). Both methods decreased MES subtype marker gene expression and increased PN subtype marker gene expression (Fig. 1G-H). TCGA data analysis further revealed a positive correlation between HDAC1 and MES subtype marker genes and a negative correlation with PN subtype marker genes (Fig. S2).

The in vivo experiments conducted on PDX models demonstrated that RG2833 is effective in treating MES subtype GBM, inducing the transformation of the MES subtype to the PN subtype.

First, after intracranial transplantation of heterologous MES GBM tissue in nude mice, RG2833 was administered by gavage, demonstrating its inhibitory effect on MES GBM proliferation and its ability to extend the survival time of nude mice (Fig. 2A-C). Immunohistochemical experiments were conducted using paraffin sections of intracranial tumor tissue, and mRNA from the tumor tissue was extracted for q-PCR analysis. The HDAC1 inhibitor reduced the expression of MES subtype marker genes and increased the expression of PN subtype marker genes in vivo (Fig. 2D-E).

The combination of RG2833 and bevacizumab inhibited the proliferation and invasion of MES GBM cells.

Analysis of survival data from 52 patients with GBM using the TCGA database revealed that MES subtype patients had a poorer prognosis and shorter median survival compared to those with PN and CL subtypes (Fig. S3A-B). This finding prompted an exploration of therapeutic strategies aimed at transforming the poorly prognostic MES subtype into other subtypes. The PN subtype, for example, shows an effective therapeutic response to bevacizumab [15]. Our experiments demonstrated that bevacizumab inhibited the proliferation and invasion of the PN subtype cell line U251 in vitro (Fig. 3A-C). Although bevacizumab had no significant effect on MES cell lines, RG2833 significantly inhibited their proliferation and invasion. Additionally, combining RG2833 with bevacizumab had a more pronounced inhibitory effect on the proliferation and invasion of the N9 cell line (Fig. 3D-F). Western blotting analysis was conducted to investigate the protein expression of MES marker genes following bevacizumab treatment in N9 cells, revealing no significant changes. However, the combination of bevacizumab and RG2833 significantly reduced the protein expression of MES marker genes. These changes were mirrored at the mRNA level, showing a consistent trend with the protein expression (Fig. 3G-H).

Perform differential analysis of RNA-seq data for MES cells before and after treatment with RG2833.

To investigate the molecular mechanism by which RG2833 induces subtype conversion, the N9 cell line was divided into a control group and an RG2833 treatment group, each with two replicates. Differential gene analysis via clustering identified the 40 genes with the most significant differences, all with p-values less than 0.0001 (Fig. 4A). The intersection of identified genes (p < 0.05) between replicates revealed gene overlap rates of 83% and 87% in the control and RG2833 treatment groups, respectively (Fig. 4B). Volcano plot analysis of overlap genes indicated that RG2833 treatment significantly upregulated 833 genes, downregulated 575 genes, and left 15,340 genes unchanged (p-adjust value of 0.05 and log fold change (log FC) of 1) (Fig. 4C). GO analysis linked the differentially expressed genes to cell growth, death, and cancer-related pathways (Fig. 4D).

HDAC1 interacts with p-SMAD3, and RG2833 induces alterations in the subcellular localization of HDAC1.

Immunofluorescence experiments using specific antibodies revealed significant co-localization of HDAC1 and p-SMAD3 in TBD0220L cells, indicating a strong correlation between these proteins (Fig. 5A). In the N9 cell line, Co-IP assays demonstrated a reciprocal recruitment interaction between HDAC1 and p-SMAD3 (Fig. 5B). Additionally, we used antibodies against H2AK5ac, H2BK5ac, H3K9ac, and H3K27ac to assess changes in histone acetylation following RG2833 treatment. The results showed increased acetylation levels within the nucleus (Fig. 5C). Furthermore, RG2833 treatment altered HDAC1 protein distribution, decreasing its presence in the nucleus while increasing its presence in the cytoplasm (Fig. 5D). Intracellular immunofluorescence staining of TBD0220L cells showed consistent alterations, confirming the observed trends (Fig. 5E).

By inhibiting HDAC1 to block histone acetylation leads to the recruitment of p-SMAD3 to the genome, thereby influencing transcription in MES GBM cells.

