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. 2024 Jan 25;56(2):174–183. doi: 10.3724/abbs.2023259

Valproic acid regulates the miR-155/Jarid2 axis by affecting miR-155 promoter methylation in glioma

VPA regulates miR-155/Jarid2 axis in glioma

Ruixuan Wang 1, Yanhong Chen 2, Weilu Kuang 1, Wuzhong Jiang 1, Wenjing Zeng 2, Yinyun Chen 3, Zhengzheng Liu 1,4,*
PMCID: PMC10984859  PMID: 38273784

Abstract

The most frequent primary brain tumor in adults is glioma, yet no effective curative treatments are currently available. Our previous study demonstrated the enhancing effects of JARID2 on glioma sensitivity to TMZ treatment. In this study, miR-155 is predicted to target JARID2. miR-155 is overexpressed in clinical glioma specimens and cell lines. miR-155 overexpression in glioma cells enhances cell viability and represses cell apoptosis. Through targeting, miR-155 inhibits JARID2 expression. miR-155 inhibition inhibits glioma cell viability and enhances cell apoptosis, whereas JARID2 knockdown enhances cell viability and inhibits cell apoptosis; JARID2 knockdown partially reverses miR-155 inhibition effects on glioma phenotypes. miR-155 inhibition reduces but knockdown of JARID2 promotes the tumor formation ability of glioma cells in vivo. Valproic acid (VPA) upregulates JARID2 expression, inhibits glioma cell viability and enhances cell apoptosis. VPA downregulates the expression level of miR-155 by increasing the methylation level of the miR-155 promoter, suggesting that the miR-155/JARID2 axis is implicated in VPA inhibition of glioma cell viability and enhancement of glioma cell apoptosis. This study demonstrates a new mechanism of VPA treatment of gliomas by affecting the miR-155/JARID2 axis, which could be regarded as a new strategy for the prevention and treatment of glioma.

Keywords: gliomas, miR-155, JARID2, methylation, valproic acid

Introduction

The most frequent primary brain tumor in adults is glioma, which can develop in either the white or gray brain matter and is likely to have a glial origin from the white brain matter [ 1, 2]. Adults are more likely to develop infiltrative astrocytoma rather than any other type of primary brain tumor. Compared to patients with the two low-grade astrocytomas, those with high-grade astrocytomas have a significantly greater mortality rate [ 3, 4].

The standard first-line chemotherapeutics for recurrent high-grade glioma and other solid neoplasias are alkylating agents such as temozolomide (TMZ) [ 5, 6]. The induction of methyl adducts at multiple DNA bases, such as O6-guanine, N7-guanine, and N3-adenine, leads to the antitumor effects of TMZ [ 7, 8]. Acquired chemotherapeutic resistance is still a major challenge. Moreover, the second chemotherapy cycle is ineffective for more than 90% of recurrent gliomas. [9]. Some mechanisms have been shown to exert crucial effects on TMZ chemoresistance [ 10– 12]. Our previous study confirmed the enhancing effects of JARID2 on glioma sensitivity to TMZ treatment. Overexpressing JARID2 in TMZ-treated glioma cells dramatically suppressed cell viability, enhanced cell apoptosis, and elevated the levels of p21, cleaved-PARP, and cleaved-caspase3 [13]. However, because JARID2 expression is markedly downregulated in gliomas, identification of the inhibitory regulator may lead to the development of new therapeutic targets in gliomas.

