miR-506-5p targets MAPK7 to impede glioma growth and invasion through suppressing epithelial-mesenchymal transition and matrix metalloproteinases

Introduction

Gliomas represent a class of highly invasive, recurrent, and therapeutically challenging primary malignant tumors emanating from glial cells in the central nervous system (1,2). Histologically, they are classified into astrocytomas, oligodendrogliomas, and ependymomas, and are further stratified by the World Health Organization (WHO) into four malignancy grades. While grades I and II are considered low-grade gliomas (LGGs) with relatively indolent progression, grades III and IV constitute high-grade gliomas (HGGs), which are characterized by aggressive growth and dismal clinical outcomes (3). Among these, glioblastoma [GBM, WHO grade IV] is recognized as one of the most deadly primary brain tumors, with a median survival of approximately two years even under standard treatment (4). In the absence of therapeutic intervention, the median survival of GBM patients is merely 6.1 months, declining sharply to 3.2 months in individuals over 70 years of age (4). The management of gliomas encounters multiple obstacles, such as the infiltrative tumor development, the blood-brain barrier restricting drug administration, and the immunosuppressive properties of the tumor environment (5,6). Current conventional treatment protocols encompass maximal safe surgical resection, succeeded by radiation and chemotherapy. Current standard-of-care involves maximal safe surgical resection followed by radiotherapy and temozolomide-based chemotherapy. However, the invasive nature of HGGs often precludes complete resection, and therapeutic efficacy remains limited by tumor recurrence and treatment resistance (7,8). The high incidence rate and poor prognosis underscores the urgent need to identify novel molecular biomarkers for early diagnosis and to develop more effective targeted therapies (9).

Non-coding RNAs have lately emerged as significant regulators of gene expression in glioma, garnering substantial scientific attention for their potential as diagnostic biomarkers and therapeutic targets. Among them, microRNAs (miRNAs) contribute significantly to glioma pathogenesis by acting as tumor suppressors or oncogenes, modulating oncogenic behaviors through the regulation of specific target genes. For instance, miR-27a originating from astrocyte exosomes can enhance glioma cell proliferation and invasion, but miR-128-3p inhibits the expression of KLHDC8A, hence reducing the malignant behavior of glioma cells (10). Moreover, glioma cells transfer miR-25-3p to macrophages by exosomes, prompting M2 polarization via the PHLPP2/PI3K-AKT pathway, consequently altering the immunosuppressive context (11). Multiple studies argue that miR-506 contributes as a tumor suppressor in various solid tumors, including renal cell carcinoma, breast cancer, ovarian cancer, and gastric cancer (12,13). By sequestering miR-506-5p, the FOXD2-AS1 facilitates tumor proliferation, migration, and EMT in gliomas (14).

MAPK7 (also known as ERK5), a key component of the MAPK family, regulates critical biological processes encompassing cell proliferation, migration, differentiation, and survival (15,16). Although its oncogenic roles have been established in several cancer types, the functional significance and regulatory mechanisms of MAPK7 in glioma remain incompletely understood. Based on bioinformatics predictions from the TargetScan database, we identified a potential targeting relationship between miR-506-5p and MAPK7. This study identifies miR-506-5p as a tumor suppressor that directly targets MAPK7, thereby inhibiting glioma cell proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) to suppress malignant progression. We present this article in accordance with the ARRIVE and MDAR reporting checklists (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2026-0761/rc).

Methods

Patients and samples

The research involved 30 glioma patients (aged 18–40 years) admitted to the Department of Neurosurgery at The First Affiliated Hospital of Kunming Medical University between September 2022 and September 2023. Glioma tissues and corresponding adjacent tissues were collected during surgery. All recruited individuals had not received preoperative radiotherapy or chemotherapy, and those with comorbidities such as diabetes or hypertension were excluded. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study protocol was approved by the Medical Ethics Committee of The First Affiliated Hospital of Kunming Medical University (Approval No. [2025] Ethics Approval L No. 324), and all patients provided written informed consent.

