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BY 4.0 license Open Access Published by De Gruyter February 8, 2023

Hypoxia-inducible factor 1α (HIF-1α)-activated Gli1 induces invasion and EMT by H3K4 methylation in glioma cells

  • Yihai Lin and Zhangyi Wu EMAIL logo
From the journal Oncologie

Abstract

Objectives

Gliomas are highly aggressive neuroepithelial-layer malignancies. Hypoxia-inducible factor 1α (HIF-1α) was revealed to be upregulated in gliomas under hypoxia. Nevertheless, its role in glioma cells remains elusive. We attempted to clarify the molecular mechanism of HIF-1 underlying glioma.

Methods

Cellular models were established to mimic the characteristics of hypoxia. RT‒qPCR was used to detect HIF-1α and Gli1 levels in glioma cells with or without hypoxic treatment. Transwell assays were used to measure the invasive ability of U87 and U251 cells. Western blotting was used to evaluate epithelial-mesenchymal transition (EMT)-associated protein abundance and H3K4 methylation (H3K4me)-associated protein abundance in U87 and U251 cells. ChIP assessed the association of HIF-1α or H3K4me with the Gli1 promoter in glioma cells.

Results

HIF-1α and Gli1 were upregulated in glioma cells relative to normal human astrocytes (NHAs). HIF-1α and Gli1 were also upregulated in hypoxia-treated glioma cells relative to untreated glioma cells. Both HIF-1α and Gli1 silencing suppressed glioma invasion and EMT under hypoxia. HIF-1α upregulated Gli1 transcriptionally via MLL1-mediated H3K4me. H3K4me mutation silencing was further demonstrated to suppress glioma cell invasion and EMT under hypoxia.

Conclusions

Both HIF-1α and Gli1 are upregulated in glioma cells and function as oncogenes in glioma cells. HIF-1α transcriptionally activates Gli1 via MLL1-mediated H3K4 methylation in glioma cells, providing ideas for seeking new therapeutic directions for glioma.

Introduction

Gliomas are highly aggressive malignancies originating from the neuroepithelial layer. The latest survey revealed that there were approximately 167,000 new cases of glioma and 121,000 deaths in China in 2017, and the trend is on the rise year by year [1]. Low-grade gliomas present a relatively high cure rate via surgery combined with chemoradiotherapy, whereas for patients with high-grade (WHO III-IV) gliomas, postoperative median survival remains low [2, 3]. Thus, clarifying the mechanism of glioma invasion and identifying molecular markers specific for glioma invasion and metastasis have crucial clinical significance for the treatment and prognosis of glioma.

Hypoxia is one of the basic features of the tumor microenvironment and a vital factor for facilitating tumor invasion and metastasis [4]. Hypoxia-inducible factor 1 (HIF-1), a major transcription factor mediating the hypoxia response, is upregulated in multiple tumors, including gliomas, and is related to glioma metastasis and prognosis [56]. HIF-1α/HIF-1β transcription complex stabilization and activation trigger downstream target genes and modulate multiple tumor biological behaviors, including cell proliferation, angiogenesis, and epithelial-mesenchymal transition (EMT) as well as tumor metastasis [7]. EMT is a process in which polar epithelial cells are converted into mesenchymal cells that are active and move freely between cell matrices, which is a major mechanism of hypoxia-triggered tumor cell invasion and migration. Its phenotypic changes include epithelial marker genes (E-cadherin, plakoglobin, etc.) expression inhibition as well as mesenchymal marker genes (N-cadherin, vimentin, etc.) expression activation [8]. HIF-1 can directly activate SNAI1 and Twist expression, triggering EMT and facilitating tumor metastasis [9]. Recently, histone deacetylase 3 (HDAC3) was identified as a direct target gene of HIF-1, and HIF-1α-activated HDAC3 under hypoxia mediates histone H3K4 deacetylation on the promoter of EMT marker genes, thus modulating the transformation of epithelial cells to a mesenchymal phenotype [10].