Recent research has demonstrated that microRNAs (miRNAs) play a crucial role in controlling gene expression in healthy and cancer cells. MiRNAs are endogenous, noncoding, and 20–23 nt in length, targeting mRNAs that may be posttranscriptionally bound by RNAs, which may cause mRNA cleavage, direct translational inhibition, mRNA degradation, or a combination of the two in some instances [ 14, 15]. An individual miRNA can modulate the expression of multiple target RNAs and thereby regulate several gene pathways, subsequently modulating almost all cellular processes, including proliferation, differentiation, apoptosis, and cell growth. MiRNAs may be important as tumor suppressors or oncogenes (oncogenic miRNAs or ‘oncomirs’). MiRNAs are believed to be promising therapeutic targets/tools for cancer therapy and serve as potential biomarkers for human cancers [ 16, 17]. Piwecka et al. [18] identified over 290 miRNAs deregulated in glioma tissues by performing a meta‐analysis of miRNA expression profiling studies. Using the extensive data generated by deep sequencing, Moore et al. [19] discovered a complex network of gene expression alterations in the miRNA biogenetic pathway that affect miRNA maturation and are connected to glioma development. Thus, JARID2 downregulation in glioma may be caused by miRNAs that target JARID2.

Moreover, valproic acid (VPA), one of the most common histone deacetylase inhibitors (HDACIs), has been detected to directly or synergistically exert inhibitory effects on glioma in vitro and in vivo [20]. For instance, Han et al. [21] reported that VPA enhanced apoptosis by promoting autophagy via Akt/mTOR signaling in glioma. Phospho-VPA suppressed glioblastoma growth in preclinical models through the inhibition of STAT3 phosphorylation [22]. In vivo efficacy experiments showed that combination treatment with MSCs-TK and VPA significantly inhibited tumor growth and prolonged the survival of glioma-bearing mice compared with single-treatment groups [23]. Since the role and mechanism of the JARID2/CCND1 axis modulating glioma cell growth and sensitivity to TMZ have been demonstrated [13], this study looked into JARID2’s potential role in VPA’s enhancement of TMZ toxicity in glioma.

In this study, we employed mirDIP, miRDB, TargetScan, and DIANA TOOLS to analyze miRNAs and found that miR-155 might target JARID2. Through the studies of miR-155 overexpression/inhibition, and miR-155 promoter methylation on VPA-treated glioma, we confirmed that the miR-155/JARID2 axis might participate in VPA’s regulation of glioma cell viability and apoptosis.

Materials and Methods

Clinical sampling

A total of 18 glioma tissue samples (6 cases of WHO grades II, III, and IV) were harvested from the tumor core region, and 6 peritumoral brain edema (PTBE) region samples were harvested as controls. All samples were harvested from patients who underwent surgical resection at Xiangya Hospital, Central South University. The sampling procedure was conducted with the approval of the Ethics Committee of Xiangya Hospital of Central South University (approval No. 201803393), and signed informed consent was collected from each patient. All samples were immediately transferred to a ‒80°C container after harvesting, prior to subsequent experiments.

Hematoxylin and eosin (H&E) staining

The tissue specimens were fixed in formalin, embedded in paraffin, and cut into 5-μm-thick cross-sections. The sample section was then washed with xylene, 100% ethanol, 95% ethanol, 80% ethanol, and PBS. After rehydration, sections were stained with H&E solution (Solarbio, Beijing, China). After staining, the sections were observed under a microscope (BX43; Olympus, Tokyo, Japan) and photographed.

Selection of JARID2 3′UTR targeted miRNAs

To identify miRNAs that might target the JARID2 3′UTR, mirDIP [24] (integrated score>0.6), miRDB [25] (target score>90), TargetScan [26] (conserved sites), and DIANA TOOLS [27] (miTG score>0.99) were used for prediction.

qRT-PCR

The reagents used for PCR-based analyses were Trizol reagent (Invitrogen, Carlsbad, USA) for total RNA extraction, a cDNA first-strand synthesis kit (Promega, Madison, USA) for reverse transcription, and SYBR Green PCR Master Mix (Qiagen, Hilden, Germany) for PCR. All reagents were used following standard protocols. GAPDH (mRNA) or U6 (miRNA) was used as an internal reference, and the Ct method was used to calculate the relative expression of each target factor. The primer sequences are listed in Supplementary Table S1. PCR condition was as follows: 95°C for 2 min followed by 40 cycle of 95°C for 5 s and 60°C for 10 s.