Cell culture

This study employed the human microglial cell line HMC3 and four human glioma cell lines (U87, HS683, SNB-19, and KNS-89) as in vitro experimental models. All cell lines were obtained from commercial sources: HMC3 (CL-0620), U87 (CL-0238), and HS683 (CL-0362) from Wuhan Procell Life Science Technology Co., Ltd. (Wuhan, China); SNB-19 (STM-CL-5319) and KNS-89 (STM-CL-5547) from Stemrecell (Shanghai, China) Biotechnology Co., Ltd. Cells were maintained in high-glucose DMEM supplemented with 10% fetal bovine serum (FBS) (HyClone, SH30070.03, Utah, USA) and 1% penicillin-streptomycin at 37 ℃ in a 5% CO₂ humidified incubator. All experiments were conducted using cells in the logarithmic growth phase, with routine subculturing and cryopreservation performed according to standard protocols.

Reverse transcription quantitative polymerase chain reaction (RT-qPCR)

Total RNA was extracted from cells using TRIzol reagent (DP419, TianGen Biotech, Beijing, China) following the manufacturer’s protocol. The concentration and purity of RNA were evaluated using a micro-spectrophotometer (840-317400, Thermo Fisher Scientific, Massachusetts, USA), and its integrity was verified using 1% agarose gel electrophoresis. Following the removal of genomic DNA, complementary DNA (cDNA) was generated from total RNA utilizing a reverse transcription kit (RR037Q, Takara, Shiga, Japan) according to the provided instructions. The RT-qPCR reaction was prepared using the TB Green Premix Ex Taq II (Tli RNaseH Plus) (RR820Q, Takara) according to the manufacturer’s instructions and amplified using an ABI 7000 series real-time PCR instrument. miR-506-5p was normalized using U6 as an internal control, while other messenger RNAs (mRNAs) were normalized using GAPDH as an internal control. The 2^–ΔΔCt method was used for analyzed triplicate experimental analysis, primer sequences are detailed in Table S1.

Construction of lentiviral vectors and establishment of stable cell lines

The overexpression and knockdown lentiviral vectors for miR-506-5p and MAPK7, along with their corresponding negative control (NC) vectors, were constructed and packaged by Shanghai Genomeditech Co., Ltd. (Shanghai, China). All interfering sequences and overexpression sequences were designed, validated, synthesized by Shanghai Genomeditech Co., Ltd., and cloned into lentiviral backbone vectors. All vectors carried the enhanced green fluorescent protein (EGFP) reporter gene. Lentiviral particles were produced in U87 cells via co-transfection, with supernatants collected at 48 and 72 h, concentrated by ultracentrifugation, and titrated for subsequent use. For stable transfection, logarithmically growing U87 cells were infected with concentrated lentivirus at a multiplicity of infection (MOI) of 2 in medium containing 5 µg/mL polybrene to enhance infection efficiency. After 48 h, infection efficiency was assessed by observing EGFP expression under an inverted fluorescence microscope (ECLIPSE Ts2, Nikon Corp., Tokyo, Japan). No fluorescence was observed in the blank control group (non-transduced cells), while green fluorescence was observed in all lentiviral-transduced groups. Infection was considered successful when the fluorescence rate exceeded 80%. Stable cell lines were then selected with puromycin, and knockdown or overexpression efficiencies were validated by RT-qPCR.

Dual-luciferase reporter assay

The targeting of miR-506-5p to the MAPK7 3'-UTR was validated by dual-luciferase reporter assay in 2,93T cells. Cells were seeded in 96-well plates and co-transfected using Lipofectamine 3000 (Yeasen) with a firefly luciferase reporter plasmid (0.2 µg/well), the pRL-TK Renilla plasmid (0.01 µg/well), and either OE-miR-506-5p or NC (100 nM). After 48 h, luciferase activity was measured using the Dual-Luciferase^® Assay System, and firefly signals were normalized to Renilla.