The zinc finger transcription factor Gli1 in the Hedgehog (HH) signaling pathway may also be involved in modulating hypoxia/HDAC3. The mammalian HH pathway is stimulated by three HH ligands, sonic hedgehog (SHH), Indian hedgehog (IHH) and desert hedgehog (DHH); HH ligand binding to receptor transmembrane protein Patched1 (PTCH1) hinders the inhibitory impact of PTCH1 on transmembrane protein SMO, releases SMO activity, and activates zinc finger ZJNSF transcription factors Gli1 and Gli2, thus modulating downstream target gene expression [11]. Recently, the association of hypoxia/HIF-1α with the HH pathway has been revealed. Hypoxia can upregulate Gli1 in breast cancer cells, and Gli1 silencing suppresses hypoxia/HIF-1-triggered EMT and tumor cell infiltration [12], [13], [14]. Hypoxia can activate HDAC3, which induces histone H3K4 deacetylation modification on the Gli1 gene promoter, while transcriptional regulation of genes through histone modification is usually the result of the combined impact of deacetylation modification and methylation modification [15, 16]. Hypoxia-triggered EMT elevates Gli1 levels in tumors such as liver cancer and is closely related to disease staging and prognosis [17], [18], [19]. Nevertheless, whether hypoxia modulates Gli1 levels in glioma by inducing H3K4 methylation (H3K4me) has not been demonstrated. We hypothesized that hypoxia/HIF-1 may activate the HH pathway (the key molecule Gli1) by inducing H3K4me.

Herein, we attempted to clarify the mechanism by which hypoxia/HIF-1 modulates Gli1 in glioma cells, which may provide ideas for seeking new therapeutic directions for glioma.

Materials and methods

Cell lines, reagents, and antibodies

Glioma cell lines (A172, U87, U251 and Hs683) as well as normal human astrocytes (NHAs) from Cell Bank, Chinese Academy of Sciences (Shanghai, China); DMEM from Thermo Fisher Scientific (Cleveland, OH, USA); FBS from Gibco (Carlsbad, CA, USA); Lipofectamine 2000 from Invitrogen (Carlsbad, CA, USA); short hairpin RNA (shRNA) targeting HIF-1α (sh-HIF-1α#1/2) and Gli1 (sh-Gli1#1/2) as well as corresponding negative control (sh-NC), and plasmid for point mutation at H3K4 site (H3K4me Mut) from GenePharma (Shanghai, China); primary antibodies including anti-E-cadherin, anti-N-cadherin, anti-vimentin, anti-HIF-1α, anti-Gli1, anti-H3K4me2, anti-H3K4me3 and anti-GAPDH as well as anti-rabbit horseradish peroxidase-labeled secondary antibody from Abcam (Shanghai, China).

RNA extraction and RT‒qPCR

Total RNA was extracted from cells using TRIzol reagent. Reverse transcription reactions were performed with the Prime Script RT Reagent Kit with GAPDH as an internal control. The PCR was run in triplicate with a 7,500 Real-Time PCR System (Applied Biosystems, Darmstadt, Germany) using SYBR Premix Ex Taq II (Takara Bio Inc. Dalian, China).

Cell culture and treatment

The glioma cell lines as well as the control cell line were cultured in DMEM containing 10% FBS. The negative control group was cultured in a 37 °C, 5% CO2 incubator, and logarithmic growth phase glioma cells (A172, U87, U251 and Hs683) were placed in a hypoxia incubator (37 °C, 5% CO2, 1% O2 and 94% N2) for culture. Cellular RNA extraction was conducted after 24 h of culture.

Cell transfection

U87 and U251 cells were cultured to approximately 80% confluence in plates and then transfected with the designated plasmids with Lipofectamine 2000 according to the manufacturer’s instructions. After 48 h of transfection, the cells were harvested for subsequent assays.