Cell culture and treatment

The normal glial cell line HEB was procured from Beinuo Life Science Company (Shanghai, China) and kept in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Helgerman Court, USA) supplemented with 10% FBS (Gibco). The glioma cell line SHG44 (3131C0001000700048) was procured from Cell Resource Center, Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China) and cultured in RPMI 1640 (Gibco) supplemented with 10% FBS (Gibco). The glioma cell line U251 (09063001) was procured from Merck (Darmstadt, Germany) and cultured in EMEM (Gibco) supplemented with 10% FBS (Gibco). The glioma cell line T98G (CRL-1690) was procured from ATCC (Manassas, USA) and cultured in Eagle’s minimum essential medium (Gibco) supplemented with 10% FBS (Gibco). All cells were cultured at 37°C in 5% CO 2. For TMZ treatment, SHG44 cells were treated with 100 μM TMZ (Sigma-Aldrich, St Louis, USA) for 48 h. For VPA treatment, SHG44 cells were treated with 5 mM VPA (Sigma-Aldrich) for 48 h.

Cell transfection

agomir-155-5p or antagomir-155-5p was designed and produced by GenePharma (Shanghai, China) for cell transfection to achieve miR-155-5p overexpression or inhibition. Based on a pLVX-shRNA2-puro plasmid, a short hairpin RNA against the JARID2 gene (sh-JARID2) and its nontarget sequence (sh-NC) were constructed to achieve JARID2 knockdown. Briefly, cell transfection was performed using Lipofectamine 3000 reagent (Thermo Fisher Scientific, Waltham, USA) following the product manual. The transfection process lasted for 48 h. The sequences for the agomir, antagomir, and shRNA vectors are listed in Supplementary Table S1.

CCK-8 for cell viability

The Cell Count Kit-8 (SKU: CK04; Dojindo, Rockville, USA) was used to determine cell viability. Cells were seeded in 96-well plates at 1×10 4 cells/well and cultured at 37°C overnight. After transfection or treatment, the cells were incubated with 10 μL CCK-8 solution and 100 μL serum-free DMEM at 37°C for 4 h. A microplate reader (xMark TM; Bio-Rad, Hercules, USA) was used to measure the absorbance at 450 nm.

Flow cytometry for cell apoptosis

Target cells were transfected, and a FITC-labelled Annexin V/Propidium Iodide (PI) Apoptosis Detection kit (Beyotime, Shanghai, China) was used to detect cell apoptosis. Flow cytometry was performed immediately after staining on the ACEA NovoCyte flow cytometer (Agilent, Santa Clara, USA). Annexin V-positive cells were identified in early apoptosis, while Annexin V- and PI-positive cells were detected in late apoptosis.

Immunoblotting

Protein samples that were extracted from cells or tumor tissues were separated by 10% or 15% SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was first blocked with 5% skimmed milk dissolved in diphenyltriazole buffer for 2 h at room temperature to prevent nonspecific binding. The membrane was subsequently incubated with the following primary antibodies: p21 (701151; Thermo Fisher Scientific), CDK4 (11026-1-AP; Proteintech, Wuhan, China), caspase3 (19677-1-AP; Proteintech), cleaved-caspase3 (ab2302; Abcam, Cambridge, USA), JARID2 (ab192252; Abcam), and CCND1 (MA5-16356; Thermo Fisher Scientific) overnight at 4°C, followed by another incubation with proper horseradish peroxidase-conjugated secondary antibodies (Proteintech) for 2 h at room temperature. Finally, an enhanced chemiluminescence (ECL) kit (Beyotime) was used to visualize the signal.

Dual-luciferase reporter assay

To confirm the binding of miR-155-5p with JARID2, wild-type and mutant JARID2 reporter vectors (wt-JARID2 and mut-JARID2) were constructed. Briefly, about 300 bp fragments including predicted binding site and mutant type were cloned by PCR and inserted into the psicheck2 reporter vector (Promega). Then, 293T cells were cotransfected with wt-JARID2/mut-JARID2 and agomir-155-5p/antagomir-155-5p with the help of Lipo3000 (Invitrogen). The luciferase activity was determined using a Dual-Luciferase Reporter System (Promega).