5-ethynyl-2'-deoxyuridine (EdU) staining

The implications of miR-506-5p on U87 cell proliferation was evaluated utilizing an EdU assay kit (C0071S, Beyotime, Shanghai, China). U87 cells were EdU-pulsed (20 µM, 2 h), fixed, permeabilized, processed via the Click reaction (30 min, dark), and counterstained with DAPI (1 µg/mL, MBD0015, Sigma-Aldrich, Steinheim am Albuch, Germany). Fluorescence microscopy was used for visualization, with EdU-positive (red) and DAPI-stained (blue) cells quantified to calculate the proliferation rate.

Scratch wound assay

Cell migration was assessed in U87 cells using a scratch wound assay. Cells were cultivated to confluence on 6-well plates, and incisions were created using a sterile pipette tip. Following PBS washing, serum-free medium was introduced, and wound closure was documented at 0, 12, and 24 h. Migration rates were computed in ImageJ using the formula: (A₀ − A_t)/A₀ × 100%.

Transwell assay

Cell invasion was assessed with Matrigel-coated Transwell inserts. U87 cells (1×10⁵/mL) were inoculated in serum-free medium in the upper chamber, with 10% FBS media beneath serving as a chemoattractant. Following a 48-hour period, non-invading cells were eliminated, and the invading cells on the bottom membrane were fixed, stained with crystal violet, and enumerated from five random fields per insert.

Xenograft tumor model in mice

Five-week-old male BALB/c nude mice were bought from Beijing spfbiotech Co., Ltd. [certificate No. SCX (Jing) 2019-0010] (Beijing, China) and acclimatized for one week in a pathogen-free environment before the study. Animal experiments were performed under a project license (BST-PZ-MICE-20240331-01) granted by the Animal Care and Use Committee of Yunnan Beisitai Biotechnology Co., Ltd., in compliance with the protocols conformed to the Guidelines for the Care and Use of Laboratory Animals published by the National Institutes of Health. A protocol was prepared before the study without registration. A collection of 24 female BALB/c nude mice were randomly allocated into four groups (n=6 per group): knockdown NC (KD-NC), knocking down miR-506-5p (KD-miR), overexpression NC (OE-NC), and overexpressing miR-506-5p (OE-miR). U87 cells, stably transfected with the appropriate lentiviral vectors, were collected, resuspended in PBS, and adjusted to a concentration of 1×10⁷ cells/mL. Each mouse received an intramuscular injection of 0.2 mL of the cell suspension in the right axilla (17). Tumor growth was assessed weekly by measuring tumor length (a) and width (b), with volume computed using the formula: V=1/2 × a × b². Five weeks post-injection, all mice were anesthetized with 5% isoflurane and euthanized using cervical dislocation, after which the xenograft tumors were carefully excised and weighed.

Immunohistochemistry (IHC)

After deparaffinization and antigen retrieval in EDTA buffer, tumor sections were blocked and incubated with Ki-67 antibody (1:500, AF0198, Affbiotech, Changzhou, China) overnight at 4 ℃, followed by horseradish peroxidase (HRP)-conjugated secondary antibody for 50 min. Signals were developed with DAB (DA1010, Solarbio, Beijing, China), counterstained with hematoxylin, and mounted. Ki-67-positive cells were counted from five random fields per section using ImageJ.

Hematoxylin and eosin (H&E) staining

Tumor tissues were fixed, paraffin-embedded, and sectioned at 4 µm. After deparaffinization and rehydration, sections were stained with H&E, dehydrated, cleared, and mounted with neutral resin for pathological evaluation.

Western blot analysis

Proteins were extracted from cells or tumor tissues using radioimmunoprecipitation assay (RIPA) buffer with protease inhibitors (P0013B, Beyotime) and quantified by bicinchoninic acid (BCA) assay (P0012, Beyotime). After denaturation, proteins were separated on FuturePAGE™ gels and transferred to polyvinylidene fluoride (PVDF) membranes. Membranes were blocked with 5% BSA and incubated overnight at 4 ℃ with primary antibodies against MAPK7 (1:2,000, DF6835, Affbiotech), MMP9 (1:3,000, AF5228, Affbiotech), MMP12 (1:1,000, DF7686, Affbiotech), E-cadherin (1:2,000, AF0131, Affbiotech), N-cadherin (1:3,000, AF6710, Affbiotech), Vimentin (1:2,000, AF7013, Affbiotech), and β-actin (1:1,000, AF7018, Affbiotech). Following incubation with HRP-conjugated secondary antibody, signals were detected usk ing ECL substrate (34094, Thermo Fisher Scientific) and quantified with ImageJ. Protein expression levels were normalized to β-actin.