CCK-8 assays

The viability of U87 and U251 cells cultured under hypoxia was evaluated using a Cell Counting Kit-8 (Beyotime, Shanghai, China) following the manufacturer’s instructions. Cells were seeded into 96-well plates at 2 × 103 cells per well. After incubation for 24, 48 and 72 h under hypoxia, 10 μL of CCK-8 reagent was added to each well and cultured for another 2 h at 37 °C. Finally, a micrometer reader (BioTek Instruments Inc., Winooski, USA) was used to detect the absorbance at 450 nm.

Transwell invasion

A total of 50 μL of Matrix gel was added to the respective well. U87 and U251 cells cultured under hypoxia were seeded in each Transwell chamber containing 200 μL of serum-free DMEM (5 × 105 cells/well). The lower compartment was filled with DMEM containing 10% FBS. The cells were cultured for 24 h, and the cells remaining on the upper surface of the filter membrane were removed. The cells on the lower surface of the filter membrane were fixed with methanol, stained with crystal violet, and photographed under a microscope.

Western blot

Total protein was extracted from cells with RIPA lysis buffer. Protein quantification was conducted using a BCA kit (Beyotime, Shanghai, China). SDS gel electrophoresis buffer (5×) was added and denatured at 100 °C for 10 min. After complete separation by electrophoresis, the protein was transferred to a PVDF membrane by a semidry method. After blocking with 5% skimmed milk powder at room temperature for 2 h, the indicated primary antibodies were added and incubated overnight at 4 °C. Then, secondary antibodies were added, incubated for another 2 h, and washed with TBS. Absorbance analysis was performed after color development to calculate the relative expression of each protein. The primary antibodies included anti-E-cadherin (ab40772, 1/10,000, Abcam, Cambridge, UK), anti-N-cadherin (ab76011, 1/5,000, Abcam, Cambridge, UK), anti-Vimentin (ab92547, 1/1,000, Abcam, Cambridge, UK), anti-HIF-1α (ab179483, 1/1,000, Abcam, Cambridge, UK), anti-Gli1 (ab134906, 1/1,000, Abcam, Cambridge, UK), anti-H3K4me2 (ab32356, 1/2,000, Abcam, Cambridge, UK), anti-H3K4me3 (ab213224, 1/1,000, Abcam, Cambridge, UK), and anti-acetyl Lysine (ab190479, 1/1,000, Abcam, Cambridge, UK) with GAPDH (ab8245, 1/1,000, Abcam, Cambridge, UK) as a loading control.

ChIP

Cultured cells were fixed with 1% formaldehyde solution for 15 min and then terminated using glycine. Cells were washed with PBS and lysed using ChIP lysis buffer (containing 1% Triton X-100 and proteinase inhibitor). DNA gels were used to ensure that isolated chromatin sheared into approximately 500–800 bp fragments. The magnetic beads were incubated for 2 h with HIF-1α antibody, H3K4me antibody (lgG as a negative control), H3ac, H4ac, MLL1, MLL2, MLL3 or MLL4. The antibodies were provided by the Abcam and Thermo Fisher company. After washing complexes with PBS and purifying DNA via a column, agarose gel analysis or RT‒qPCR was used to detect the condensed DNA fraction.

Statistical analysis

SPSS 20.0 software was used to process the data. The data are expressed as the mean ± standard deviation (m ± s). The mean of samples between groups was compared using a t test, and that of multiple groups was compared through one-way analysis of variance followed by Tukey’s post-hoc test. The difference was considered statistically significant at p<0.05.