Methylation specific PCR (MSP)

Cellular DNA isolation, genomic DNA methylation, and MSP were performed to determine the methylated and unmethylated alleles of the miR-155-5p promoter region as described previously [28]. The MSP reaction was carried out in a 10 μL system, and each reaction used 1 μL (20 ng) of template DNA. Water was used as a negative control. The methylated and unmethylated band intensities were quantified using ImageJ software (NIH, Bethesda, USA). The PCR primers used for MSP are listed in Supplementary Table S1.

Xenograft mouse assay

Animal experiment procedures were carried out under the approval of the Ethics Committee of Xiangya Hospital of Central South University. Twenty-four male BALB/c nude mice (6 weeks old) were purchased from the Hunan SLA Laboratory Animal Co., Ltd (Changsha, China). The mice were maintained under specific pathogen-free conditions (22°C, ~50% humidity). The mice were randomly divided into 4 groups: antagomir-NC+lv-shNC group, antagomir-155+lv-shNC group, antagomir-NC+lv-shJARID2 group, and antagomir-155+lv-shJARID2 group. SHG44 cells were infected with lentivirus constructed with shJARID2 vectors and/or antagomir-155. After infection for 48 h, the infected SHG44 cells were harvested for injection. A total of 1×10 6 infected SHG44 cells were suspended in 50 μL PBS and injected subcutaneously into the left anterior armpits of the nude mice. Tumor growth was observed and recorded for 28 days. A calliper was used to measure tumor size, and tumor volume was calculated using the following formula: volume=length×width 2/2. At the termination of the experiment (the 28 th day), the mice were sacrificed, and the tumor was excised from each mouse to measure the protein levels of JARID2, p21, CDK4, cleaved-caspase3, and caspase3 by immunoblotting.

Statistical analysis

The data for each experiment were analyzed by GraphPad software (San Diego, USA) and are expressed as the mean±SD of three independent experiments. One-way analysis of variance (ANOVA), followed by Tukey’s multiple comparison test, was used to assess the differences among multiple groups; Student’s t-test was used to assess the differences between two groups. Kaplan-Meier survival analysis and Cox proportional hazards models were applied to determine the link between the expression levels of miR-155 and the prognosis of glioma patients. The association between miR-155 expression and JARID2 expression levels was analyzed by Pearson’s correlation analysis. A P value of less than 0.05 was considered statistically significant.

Results

miR-155 is upregulated in glioma tissues and cells

mirDIP, miRDB, TargetScan, and DIANA TOOLS were used to identify miRNAs that might target JARID2 ( Supplementary Figure S1). Among candidate miRNAs (miR-3666, miR-454-3p, and miR-155-5p), miR-155 is one of the most conserved and multifunctional miRNAs frequently overexpressed in a variety of diseases, including malignancies [29]. Therefore, miR-155 was chosen for subsequent experiments. First, clinical samples were collected, including normal and grade II, III, and IV glioma samples; next, H&E staining was performed to confirm the histopathological properties ( Figure 1A). As revealed by qRT-PCR, miR-155 expression was dramatically increased in glioma tissue samples with increasing grade ( Figure 1B) and increased in glioma cells compared with HEB cells ( Figure 1C). Moreover, glioma patients from The Cancer Genome Atlas (TCGA)-GBMLGG and Chinese Glioma Genome Atlas (CGGA) databases were separated into high- and low-miR-155 expression groups using the median value as the cutoff; the correlation between miR-155 expression and overall survival of patients was analyzed. The results showed that higher expression of miR-155 could predict poorer prognosis in glioma patients ( Figure 1D).

Figure 1 .

Figure 1

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miR-155 is upregulated in glioma tissues and cell lines

(A) The histopathological characteristics of normal and glioma samples (Grade II, III, and IV) were evaluated by H&E staining. (B) miR-155 expression was determined in normal and glioma samples by qRT-PCR. *P<0.05, **P<0.01 compared to the normal group. (C) miR-155 expression in the normal glial cell line HEB and glioma cell lines (U251, TG98, and SHG44) was determined by qRT-PCR. **P<0.01 compared to HEB cells. (D) The correlation between miR-155 expression and the overall survival of glioma patients was analyzed based on the TCGA-GBMLGG and CGGA databases.