Statistical analysis

Data are expressed as mean ± standard deviation (SD) and analyzed in GraphPad Prism 9.0. Student’s t-test (two groups) or one-way analysis of variance (ANOVA) with Tukey’s test (multiple groups) was applied, with P<0.05 defined as statistically significant.

Results

Expression of miR-506-5p and its potential target gene MAPK7 in Glioma

In accordance with its presumed tumor-suppressive function, miR-506-5p expression was considerably diminished in glioma tissues (Figure 1A). Bioinformatics analysis using TargetScan identified a putative binding site for miR-506-5p within the 3'-UTR of MAPK7, which is complementary to the region of the miRNA (Figure 1B). To experimentally validate this interaction, a luciferase reporter vector containing the predicted binding site was constructed. Dual-luciferase reporter assays exhibited a significant decline in luciferase activity, confirming that miR-506-5p directly targets MAPK7 (Figure 1C).

Figure 1 Low expression of miR-506-5p in glioma and its targeted regulation of MAPK7. (A) miR-506-5p expression in paired glioma clinical specimens (n=30). (B) Schematic of the predicted miR-506-5p binding site within the 3'-UTR of MAPK7, as identified by TargetScan. (C) Dual-luciferase reporter assays validating direct targeting of MAPK7 by miR-506-5p (n=3). (D) MAPK7 mRNA expression in 30 paired glioma and normal adjacent tissues measured via RT-qPCR. (E,F) Expression levels of miR-506-5p (E) and MAPK7 (F) in human normal glial (HMC3) and glioma cell lines (n=3). (G) Knockdown efficiency of three miR-506-5p knockdown lentiviral constructs (KD-miR#1/2/3) in U87 cells (n=3). (H) Overexpression efficiency of miR-506-5p in U87 cells transfected with the overexpression lentiviral construct (OE-miR) (n=3). *, P<0.05; **, P<0.01; ***, P<0.001. KD-miR, knocking down miR-506-5p; KD-NC, knockdown negative control; mRNA, messenger RNAs; MUT, mutant; OE-miR, overexpression of miR-506-5p; OE-NC, overexpression negative control; RT-qPCR, reverse transcription quantitative polymerase chain reaction; WT, wild type.

Consistent with miR-506-5p-mediated regulation, MAPK7 mRNA levels were markedly elevated in glioma tissues (Figure 1D). Similarly, in glioma cell lines U87 and KNS89, miR-506-5p expression was significantly lower, while MAPK7 expression was higher, relative to the HMC3 (Figure 1E,1F). Based on the pronounced low miR-506-5p and high MAPK7 expression profile, U87 cells were selected for subsequent functional experiments.

To systematically investigate the biological function of miR-506-5p in gliomas, we constructed lentiviral vectors for KD-miR and for OE-miR, using empty lentiviral vectors as NCs (KD-NC/OE-NC). After transducing U87 cells with the aforementioned lentiviruses, we successfully established stably transduced cell lines through puromycin selection. RT-qPCR validation results showed that the KD-miR construct achieved the highest knockdown efficiency, while OE-miR effectively induced the upregulation of miR-506-5p (Figure 1G,1H).

miR-506-5p suppresses malignant phenotypes in glioma (Figure 2)