Results

Hypoxia triggers HIF-1α and Gli1 upregulation in glioma cells

HIF-1, a major transcription factor mediating the hypoxia response, is upregulated in gliomas [6]. HIF-1α transcription complex stabilization and activation trigger downstream target genes and modulate biological behaviors [7]. However, the explicit function and downstream regulatory mechanism of HIF-1α in glioma cells under hypoxia have not been fully investigated. First, we measured HIF-1α expression status in glioma cell lines and control NHA cells via RT‒qPCR. HIF-1α expression was elevated in glioma cell lines (A172, U87, U251 and Hs683) relative to NHAs, and HIF-1α expression was most significantly upregulated in U87 and U251 cells, which were chosen for further analysis (Figure 1A). Then the expression of HIF-1α in hypoxia-treated glioma cells was examined, and we found that hypoxia treatment significantly elevated HIF-1α expression in glioma cells relative to untreated controls (Figure 1B). The association of hypoxia/HIF-1α with the HH pathway (key gene Gli1) has been revealed in other cancers [12]. The expression of Gli1 in glioma cells under normal or hypoxic conditions was examined. Through RT‒qPCR, we revealed that Gli1 was elevated in glioma cell lines relative to NHA cells (Figure 1C). Furthermore, Gli1 was elevated in hypoxia-treated glioma cells relative to untreated controls (Figure 1D). Collectively, both HIF-1α and Gli1 are upregulated in glioma cells under hypoxia.

Figure 1: 
Hypoxia triggered HIF-1α and Gli1 upregulation in glioma cells. (A) RT‒qPCR was used to measure HIF-1α levels in glioma cell lines and (A172, U87, U251 and Hs683) relative to normal human astrocytes (NHAs). (B) RT‒qPCR measured HIF-1α levels in glioma cell lines (A172, U87, U251 and Hs683) with or without hypoxia. (C) RT‒qPCR measured Gli1 levels in glioma cell lines (A172, U87, U251 and Hs683) relative to NHAs. (D) RT‒qPCR measured Gli1 levels in glioma cell lines (A172, U87, U251 and Hs683) with or without hypoxia. **indicates p<0.01, and ***indicates p<0.001 compared to the NHA or control group.
Figure 1:

Hypoxia triggered HIF-1α and Gli1 upregulation in glioma cells. (A) RT‒qPCR was used to measure HIF-1α levels in glioma cell lines and (A172, U87, U251 and Hs683) relative to normal human astrocytes (NHAs). (B) RT‒qPCR measured HIF-1α levels in glioma cell lines (A172, U87, U251 and Hs683) with or without hypoxia. (C) RT‒qPCR measured Gli1 levels in glioma cell lines (A172, U87, U251 and Hs683) relative to NHAs. (D) RT‒qPCR measured Gli1 levels in glioma cell lines (A172, U87, U251 and Hs683) with or without hypoxia. **indicates p<0.01, and ***indicates p<0.001 compared to the NHA or control group.

HIF-1α facilitates glioma cell invasion and EMT under hypoxia

Then we carried out a series of loss-of-function assays to clarify the impact of HIF-1α silencing on glioma cell invasive phenotype and EMT process under hypoxic conditions. The results of RT‒qPCR showed that HIF-1α was successfully silenced through transfection with sh-HIF-1α#1/2 in glioma cells (Figure 2A). The effects of HIF-1α silencing on glioma cell viability were examined using CCK-8 assays, and the results revealed that the viability of glioma cells cultured under hypoxia was relatively lower in HIF-1α-silenced glioma cells but without statistical significance (Figure 2B). Then we explored whether HIF-1α silencing affected glioma invasion using Transwell invasion assays, and the results demonstrated a marked reduction in the number of invading U87 and U251 cells under HIF-1α knockdown (Figure 2C and D). Additionally, western blotting demonstrated that HIF-1α silencing led to downregulation of E-cadherin protein abundance and upregulation of N-cadherin and vimentin protein abundance in U87 and U251 cells under hypoxic conditions (Figure 2E and F). Overall, these findings suggest that HIF-1α exerts an oncogenic role by facilitating the glioma cell invasive phenotype and EMT process.