Specific effects of miR-155 on glioma cell phenotypes

As we confirmed that miR-155 was upregulated in glioma tissues and cells, agomir-155/antagomir-155 was transfected to achieve miR-155 expression within SHG44 cells, and the specific effect of miR-155 on glioma was investigated. qRT-PCR was performed to confirm miR-155 overexpression or inhibition ( Figure 2A). In normal SHG44 cells, miR-155 overexpression significantly promoted cell viability ( Figure 2B) and inhibited cell apoptosis ( Figure 2C,D), whereas miR-155 inhibition repressed cell viability ( Figure 2B) and promoted cell apoptosis ( Figure 2C,D). miR-155 overexpression dramatically decreased p21 level and the cleaved-caspase3/caspase3 ratio but increased CDK4 level, whereas miR-155 inhibition exerted opposite effects on these factors ( Figure 2E).

Figure 2 .

Figure 2

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Specific effects of miR-155 on glioma cell viability and apoptosis

(A) miR-155 expression was achieved in SHG44 cells by transfecting agomir-155 or antagomir-155; miR-155 overexpression or inhibition was confirmed by qRT-PCR. (B–E) SHG44 cells were examined for cell viability by CCK-8 assay (B); cell apoptosis by flow cytometry (C) and TUNEL (D) assays; the protein levels of p21, CDK4, cleaved-caspase3, and caspase3 by immunoblotting (E). *P<0.05, **P<0.01 compared to the agomir-NC group; #P<0.05, ##P<0.01 compared to the antagomir-NC group.

miR-155 directly targets JARID2 and inhibits JARID2 expression

According to the TCGA and CGGA databases, miR-155 was negatively correlated with JARID2 ( Figure 3A,B). In SHG44 cells, miR-155 negatively modulated JARID2 protein level ( Figure 3C). Subsequently, dual-luciferase reporter assay was conducted; JARID2 luciferase reporter vectors, wild-type, and mutant-type, were designed and produced, and these vectors were cotransfected with agomir-155 or antagomir-155 into 293T cells. Figure 3D shows that when cotransfected with wt-JARID2 and agomir155/antagomir-155, miR-155 overexpression inhibited, but miR-155 inhibition enhanced, the luciferase activity of wt-JARID2; when 293T cells were cotransfected with mut-JARID2 and agomir155/antagomir-155, miR-155 overexpression/inhibition caused no changes in luciferase activity. Thus, miR-155 directly targets JARID2 and inhibits JARID2 expression.

Figure 3 .

Figure 3

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miR-155 directly targets JARID2 and inhibits JARID2 expression

(A,B) The correlation between miR-155 and JARID2 expression in glioma patients was analyzed using Pearson’s correlation analysis based on the TCGA and CGGA databases. (C) SHG44 cells were transfected with agomir-155 or antagomir-155 and examined for JARID2 protein levels by immunoblotting. (D) Wild-type and mutant JARID2 luciferase reporter vectors were constructed and cotransfected with agomir-155 or antagomir-155 into 293T cells. Luciferase activity was determined. *P<0.05, **P<0.01 compared to the agomir-NC group; ##P<0.01 compared to the antagomir-NC group.

Dynamic effects of the miR-155/JARID2 axis on glioma cell viability and apoptosis

The dynamic effects of the miR-155/JARID2 axis on glioma cells were evaluated after confirming the association between miR-155 and JARID2. SHG44 cells were cotransfected with antagomir-155 and sh-JARID2. MiR-155 inhibition restrained cell viability ( Figure 4A) but promoted cell apoptosis ( Figure 4B,C). However, silencing of JARID2 exerted opposite effects on glioma cells; silencing of JARID2 partially reversed the effects of miR-155 inhibition on glioma cells. Moreover, devoid of TMZ treatment, miR-155 inhibition notably increased p21 and cleaved-caspase3/caspase3 protein levels but decreased CDK4 protein level, whereas silencing of JARID2 observably decreased p21 and cleaved-caspase3/caspase3 protein levels but increased CDK4 protein level ( Figure 4D).