Figure 2 miR-506-5p suppresses glioma malignancy by regulating MAPK7 and EMT. (A,B) Proliferation assessed by EdU assay. (A) Representative images (scale bar =50 µm). (B) Quantification of EdU-positive cells (n=5). (C,D,F) Migration analyzed by wound healing assay. (C) Representative images at 0, 12, and 24 h (scale bar =100 µm). (D) Migration rate quantification. (E,G) Invasion evaluated by Transwell assay. (E) Images of invaded cells (scale bar =100 µm; crystal violet stained). (G) Quantification of invaded cells (n=3). (H,I) Protein expression analyzed by Western blotting. (H) Representative blots of MAPK7, MMP9, MMP12, E-cadherin, N-cadherin, and Vimentin. (I) Quantitative analysis of protein level (n=3). *, P<0.05; **, P<0.01; ***, P<0.001. EdU, 5-ethynyl-2'-deoxyuridine; EMT, epithelial-mesenchymal transition; KD-miR, knocking down miR-506-5p; KD-NC, knockdown negative control; OE-miR, overexpression of miR-506-5p; OE-NC, overexpression negative control.

To determine the functional impact of miR-506-5p on glioma malignancy, we performed EdU proliferation assays. KD-miR significantly promoted cell proliferation, whereas overexpression (OE-miR) strongly suppressed proliferation (Figure 2A,2B). In scratch wound assays, miR-506-5p silencing enhanced cell migration at both 12 and 24 h, while its overexpression impeded migration (Figure 2C,2D,2F). Similarly, transwell invasion experiments demonstrated a large increase in the amount of invading cells after miR-506-5p knockdown and a notable decrease following miR-506-5p overexpression (Figure 2E,2G). Western blot analysis further demonstrated that miR-506-5p knockdown upregulated its direct target MAPK7, along with MMP9, MMP12, N-cadherin, and vimentin, and downregulated the epithelial marker E-cadherin. Conversely, miR-506-5p overexpression suppressed MAPK7, MMP9, MMP12, N-cadherin, and vimentin expression, while elevating E-cadherin levels (Figure 2H,2I). Collectively, these findings suggest that miR-506-5p functions as a tumor suppressor in glioma by restraining proliferation, migration, and invasion, presumably by targeting MAPK7 and regulating EMT-related protein expression.

miR-506-5p suppresses glioma malignancy by targeting MAPK7 (Figure 3)

Figure 3 miR-506-5p suppresses malignant phenotypes in glioma by targeting MAPK7. (A,D) Cell proliferation evaluated by EdU assay under rescue conditions. (A) Fluorescence images (scale bar =50 µm). (D) Quantification of EdU-positive cells (n=5). (B,E,F) Cell migration assessed by wound healing assay. (B) Bright-field images at 0, 12, and 24 h (scale bar =100 µm). (E,F) Quantification of wound closure rates at 12 h (E) and 24 h (F) (n=3). (C,G) Cell invasion analyzed by Transwell assay. (C) Images of invaded cells (scale bar =100 µm; crystal violet stained). (G) Quantification of invaded cells per field (n=3). *, P<0.05; **, P<0.01; ***, P<0.001. EdU, 5-ethynyl-2'-deoxyuridine; KD-miR, knocking down miR-506-5p; KD-NC, knockdown negative control; OE-M7, overexpression of MAPK7; OE-miR, overexpression of miR-506-5p; OE-NC, overexpression negative control; sh-M7, short hairpin RNA targeting MAPK7.

To determine whether miR-506-5p exerts its tumor-suppressive effects by directly targeting MAPK7, we performed rescue experiments in U87 cells that had been transfected with miR-506-5p knockdown or overexpression vectors. EdU proliferation experiments found that MAPK7 knockdown considerably diminished the increased proliferation resulting from miR-506-5p silencing, as indicated by a substantial decrease in proliferating cells in the KD-miR + sh-M7 group relative to KD-miR alone. Conversely, restoring MAPK7 expression in OE-miR cells partially reversed the anti-proliferative effect, leading to a significant increase in EdU-positive cells (OE-miR + OE-M7 vs. OE-miR; Figure 3A,3D). Wound healing experiments clarified that the concurrent knockdown of MAPK7 considerably mitigated the pro-migratory phenotype induced by miR-506-5p suppression, resulting in markedly reduced migration rates at 12 and 24 h in the KD-miR + sh-M7 group compared to KD-miR alone. Similarly, MAPK7 co-overexpression restored migration ability in OE-miR cells (Figure 3B,3E,3F). Transwell invasion assays further demonstrated that MAPK7 modulation reversed miR-506-5p-mediated invasion phenotypes. The quantity of infiltrating cells was markedly diminished in the KD-miR + sh-M7 group relative to KD-miR, whereas MAPK7 overexpression partially rescued invasion suppression in OE-miR cells (Figure 3C,3G). Western blot analysis displayed that MAPK7 co-knockdown in miR-506-5p-silenced cells downregulated MAPK7, MMP9, MMP12, N-cadherin, and vimentin, and increased E-cadherin. In contrast, MAPK7 co-overexpression in OE-miR cells upregulated these mesenchymal markers and reduced E-cadherin (Figure 4).