Figure 2: 
HIF-1α facilitated glioma cell invasion and EMT under hypoxia. (A) RT‒qPCR was used to measure HIF-1α levels in U87 and U251 cells transfected with sh-HIF-1α#1/2 or sh-NC. (B) CCK-8 assays were used to measure the viability of HIF-1α-silenced glioma cells under hypoxia. (C–D) Transwell invasion assays were used to evaluate glioma cell invasive ability under hypoxia and indicated transfection conditions. (E–F) Western blotting assessed glioma cell EMT-related protein abundance under hypoxia and the indicated transfection conditions. **indicates p<0.01 compared to the sh-NC group. ***indicates p<0.001 compare to sh-NC group.
Figure 2:

HIF-1α facilitated glioma cell invasion and EMT under hypoxia. (A) RT‒qPCR was used to measure HIF-1α levels in U87 and U251 cells transfected with sh-HIF-1α#1/2 or sh-NC. (B) CCK-8 assays were used to measure the viability of HIF-1α-silenced glioma cells under hypoxia. (C–D) Transwell invasion assays were used to evaluate glioma cell invasive ability under hypoxia and indicated transfection conditions. (E–F) Western blotting assessed glioma cell EMT-related protein abundance under hypoxia and the indicated transfection conditions. **indicates p<0.01 compared to the sh-NC group. ***indicates p<0.001 compare to sh-NC group.

HIF-1α binds to Gli1 and transcriptionally activates Gli1 via H3K4me

The downstream HIF-1α mechanism in glioma cells under hypoxic conditions was further investigated. RT‒qPCR was used to explore the regulation of HIF-1α on Gli1, and the results demonstrated that HIF-1α silencing reduced Gli1 levels in U87 and U251 cells (Figure 3A), suggesting that HIF-1α positively regulates Gli1 expression. ChIP assays were conducted to explore the regulatory mechanism of HIF-1α on Gli1. The results showed that the Gli1 promoter showed enrichment in complexes binding to anti-HIF-1α in glioma cells (Figure 3B), suggesting that HIF-1α transcriptionally activated Gli1. Previously, HDAC3a, a direct target gene of HIF-1 [10], triggered histone H3K4 deacetylation modification on the Gli1 gene promoter, while transcriptional regulation of genes through histone modification is usually the result of the combined impact of deacetylation modification and methylation modification [15]. We then examined the impact of HIF-1α silencing on the acetylation and methylation of Gli1. The results showed that Gli1 acetylation was not significantly affected by HIF-1α in glioma cells under hypoxia (Figure 3C). The histone acetylation levels of total H3 and H4 in Gli1 promoter exhibited no significant changes after silencing HIF-1α in glioma cells under hypoxia (Figure 3D). Therefore, we hypothesized that HIF-1α may positively modulate Gli1 levels via methylation modification. Gli1 promoter presented a marked enrichment in complexes binding to anti-H3K4me in both mock glioma cells and H3K4me-Mut-transfected glioma cells, whereas Gli1 promoter enrichment intensity presented depletion in H3K4me-Mut-transfected glioma cells relative to respective mock glioma cells (Figure 3E), verifying the existence of methylation modification. Previous studies have reported that mixed lineage leukemia (MLL) family of histone H3K4-specific methyl-transferases plays a key role in basal gene regulation and activation, and MLL histone methylases are also related to hypoxia signaling and tumor growth [20, 21]. Thus, we further explored whether MLLs were implicated in the regulation of HIF-1α on Gli1 via H3K4 methylation. Based on the results of ChIP assays, we found that only the enrichment of MLL1 showed a significant decrease in complex binding to Gli1 promoter in HIF-1α-silenced glioma cells under hypoxia (Figure 3F). Furthermore, western blotting depicted a remarkable decline in HIF-1α, Gli1, H3K4me2 and H3K4me3 protein abundances in U87 and U251 cells under HIF-1α knockdown and hypoxia (Figure 3G). These results demonstrate that HIF-1α upregulates Gli1 in glioma cells transcriptionally through MLL1-mediated H3K4me under hypoxic conditions.