Figure 4 .

Figure 4

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Dynamic effects of the miR-155/JARID2 axis on glioma cells

SHG44 cells were cotransfected with antagomir-155 and sh-JARID2, and cells were examined for cell viability by CCK-8 assay (A); cell apoptosis by flow cytometry (B) and TUNEL (C) assays; and the protein levels of p21, CDK4, cleaved-caspase3, and caspase3 by immunoblotting (D). *P<0.05, **P<0.01 compared to the antagomir-NC+sh-NC group; #P<0.05, ##P<0.01 compared to the antagomir-155+sh-NC group.

Our previous research confirmed that JARID2 could promote apoptosis under TMZ treatment; therefore, the dynamic effects of the miR-155/JARID2 axis on glioma cell viability and apoptosis with TMZ treatment were subsequently evaluated. Under TMZ treatment, miR-155 inhibition reduced CCND1 and CDK4 protein levels and cell viability ( Supplementary Figure S2A,B) but promoted JARID2, p21, and cleaved-caspase3/caspase3 protein contents and cell apoptosis ( Supplementary Figure S2C,D), whereas JARID2 knockdown exerted opposite effects on glioma cells; JARID2 knockdown partially reversed the effects of miR-155 inhibition on glioma cells ( Supplementary Figure S2). These results suggest that the miR-155/JARID2 axis is involved in glioma cell viability and apoptosis under TMZ treatment.

Dynamic effects of the miR-155/JARID2 axis on the growth and tumorigenesis of glioma in vivo

The effects of the miR-155/JARID2 axis on the growth and tumorigenesis of glioma in vivo were evaluated in a xenograft nude mouse model. SHG44 cells pretransfected with antagomir-155 and/or lv-sh-JARID2 were subcutaneously injected into the armpit of nude mice. At the end of the experiment (the 28th day), mice were painlessly sacrificed, and tumor tissues were excised and images were captured ( Figure 5A). Tumor volume and weight in the antagomir-155 group were dramatically smaller than those in the control group, whereas silencing of JARID2 exerted opposite effects on tumor volume and weight; silencing of JARID2 partially reversed the effects of miR-155 inhibition on glioma cells ( Figure 5B). In tumor tissues, miR-155 inhibition markedly increased JARID2, p21, and cleaved-caspase3/caspase3 protein levels but decreased CDK4 protein level, whereas JARID2 knockdown significantly decreased JARID2, p21, and cleaved-caspase3/caspase3 protein levels but increased CDK4 protein level ( Figure 5C). These results indicate that miR-155 inhibition reduces, but knockdown of JARID2 promotes, the tumor formation ability of glioma cells in vivo.

Figure 5 .

Figure 5

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In vivo effects of the miR-155/JARID2 axis on xenotransplant tumor growth

SHG44 cells were pretransfected with antagomir-155 and/or lv-sh-JARID2 and then subcutaneously injected into the armpit of nude mice. At the end of the experiment, mice were painlessly sacrificed, and tumor tissues were excised. (A) Images of mice and tumors. (B) Tumor volume and weight were examined. (C) The protein levels of JARID2, p21, CDK4, cleaved-caspase3, and caspase3 in tumor tissues were detected by immunoblotting. *P<0.05, **P<0.01 compared to the antagomir-NC+lv-sh-NC group; #P<0.05, ##P<0.01 compared to the antagomir-155+lv-sh-NC group.