The N9 cell line was used for ChIP-seq experiments, enriching proteins with the p-SMAD3 ChIP-grade antibody, precipitating genomic DNA, and conducting high-throughput sequencing analysis [16]. The p-SMAD3 binding sequence was identified (https://jaspar.elixir.no/) (Fig. S4). Cell samples were divided into a DMSO control group and an RG2833 treatment group, each with two replicates. Post-treatment comparisons revealed a significant increase in p-SMAD3 enrichment around the transcription start site (TSS) following RG2833 treatment (Fig. 6A). Analysis of p-SMAD3 distribution in different coding DNA regions showed an increased distribution ratio of p-SMAD3 in the promoter region, particularly the regions within 1kb base pairs of the actual TSS after RG2833 treatment (Fig. 6B). Comparing sequencing coding gene data from the control and treatment groups, we found 89% gene overlap in the control repeated group and 93% in the treatment repeated group (Fig. 6C). Combining differential genes between the control and treatment groups with genes showing significant binding changes, we performed clustering analysis to identify genes with p-adjust values < 0.0001. The results indicated that 18 genes showed a significant decrease in binding, while 31 genes showed a significant increase in binding (Fig. 6D). In summarizing the RNA-seq results before and after RG2833 treatment (Fig. 4C), we took the intersection to identify genes with increased mRNA expression (p-adjust value < 0.05) and increased p-SMAD3 binding with ChIP-seq analysis (p-adjust value < 0.05), and obtained 107 potential downstream target genes (Fig. 6E). Targeted gene GO analysis revealed associations with axon guidance, ECM-receptor interaction, and cell adhesion molecules (Fig. S5).

TP53I11 maintains the characteristics of PN subtype GBM and inhibits the proliferation and invasion of MES GBM both in vivo and in vitro.

TP53I11 emerged as a significant downstream regulated gene cluster from our previous analyses (Fig. 4A and 6D). ChIP-qPCR experiments revealed that the p-SMAD3 antibody effectively enriched TP53I11, with enrichment levels increasing following RG2833 treatment (Fig. 7A, Table S6). Analysis of TCGA GBM patient data indicated that patients with higher TP53I11 mRNA expression in tumor tissue had significantly longer survival times compared to those with lower TP53I11 mRNA expression (Fig. 7B).

By utilizing the subtype-specific TCGA GBM data, we observed a significant elevation of TP53I11 expression in the PN subtype compared to the MES subtype at the mRNA level (Fig. 7C). Gene set enrichment analysis (GSEA) based on the Verhaak GBM PN gene set showed that patients with GBM exhibiting high TP53I11 expression had higher enrichment scores, while those with low TP53I11 expression had lower enrichment scores (Fig. 7D). Receiver operating characteristic (ROC) curve analysis of TCGA GBM data indicated that TP53I11 has high sensitivity in distinguishing PN patients from other subtypes, with an area under the curve (AUC) of 84% (Fig. 7E). TP53I11-specific interfering RNA (siRNA) was constructed and applied to the PN subtype GBM cell line U251, resulting in increased cell proliferation and invasion as shown by CCK-8 and Transwell assays (Fig. 7F-H). Additionally, a shTP53I11 lentiviral vector was developed, transfected into U251 cells, and then injected into the brains of mice to form tumors. Compared to the control group, the shTP53I11-treated group displayed larger tumor volumes and weights (Fig. 7I-L). Enhanced Ki-67 positivity, detected by immunohistochemistry, indicated increased proliferative capacity in shTP53I11-treated tumors (Fig. 7M and N).

GBM, comprising both newly diagnosed and recurrent cases, accounts for 60% of all gliomas [17]. Among these GBM patients, 98.03% are IDH wild type, while only 1.97% are IDH mutant type. In recent years, there have been significant advancements in the treatment of IDH mutation gliomas (including WHO grades 1–4), but effective breakthroughs in the treatment of IDH wild-type GBM remain elusive [18–20]. Based on transcriptome differences, GBM with IDH wild type was classified into PN, CL, and MES types [3, 21]. These subtypes exhibit distinct genetic and phenotypic characteristics, with specific mRNA clusters influencing tumor development and prognosis [22, 23]. By performing RNA-seq analysis on a particular tumor, we can determine its overall tendency towards a particular type. At a more microscopic level, we can obtain molecular subtypes for different parts of the same tumor, even different cells [24]. Our previous studies, the analysis of sequencing data from different parts of a single GBM patient's tumor revealed that in a single lesion from one patient, two of five tissue directions were classified as MES subtype, two as PN subtype, and one as CL subtype (Fig. S6A). Single cell sequencing data from four patients with glioma revealed significant variation in the proportion of cells in each subtype, with MES cells accounting for 80% in the highest case and 32% in the lowest (Fig. S6B). This observation highlights substantial mRNA heterogeneity present in glioma. This provides us with a basis for implementing treatment at the mRNA level to control the proliferation and invasion of GBM.