Figure 3: 
HIF-1α binds to Gli1 and transcriptionally activates Gli1 via MLL1-mediated H3K4me under hypoxia. (A) RT‒qPCR was used to measure Gli1 levels in U87 and U251 cells transfected with sh-HIF-1α#1/2 or sh-NC. (B) ChIP was used to assess the association of HIF-1α with the Gli1 promoter in U87 and U251 cells under hypoxia. (C) Acetylation of GLi1 in HIF-1α-silenced glioma cells under hypoxia. (D) The histone acetylation levels of total H3, H4 in Gli1 promoter in glioma cells under hypoxia and indicated transfection were detected using ChIP assays. (E) ChIP was used to assess the association of H3K4me with the Gli1 promoter in mock glioma cells or H3K4me-Mut-transfected glioma cells under hypoxia. (F) The enrichment of MLLs in Gli1 promoter in glioma cells under hypoxia and indicated transfection was detected using ChIP assays. (G) Western blotting assessed HIF-1α, Gli1 and H3K4me-related protein abundance under hypoxia and indicated transfection conditions. **indicates p<0.01 compared to the sh-NC group. ***means p<0.001 compared to the sh-NC or anti IgG or indicated group.
Figure 3:

HIF-1α binds to Gli1 and transcriptionally activates Gli1 via MLL1-mediated H3K4me under hypoxia. (A) RT‒qPCR was used to measure Gli1 levels in U87 and U251 cells transfected with sh-HIF-1α#1/2 or sh-NC. (B) ChIP was used to assess the association of HIF-1α with the Gli1 promoter in U87 and U251 cells under hypoxia. (C) Acetylation of GLi1 in HIF-1α-silenced glioma cells under hypoxia. (D) The histone acetylation levels of total H3, H4 in Gli1 promoter in glioma cells under hypoxia and indicated transfection were detected using ChIP assays. (E) ChIP was used to assess the association of H3K4me with the Gli1 promoter in mock glioma cells or H3K4me-Mut-transfected glioma cells under hypoxia. (F) The enrichment of MLLs in Gli1 promoter in glioma cells under hypoxia and indicated transfection was detected using ChIP assays. (G) Western blotting assessed HIF-1α, Gli1 and H3K4me-related protein abundance under hypoxia and indicated transfection conditions. **indicates p<0.01 compared to the sh-NC group. ***means p<0.001 compared to the sh-NC or anti IgG or indicated group.

H3K4me facilitates glioma cell invasion and EMT under hypoxia

As we have demonstrated that HIF-1α transcriptionally activated Gli1 in glioma cells via MLL1-mediated H3K4me, the effects of H3K4me on the glioma cell invasion phenotype and EMT process were further investigated. We conducted functional assays through H3K4me mutation in U87 and U251 cells under hypoxia. Transwell invasion assays demonstrated a marked reduction in the number of invading glioma cells under the K3K4me mutation (Figure 4A and B). Moreover, western blotting illustrated that H3K4me mutation led to E-cadherin protein abundance depletion but resulted in N-cadherin and vimentin protein abundance elevation in glioma cells (Figure 4C and D). Therefore, H3K4me facilitated the glioma cell invasive phenotype and EMT process under hypoxia.

Figure 4: 
H3K4me facilitated glioma cell invasion and EMT under hypoxia. (A–B) Transwell invasion assays evaluated glioma cell invasive ability with or without transfection of H3K4me-Mut under hypoxia. (C–D) Western blotting assessed glioma cell EMT-related protein abundance under hypoxia and indicated transfection conditions. **indicates p<0.01 compare to control group. ***means p<0.001 compare to control group.
Figure 4:

H3K4me facilitated glioma cell invasion and EMT under hypoxia. (A–B) Transwell invasion assays evaluated glioma cell invasive ability with or without transfection of H3K4me-Mut under hypoxia. (C–D) Western blotting assessed glioma cell EMT-related protein abundance under hypoxia and indicated transfection conditions. **indicates p<0.01 compare to control group. ***means p<0.001 compare to control group.