Figure 4 MAPK7 restoration reverses miR-506-5p-mediated regulation of EMT and invasion-related proteins. (A) Representative Western blots of indicated proteins in U87 cells under rescue conditions. (B,C) Quantification of protein level (n=3). *, P<0.05; **, P<0.01; ***, P<0.001. EMT, epithelial-mesenchymal transition; KD-miR, knocking down miR-506-5p; KD-NC, knockdown negative control; OE-M7, overexpression of MAPK7; OE-miR, overexpression of miR-506-5p; OE-NC, overexpression negative control; sh-M7, short hairpin RNA targeting MAPK7.

miR-506-5p suppresses glioma progression in vivo

The U87 xenograft model was created in BALB/c nude mice to assess the in vivo anticancer efficacy of miR-506-5p. Tumors derived from KD-miR cells exhibited significantly increased growth, as reflected by larger tumor volumes and higher weights compared to the KD-NC group. In contrast, OE-miR strongly suppressed tumor growth, resulting in markedly reduced tumor volume and weight relative to the OE-NC group (Figure 5A-5C). Histological analysis by H&E staining revealed that KD-miR tumors displayed increased nuclear size and density, along with substantial inflammatory infiltration, whereas OE-miR tumors showed more regular nuclear morphology and diminished inflammatory responses (Figure 5D). Ki-67 IHC further indicated that the reduction of miR-506-5p considerably elevated the quantity of Ki-67-positive cells, while its overexpression reduced Ki-67 positivity (Figure 5E,5F). Western blot analysis of tumor lysates indicated that sh-miR-506-5p upregulated MAPK7, MMP9, MMP12, N-cadherin, and vimentin, and downregulated E-cadherin. Conversely, OE-miR downregulated these mesenchymal and invasion-related proteins and increased E-cadherin expression (Figure 5G,5H). Collectively, these in vivo findings demonstrate that miR-506-5p constrains glioma growth and malignant progression by targeting MAPK7 and modulating downstream effectors involved in invasion and EMT.

Figure 5 miR-506-5p impedes glioma development in vivo. (A) Representative xenograft tumors (n=6). (B) Final tumor weights. (C) Tumor growth curves. (D) H&E staining of tumor sections (scale bar =50 µm). (E,F) Ki-67 IHC staining and quantification of positive cells (scale bar =50 µm). (G) Western blot analysis of MAPK7, MMP9, MMP12, and EMT-related proteins in xenograft tissues. (H) Quantification of protein expression level (n=3). *, P<0.05; **, P<0.01; ***, P<0.001. EMT, epithelial-mesenchymal transition; H&E, hematoxylin and eosin; IHC, immunohistochemistry; KD-miR, knocking down miR-506-5p; KD-NC, knockdown negative control; OE-miR, overexpression of miR-506-5p; OE-NC, overexpression negative control.