VPA upregulates JARID2 expression

SHG44 cells were treated with VPA or PBS and examined for JARID2 mRNA and protein levels. As shown in Figure 6A,B, VPA upregulated JARID2 mRNA expression level and increased JARID2 protein level. SHG44 cells were subsequently treated with VPA or PBS and examined for cell phenotypes. VPA inhibited cell viability ( Figure 6C) and promoted cell apoptosis ( Figure 6D). In addition, VPA has been reported to sensitize glioma cells to TMZ treatment [ 30– 32]. Here, the possible involvement of JARID2 in VPA sensitizing glioma cells to TMZ was investigated. Under TMZ treatment, VPA treatment also inhibited cell viability ( Supplementary Figure S2E) and promoted cell apoptosis ( Supplementary Figure S2F).

Figure 6 .

Figure 6

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VPA upregulates JARID2 and regulates cell viability and apoptosis

(A,B) SHG44 cells were treated with VPA (5 mM for 48 h) or PBS and examined for JARID2 mRNA by qRT-PCR and JARID2 protein level by immunoblotting. SHG44 cells were subsequently treated with VPA or PBS and examined for cell viability by CCK-8 assay (C); cell apoptosis by flow cytometry (D); and the protein levels of p21, CDK4, cleaved caspase3, and caspase3 by immunoblotting (E). **P<0.01 compared to the PBS group.

VPA promotes miR-155 promoter methylation and inhibits miR-155 expression

Methylation is one main mechanism of TMZ toxicity [ 33, 34]. Thus, the miR-155 promoter methylation level was detected in normal and glioma tissues using MSP (methylation specific PCR). It was found that the unmethylated level of the miR-155 promoter was almost the same in normal and glioma tissues, whereas the methylated level of the miR-155 promoter was dramatically lower in glioma tissue samples than in normal tissue samples ( Figure 7A). Similarly, in HEB and SHG44 cells, the unmethylated level of the miR-155 promoter remained similar, whereas the methylated level was dramatically reduced in SHG44 cells ( Figure 7B). SHG44 cells were treated with VPA or PBS and examined for miR-155 level by qRT-PCR. The results showed that VPA significantly downregulated miR-155 expression ( Figure 7C), suggesting that VPA might modulate the methylation level of miR-155. Thus, SHG44 cells were treated with VPA or PBS and examined for miR-155 promoter methylation level. PBS or VPA treatment caused no change in the unmethylated level of the miR-155 promoter, whereas VPA treatment dramatically increased the methylated level of the miR-155 promoter ( Figure 7D). Thus, VPA promotes miR-155 promoter methylation to inhibit its expression.

Figure 7 .

Figure 7

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VPA promotes miR-155 promoter methylation and inhibits miR-155 expression

(A) MSP (Methylation Specifc PCR) was used to detect miR-155 promoter methylation in normal and glioma tissues. **P<0.01 compared to the normal group. (B) miR-155 promoter methylation levels were determined using MSP in normal and cancer cells. **P<0.01 compared to the HEB group. (C) SHG44 cells were treated with VPA or PBS and examined for miR-155 level by qRT-PCR. (D) SHG44 cells were treated with VPA or PBS and examined for miR-155 promoter methylation level using MSP. M: methylation; U: unmethylation. **P<0.01 compared to the PBS group.

Discussion

In this study, miR-155 was predicted to target JARID2. miR-155 was overexpressed in clinical glioma specimens and cell lines. miR-155 overexpression in glioma cells enhanced cell viability and repressed cell apoptosis. Through targeting, miR-155 inhibited JARID2 expression. In glioma cells, miR-155 inhibition inhibited cell viability and enhanced cell apoptosis, whereas JARID2 knockdown enhanced cell viability and inhibited cell apoptosis; JARID2 knockdown partially reversed miR-155 inhibition effects on glioma phenotypes. miR-155 inhibition was reduced, while knockdown of JARID2 promoted the tumor formation ability of glioma cells in vivo. VPA upregulated JARID2 expression, inhibited glioma cell viability and enhanced cell apoptosis. VPA downregulated the expression level of miR-155 by increasing the methylation level of the miR-155 promoter.