Gli1 facilitates glioma cell invasion and EMT

The impact of Gli1 on the glioma cell invasive phenotype and EMT process was further investigated using loss-of-function assays. The results of CCK-8 assays revealed that Gli1 silencing did not significantly affect glioma cell viability under hypoxic conditions (Figure 5A). Transwell invasion assays demonstrated a marked reduction in the number of invading glioma cells under Gli1 downregulation (Figure 5B and C). Moreover, western blotting illustrated that Gli1 silencing caused E-cadherin protein abundance depletion but resulted in N-cadherin and vimentin protein abundance elevation in glioma cells (Figure 5D). Collectively, Gli1 exerts a promoting role in the glioma cell invasive phenotype and EMT process.

Figure 5: 
Gli1 facilitated glioma cell invasion and EMT under hypoxia. (A) CCK-8 assays were used to evaluate the effects of Gli1 silencing on the viability of glioma cells under hypoxia. (B–C) Transwell invasion assays were used to evaluate glioma cell invasive ability after transfection with sh-Gli1#1/2 or sh-NC. (D) Western blotting assessed glioma cell EMT-related protein abundance under hypoxia and the indicated transfection conditions. **indicates p<0.01 compared to the sh-NC group. ***indicates p<0.001 compared to the sh-NC group.
Figure 5:

Gli1 facilitated glioma cell invasion and EMT under hypoxia. (A) CCK-8 assays were used to evaluate the effects of Gli1 silencing on the viability of glioma cells under hypoxia. (B–C) Transwell invasion assays were used to evaluate glioma cell invasive ability after transfection with sh-Gli1#1/2 or sh-NC. (D) Western blotting assessed glioma cell EMT-related protein abundance under hypoxia and the indicated transfection conditions. **indicates p<0.01 compared to the sh-NC group. ***indicates p<0.001 compared to the sh-NC group.

Discussion

Hypoxia is a fundamental feature in the tumor growth microenvironment [22]. Tumor cells can continue to survive under hypoxia by activating adaptive responses [23]. Hypoxia-inducible factors (HIFs) are the most crucial transcription factors related to hypoxia. Under hypoxia, they exert a vital role in transmitting hypoxic signals and mediating hypoxic effects [24, 25]. HIFs are a heterodimeric complex consisting of HIF-α and HIF-β, among which HIF-β is a stable subunit and HIF-α is an oxyphilic subunit [26]. Mammalian HIF-α has three subtypes, namely, HIF-1α, HIF-2α and HIF-3α [27]. HIF-1α is a hypoxia-triggered transcription factor with DNA binding activity first isolated from hypoxic Hep-3 nuclei by Wang and Semenza in 1992 [28]. Tumor malignant proliferation makes oxygen consumption in tissue and the formation of blood vessels incomplete, resulting in insufficient oxygen content in the microenvironment, so that each area in the tumor is in a different hypoxic microenvironment [29]. In response to the hypoxic microenvironment, HIF-1α can regulate a variety of target genes participating in cell adaptation and survival under hypoxic stress, elevating its transcriptional activity and expressing corresponding products to adapt to the hypoxic stress response [30]. Currently, high expression of HIF-1α is related to glioma invasion and metastasis, and the mechanism may be that HIF induces EMT indirectly or directly [31, 32]. Herein, HIF-1α was elevated in glioma cell lines relative to control cells. Moreover, HIF-1α was elevated in hypoxia-treated glioma cells relative to untreated controls. Furthermore, HIF-1α silencing suppressed glioma cell invasion and EMT. These findings suggested that HIF-1α exerts an oncogenic role by facilitating the glioma cell invasive phenotype and EMT process, which is consistent with previous reports.