Discussion

The consistently decreased expression of miR-506-5p across multiple malignancies strongly indicates its fundamental role as a pan-cancer tumor suppressor. In gastric cancer, a luciferase reporter assay confirmed the direct binding of miR-506-5p to the 3'-UTR of PAK1; furthermore, in the gastric cancer cell lines AGS and SGC-7901, overexpression of miR-506-5p significantly reduced both the mRNA and protein levels of PAK1 (18). Similarly, in colorectal cancer, the long non-coding RNA LINC00473 promotes tumor progression by acting as a molecular sponge for miR-506-5p, thereby upregulating the oncogenic RNA helicase DDX5—a mechanism that further substantiates the tumor-suppressive function of miR-506-5p in gastrointestinal malignancies (19). In gliomas, accumulating evidence from multiple independent studies has consistently documented miR-506-5p downregulation, with its expression levels showing inverse correlation with tumor grade and positive association with patient survival (20). Accumulating evidence has established that miR-506-5p is frequently downregulated in glioma tissues, with its expression levels closely correlated with pathological grade, proliferation, invasion, and apoptotic activity. Functional researches have proven that that miR-506-5p overexpression suppresses glioma cell proliferation, migration, and invasion, while promoting apoptosis and inducing cell cycle arrest (21,22). Additionally, miR-506-5p has been reported to inhibit glioma progression through targeting GPNMB (23). In line with these findings, we identify miR-506-5p as a frequently downregulated tumor suppressor in glioma. Functional restoration of miR-506-5p suppresses malignancy and in vivo growth, validating its therapeutic relevance.

Through dual-luciferase reporter assays, we provided direct experimental evidence that miR-506-5p targets the 3'-UTR of MAPK7, consistent with the observed reduction in MAPK7 expression following miR-506-5p overexpression. MAPK7 (ERK5), a crucial component of the MAPK signaling cascade, functions as a potent oncogene across various tumor types. In osteosarcoma, MAPK7 overexpression correlates with advanced disease progression, therapy resistance, and poor survival outcomes, while its inhibition effectively suppresses cancer cell proliferation, migration, and invasion (24). Similarly, in osteosarcoma, oral squamous cell carcinoma (OSCC), prostate cancer, triple-negative breast cancer, MAPK7 drives malignant phenotypes including proliferation and invasion, and its knockdown significantly attenuates these oncogenic behaviors (25). Supporting these findings, fluorescence in situ hybridization (FISH) analyses have identified MAPK7 gene amplification in subsets of lung squamous cell carcinomas (4%, 3/74) and esophageal cancers (2%, 1/54), further implicating MAPK7 signaling in the pathogenesis of these malignancies (26). Our study extends these observations to glioma, demonstrating significant MAPK7 upregulation in clinical specimens and cell lines. More importantly, we provide compelling mechanistic evidence that miR-506-5p suppresses multiple malignant behaviors of glioma cells primarily through direct targeting of MAPK7.

During the EMT process, molecular changes occur in epithelial cells, including downregulation of E-cadherin and upregulation of beta-catenin and N-cadherin, leading to loss of polarity and enhanced migration and invasion capabilities (27). During malignant progression, elevated N-cadherin expression emerges as a hallmark of EMT, correlating strongly with enhanced invasiveness and metastatic potential. Vimentin maintains cytoskeletal integrity and facilitates cell motility during EMT-driven remodeling (28). Its upregulation accompanies the characteristic loss of E-cadherin during EMT. E-cadherin, a key epithelial cadherin, maintains intercellular adhesion in epithelial tissues; its functional impairment facilitates tumor cell dissociation from primary sites and promotes invasion and metastasis (29). Our findings demonstrate that miR-506-5p overexpression significantly downregulated N-cadherin and vimentin while upregulating E-cadherin, implying that miR-506-5p may suppress glioma malignancy by reversing EMT. Furthermore, MAPK7 co-overexpression in miR-506-5p-overexpressing cells counteracted these effects, restoring N-cadherin and vimentin levels and reducing E-cadherin expression. These results confirm that miR-506-5p regulates the EMT phenotype primarily through targeted inhibition of MAPK7.