Analysis of TCGA GBM data and GBM tissues and cells revealed elevated HDAC1 expression in the MES subtype. This was accompanied by an increase in MES-related markers and a decrease in PN-related markers following HDAC1 downregulation. This led us to hypothesize that HDAC1 plays a pivotal role in distinguishing MES and PN types. Prior research has highlighted the effectiveness of HDAC inhibitors in treating hematological tumors [25–27]. Moreover, combining HDAC inhibitors with PD-1 monoclonal antibodies has improved overall survival in patients with solid tumors [28–30]. Building on these preliminary findings, comparative experiments using a nude mouse PDX model of mesenchymal tumor cells and selected RG2833—a potent HDAC1 inhibitor capable of crossing the blood-brain barrier [31]—were conducted. Results demonstrated significant inhibition of MES intracranial tumor growth by RG2833, along with reductions in MES-related marker expression.

Bevacizumab, a monoclonal antibody targeting VEGF, is commonly used in combination with temozolomide or lomustine/carmustine for treating adults with progressive or recurrent GBM [1, 32]. The therapeutic effects of combining bevacizumab with RG2833 on MES GBM were investigated and achieved a good therapeutic response. Given heterogeneity of GBM, the strategy of drug combination provided a significant theoretical basis for targeted GBM treatment.

Next, we conducted a prediction of potential protein interactions with HDAC1, focusing particularly on transcriptional regulatory factors. Experimental results revealed a significant interaction between HDAC1 and p-SMAD3, both highly enriched in the nuclei of GBM cells. SMAD3, a component of the transforming growth factor-beta (TGF-β) signaling pathway, transmits signals from the cell surface to the nucleus, regulating gene activity and cell proliferation [33–35]. Our study identified the co-localization and binding of HDAC1 and p-SMAD3 within the nuclei of MES subtype GBM cell lines. RG2833, a small molecule inhibitor of protease, binds to HDAC1 protein to prevent it from performing histone deacetylation, thus reducing the binding of HDAC1 and p-SMAD3 and the nuclear distribution of HDAC1. Therefore, we investigated the impact on genome transcription by conducting RNA-seq and ChIP-seq [36, 37]. Both experiments were conducted using the MES cell line N9, which was selected for its stable division and no more than 8 passages. In the RNA-seq experiment, the application of RG2833 resulted in a marked increase in the number of upregulated mRNA genes (833 vs. 575) compared to downregulated genes, indicating that inhibition of HDAC1 enhances histone acetylation, allowing chromatin increased possibility to interact with transcription factors and initiate transcriptional activation. The distribution of ChIP-seq signals near the TSS is of particular interest due to the frequent binding of transcription factors or histone modifications that serve as regulatory "markers" in the promoter region, influencing gene expression [38]. Our findings revealed a significant increase in p-SMAD3 binding at the TSS region after RG2833 treatment, particularly within the 1kb promoter region. These results suggest that RG2833 induces a relaxation of the chromatin-histone interaction, thereby facilitating the binding of p-SMAD3 to chromatin and enabling its transcriptional regulatory function. RNA-seq detected changes in mRNA levels of coding genes, while ChIP-seq annotated peaks with genes, defining the associated gene of a peak as the gene represented by the TSS closest to the peak, and detecting the changes in the enrichment fold of associated gene peaks. Differential gene analysis between these methods identified intersecting gene sets, leading to the identification of TP53I11, a key gene potentially involved in the MES to PN subtype transition. TP53I11, also known as p53-Induced Gene 11 Protein (PIG11), located on chromosome 11p11.2, was involved in apoptosis induced by p53 [39–42]. TP53I11 also suppresses epithelial-mesenchymal transition and metastasis in breast cancer cells [43]. The study identifies HDAC1 as a novel target for MES subtype GBM cells. The small-molecule inhibitor RG2833 effectively induces the transition of GBM from MES to PN subtype by modulating the HDAC1/p-SMAD3-TP53I11 axis.

Data availability

All data generated or analyzed during this study are available within the article or from the corresponding author upon request.

Funding

This work was supported by National Natural Science Foundation of China (NO. 82002996).

Authors' contributions

KZ supervised this work and designed the research. HZ and RS performed the analyses. HZ, RS, HL, CW, MZ and WC performed the experiments. HZ and KZ wrote the manuscript. All authors read and approved the final manuscript.

Ethics declarations

Competing interests

The authors declare no competing interests.

Ethics approval and consent

The study was performed on account of the Declaration of Helsinki.

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Unveiling the conversion mechanism of glioblastoma from the mesenchymal to the proneural subtype driven by HDAC1/p-SMAD3-TP53I11 axis

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Abstract

Figures

Facts

Introduction

Materials and methods

SiRNA, reagents, plasmids, Lentiviru and transfection

RNA sequencing and ChIP sequencing

Western blotting analysis and Co-IP

Immunohistochemistry, HE staining, and Quantitative real-time PCR

Immunofluorescence and confocal imaging

Cell proliferation and Transwell assay

ChIP

Animals

Statistical analysis

Results

Discussion

Declarations

References

Additional Declarations

Supplementary Files

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