The glioma-associated oncogene homolog (Gli) family has three homologous isoform genes (Gli1, Gli2, Gli3) in mammals, all of which are protein transcription factors with zinc finger structures [33, 34]. During embryonic development, Gli1 protein is expressed in both the nucleus and cytoplasm. Gli1 can activate downstream target genes of the HH signaling pathway, facilitate cell proliferation and differentiation, and affect embryonic development [35, 36]. In tumors, Gli1, as a tumor-promoting gene, is markedly upregulated in most tumor cells and tumor tissues [37, 38]. Herein, Gli1 was elevated in glioma cell lines relative to control cells. Gli1 was elevated in hypoxia-treated glioma cells relative to untreated controls. Additionally, there is a crucial association of Gli1 with EMT, and Gli1 overexpression can facilitate EMT in cells [39, 40]. In a variety of cancer cells, whether by interfering or overexpressing Gli1, corresponding changes in vimentin, E-cadherin, N-cadherin, p-catenin, and twist levels can be observed via western blotting [41]. Herein, Gli1 silencing suppressed glioma cell invasion and EMT. These findings suggest that Gli1 exerts an oncogenic role by facilitating the glioma cell invasive phenotype and EMT process.

HIF-1α/HIF-1β transcription complex stabilization and activation trigger downstream target genes and modulate multiple tumor biological behaviors, including EMT and tumor metastasis [7]. Herein, HIF-1α silencing reduced Gli1 levels in glioma cells, suggesting that HIF-1α positively regulates Gli1 expression. Previously, in addition to histone deacetylation, histone demethylation has also been demonstrated to be an existing epigenetic mechanism during EMT [42, 43]. EMT-TFs interact with a series of histone modifiers to form a finely regulated network to modulate related gene expression; the EMT-related gene promoter region can have both transcriptional repressive histone modification H3K27me3 and transcriptional activation modification H3K4me3 simultaneously; and cells can quickly modulate gene expression by changing “one-side” modification status according to stimulation of external signals [4445]. Herein, HIF-1α protein abundantly bound to the Gli1 promoter in glioma cells. Gli1 promoter binding to H3K4me was depleted in H3K4me-Mut-transfected glioma cells relative to the respective mock glioma cells, and HIF-1α silencing also led to Gli1 protein and H3K4me-associated protein levels, verifying that HIF-1α upregulates Gli1 in glioma cells transcriptionally through H3K4me. Furthermore, H3K4me mutation suppressed glioma cell invasion and EMT. These findings suggested that H3K4me exerts a facilitating impact on the glioma cell invasive phenotype and EMT process.

In conclusion, both HIF-1α and Gli1 are upregulated in glioma cells and function as oncogenes in glioma cells. HIF-1α transcriptionally activates Gli1 via MLL1-mediated H3K4 methylation in glioma cells, providing ideas for seeking new therapeutic directions for glioma.


Corresponding author: Zhangyi Wu, Department of Neurosurgery, Zhejiang Provincial Tongde Hospital, No. 234, Gucui Road, Hangzhou, Zhejiang 310012, China, E-mail:

Funding source: Zhejiang Basic Public Welfare Research Program

Award Identifier / Grant number: GF21H090020

  1. Research funding: This work was supported by the Zhejiang Basic Public Welfare Research Program (No. GF21H090020).

  2. Author contributions: Y.L. and Z.W. were involved in the conception and design of this study. Y.L. and Z.W. performed the data analysis and interpretation of the results. Y.L. prepared the first draft of the manuscript. Z.W. performed a critical revision of the manuscript. Z.W. supervised the study. All authors have accepted responsibility for the entire content of this manuscript and approved its submission.

  3. Competing interests: Authors state no conflict of interest.

  4. Informed consent: Not applicable.

  5. Ethical approval: All experimental procedures were approved by the Ethics Committee of Zhejiang Provincial Tongde Hospital (approval No. 2021-043).

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Received: 2022-11-14
Accepted: 2022-12-30
Published Online: 2023-02-08

© 2023 the author(s), published by De Gruyter, Berlin/Boston

This work is licensed under the Creative Commons Attribution 4.0 International License.

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