Matrix metalloproteinases (MMPs), particularly MMP9 and MMP12, play pivotal roles in tumor invasion by degrading extracellular matrix (ECM) components. MMP9 (gelatinase B) cleaves type IV collagen, gelatin, and elastin, facilitating tumor cell dissemination, and its overexpression is linked to poor prognosis in multiple cancers (29). Upregulation of MMP2/MMP9 expression can enhance the invasion and metastatic abilities of OSCC (30). The MAPK7/MMP9 axis has been implicated in bone cancer metastasis (31), while MMP12, mainly secreted by macrophages, degrades elastin and other ECM proteins and is associated with tumor progression. Studies also indicate that MMP9 upregulation correlates with advanced breast cancer (32), and warfarin suppresses colorectal cancer metastasis by inhibiting MMP2/MMP9 and EMT (33). Similarly, TLR2 signaling upregulates the expression of MMP2 and MMP9 in glial stem cells, promoting glioma invasion (34), and the miR-4516/OTX1 axis inhibits pancreatic cancer progression by downregulating MMP2/MMP9 (35). This study revealed that the suppression of miR-506-5p elevated the expression of MMP9 and MMP12, whereas its overexpression suppressed both. Critically, MAPK7 co-knockdown reversed the upregulation of MMP9 and MMP12 induced by miR-506-5p silencing, while MAPK7 co-overexpression restored their expression in miR-506-5p-overexpressing cells. These data support that miR-506-5p modulates MMP9 and MMP12 expression via MAPK7 targeting.

Despite these findings, several limitations should be acknowledged. First, the functional experiments were primarily conducted in U87 cells; validation in additional glioma cell lines and patient-derived models would enhance the generalizability of our conclusions. Second, although we demonstrated that miR-506-5p directly targets the 3'-UTR of MAPK7, the downstream signaling pathways through which MAPK7 mediates EMT and MMP regulation warrant further investigation. Third, the in vivo findings were obtained using xenograft models; future studies employing orthotopic models or genetically engineered mouse models would better recapitulate the tumor microenvironment and provide more definitive evidence of therapeutic efficacy. Finally, the clinical relevance of the miR-506-5p/MAPK7 axis requires validation in larger cohorts with longitudinal follow-up to assess its potential as a prognostic biomarker or therapeutic target. Although our findings are primarily derived from U87 cells and xenograft models, the evolutionary conservation of the miR-506-5p seed region and the MAPK7 3'-UTR binding site suggests that this regulatory axis may be functionally relevant across mammalian species. Moreover, the consistent downregulation of miR-506-5p and upregulation of MAPK7 in human glioma specimens supports the translational potential of targeting this pathway for therapeutic intervention in patients.

Conclusions

This research illustrates that miR-506-5p works as a tumor suppressor in glioma, where it is significantly downregulated in clinical specimens. Mechanistically, miR-506-5p directly targets MAPK7, leading to the suppression of downstream MMPs and inhibition of the EMT process, thereby attenuating tumor malignant phenotypes. We acknowledge several limitations of this work. The reliance on the U87 glioma cell line and a subcutaneous xenograft model may not fully capture the heterogeneity of glioma or its native brain microenvironment. In addition, the downstream signaling network of MAPK7 remains incompletely elucidated. Future investigations should employ more diverse cellular models, orthotopic xenograft systems, and expanded proteomic or transcriptomic approaches to further dissect the MAPK7-regulated effector pathways. Such efforts will enhance the translational relevance of targeting the miR-506-5p/MAPK7 axis in glioma therapy.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the ARRIVE and MDAR reporting checklists. Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2026-0761/rc

Data Sharing Statement: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2026-0761/dss

Peer Review File: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2026-0761/prf

Funding: The study was supported by a grant from the Yunnan Provincial Basic Research Program (Kunming Medical University Joint Special Project, No. 202201AY070001-084).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2026-0761/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Medical Ethics Commitee of The First Affiliated Hospital of Kunming Medical University (Approval No. [2025] Ethics Approval L No. 324) and informed consent was obtained from all patients. Animal experiments were performed under a project license (BST-PZ-MICE-20240331-01) granted by the Animal Care and Use Committee of Yunnan Beisitai Biotechnology Co., Ltd., in compliance with the Guidelines for the Care and Use of Laboratory Animals published by the National Institutes of Health.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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