The oncogenic features of miR-155 are widely conspicuous in malignancies in the oral cavity [35], cervix [36], breast [ 37, 38], colon [39], and brain [40]. Deshpande et al. [41] reported that miR-210-3p, miR-155-5p, miR-UL-112-3p, miR-183-5p, and miR-223-5p were upregulated in high-grade astrocytic tumor tissues compared with those in low-grade tumors. Functionally, Wu et al. [40] demonstrated that inhibition of MIR155HG using small interfering RNA repressed the generation of its derivatives miR-155-5p and miR-155-3p to inhibit the capacity of cells to proliferate, migrate and invade, as well as the tumor growth of orthotopic glioma. In addition, Milani et al. [42] reported that targeting miR-155-5p TMZ-resistant T98G glioma cells induced caspase-3 cleavage and tumor cell apoptosis. Consistent with previous studies, miR-155-5p upregulation was found in glioma tissue samples, especially in high-grade tissues, as well as in cell lines. Functionally, miR-155 overexpression dramatically promoted the viability and inhibited the apoptosis of glioma cells, indicating that miR-155-5p plays a tumor-promoting role in glioma.

miRNAs play their biological roles by binding to mRNAs at a posttranscriptional level, leading to mRNA cleavage or, more commonly, direct translational inhibition, mRNA degradation or a combination of the two [ 14, 15]. In this study, JARID2, which has been reported to enhance the TMZ sensitivity of glioma cells [13], was recognized as a direct downstream target of miR-155-5p. Herein, miR-155 was shown to target and inhibit JARID2 expression. In glioma cells, miR-155 inhibition played an antitumor role, whereas JARID2 knockdown promoted the viability and suppressed apoptosis of glioma cells. Moreover, JARID2 knockdown significantly attenuated the effects of miR-155 inhibition on glioma cells.

VPA is an antiepileptic drug that prevents seizures and treats patients with brain tumors. VPA has been found to suppress cell proliferation, elicit cell differentiation and apoptosis, and exert antiepileptic effects [ 14, 15]. Notably, VPA has been reported to exert antitumor activity in glioma cells [ 43, 44]. In this study, in VPA-treated glioma cells, miR-155 expression was indeed downregulated, and JARID2 expression was upregulated. Although multiple mechanisms of VPA exerting anticancer effects have been reported, VPA-induced changes in miR-155 and JARID2 expression suggest the involvement of the miR-155/JARID2 axis. As one of the most recognized epigenetic agents, VPA can affect DNA and histone methylation status and trigger histone acetylation, thereby influencing gene expression [ 45– 47]. Herein, the unmethylated level of the miR-155 promoter showed almost no difference between normal and glioma specimens; however, the methylated level of the miR-155 promoter was sharply increased in glioma tissues. Mechanically, MSP assay showed that VPA treatment caused no changes in the unmethylated level of the miR-155 promoter but significantly increased the methylated level of the miR-155 promoter, suggesting that VPA downregulates miR-155 expression by inducing methylation in the miR-155 promoter.

In conclusion, VPA regulates miR-155 expression by inducing methylation in the miR-155 promoter, and the miR-155/JARID2 axis is implicated in VPA-mediated inhibition of glioma cell viability and enhancement of glioma cell apoptosis ( Figure 8). This study illustrates a new mechanism of VPA treatment of gliomas, providing a novel strategy for the prevention and treatment of gliomas and related pathological processes.

Figure 8 .

Figure 8

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A schematic diagram illustrates the mechanism of VPA on glioma growth inhibition

VPA modulates the miR-155/JARID2 axis by enhancing the methylation level of the miR-155 promoter, thereby inhibiting glioma cell viability and enhancing glioma cell apoptosis.

Supporting information

227FigureS1-S2-TableS1

Supplementary Data

Supplementary Data is available at Acta Biochimica et Biophysica Sinica online.

COMPETING INTERESTS

The authors declare that they have no conflict of interest.

Funding Statement

This work was supported by the grants from the National Natural Science Foundation of China (No. 81803582) and the Hunan Provincial Natural Science Foundation of China (No. 2020JJ4896).

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