Mannose Receptor C Type 2 Predicts Poor Outcome in Glioma and is Associated With Invasive Phenotypes and β-Catenin/EMT-related Changes

Summary

Public-cohort analyses and glioma cell–based experiments identify MRC2 as a poor-prognosis marker associated with enhanced proliferation, migration, invasion, and β-catenin/EMT-related changes in glioma.

Abstract

Glioma remains a lethal malignancy of the central nervous system, and additional biomarkers that improve prognostic stratification and inform therapeutic exploration are still needed. This study evaluated mannose receptor C-type 2 (MRC2/Endo180) by integrating public transcriptomic cohorts with in vitro validation. Expression profiles and matched clinical annotations were obtained from The Cancer Genome Atlas (TCGA) and the Genotype-Tissue Expression (GTEx) project for tumor–normal comparisons and pan-cancer screening, and prognostic associations were further examined in the Chinese Glioma Genome Atlas (CGGA) cohort. MRC2 expression was assessed in glioma cell lines by reverse transcription quantitative PCR (RT-qPCR) and immunoblotting, and stable MRC2 knockdown was established in U87 and U251 cells using lentiviral short hairpin RNAs (shRNAs). Cell growth and clonogenicity were evaluated using CCK-8 and colony formation assays, whereas migration and invasion were assessed using porous-membrane insert assays. CDK4, CDK6, and β-catenin/epithelial–mesenchymal transition (EMT)-related markers were analyzed by immunoblotting. Across datasets, MRC2 was elevated in glioma, and higher expression was associated with worse overall survival, disease-specific survival, and progression-free interval. In vitro, MRC2 depletion reduced proliferation, clonogenicity, migration, and invasion, accompanied by decreased CDK4 and CDK6 protein levels and changes in β-catenin and EMT-related markers. Together, these findings support MRC2 as a candidate prognostic biomarker associated with aggressive glioma phenotypes and warrant further mechanistic and in vivo investigation.

Introduction

Gliomas represent the most common primary tumors arising in the central nervous system and account for a substantial fraction of intracranial neoplasms^1,2. Epidemiologic studies estimate an incidence on the order of a few cases per 100,000 people each year, with geographic and sex-related differences reported across populations^3,4. Clinically, gliomas span a broad spectrum of behavior: lower-grade gliomas can follow a prolonged course, whereas high-grade disease—particularly glioblastoma—remains rapidly fatal for most patients^5,6,7,8. Modern WHO classifications integrate histology with key molecular features (e.g., IDH mutation, TERT promoter alteration, MGMT promoter methylation, and 1p/19q co-deletion), which refine diagnosis and inform prognosis^9,10. Despite multimodal management combining surgery, radiotherapy, and chemotherapy, recurrence is common, and long-term outcomes for high-grade tumors remain poor. Therefore, identifying additional prognostic markers and targetable vulnerabilities remains an urgent priority.

MRC2 (mannose receptor C-type 2; also referred to as Endo180/CD280/uPARAP) is a transmembrane member of the C-type lectin receptor family¹¹. It is enriched in stromal and immune compartments, such as fibroblasts and macrophages, and participates in extracellular matrix (ECM) turnover, fibrosis-related remodeling, and tumor–microenvironment interactions^12,13. Structurally, MRC2 contains an N-terminal cysteine-rich region, a fibronectin type II domain that binds collagen, multiple C-type lectin-like domains, a transmembrane segment, and a cytoplasmic tail^14,15. Through collagen internalization and remodeling, MRC2 can facilitate cell motility and tissue invasion, and elevated MRC2 has been linked to aggressive behavior in several cancers, including breast, pancreatic, prostate, melanoma, and glioblastoma^{11,14,16,17,18}. Prior work also suggests connections between MRC2 and pro-invasive signaling programs (e.g., TGF-β–associated pathways), matrix metalloproteinase activity, and EMT-like phenotypic changes, as well as roles in cancer-associated fibroblast and macrophage states^{17,18,19,20,21}. However, the clinical significance of MRC2 in glioma and the functional consequences of MRC2 dysregulation in glioma cells remain unclear.

Although MRC2 has been implicated in extracellular matrix remodeling and invasive behavior in several malignancies, its clinical significance in glioma has not been systematically established across independent transcriptomic cohorts, and its phenotype-associated effects in glioma cells have not been evaluated within an integrated framework combining public dataset analysis with functional validation. This study, therefore, hypothesized that MRC2 is associated with adverse clinical outcomes and malignant phenotypes in glioma. To address this question, pan-cancer screening, independent glioma-cohort validation, and stable knockdown experiments in glioma cell lines were integrated to evaluate the prognostic significance of MRC2 and its association with proliferation, migration, invasion, and changes in β-catenin/EMT-related markers. Because MRC2 belongs to the mannose receptor family and glioma progression is also shaped by the tumor microenvironment, an additional exploratory analysis of MRC1 expression by IDH status was included to provide contextual information on immune-related differences across glioma subtypes.

The novelty of this work lies in the combined assessment of MRC2 across public glioma datasets and in vitro functional assays, thereby providing complementary clinical and experimental evidence for its relevance in glioma. Compared with single-cohort analyses or cell-only studies, this integrated workflow offers several practical advantages. The incorporation of TCGA, GTEx, and CGGA enables cross-cohort validation and reduces dependence on a single dataset. In addition, coupling public-cohort analyses with glioma cell knockdown assays links clinical associations to phenotype-level evidence. Finally, the use of complementary readouts, including RT-qPCR, immunoblotting, CCK-8, colony formation, and porous-membrane insert assays, enables the assessment of transcriptional, protein-level, proliferative, and motility-related changes in parallel. The overall study design and analytical workflow are summarized in Figure 1. Together, this strategy improves the robustness and biological interpretability of the findings relative to bioinformatics-only or single-assay approaches.

Protocol

This study used publicly available, de-identified datasets (TCGA, GTEx, and CGGA) and established commercial cell lines. No newly recruited human participants, identifiable patient information, or patient-derived specimens were involved. All analyses were conducted in accordance with relevant institutional and international guidelines for the use of publicly available data. Therefore, no additional institutional ethics approval or informed consent was required for this study.

1. Bioinformatic workflow and data preprocessing

Gene-expression profiles and matched clinical annotations were obtained from publicly available resources, including The Cancer Genome Atlas (TCGA), the Genotype-Tissue Expression (GTEx) project, and the Chinese Glioma Genome Atlas (CGGA). Expression matrices and clinical metadata were imported into R and harmonized according to sample identifiers. Duplicate or unmatched entries were removed, and samples lacking essential survival information were excluded from survival analyses. MRC2 expression values and corresponding clinicopathologic variables were extracted for downstream analyses. For tumor-normal comparisons, MRC2 expression was compared across cancer types using TCGA-only data when matched normal tissues were available and using integrated TCGA–GTEx data when additional normal controls were required. For glioma-focused analyses, patients in the TCGA-LGG and CGGA cohorts were stratified into high- and low-expression groups according to the cohort median MRC2 expression level. Kaplan-Meier survival analysis, time-dependent receiver operating characteristic (ROC) analysis, and Cox proportional-hazards regression were then performed to evaluate the prognostic relevance of MRC2. Cox models included available covariates such as age, sex, WHO grade, and other clinicopathologic variables, depending on data completeness within each cohort. Subgroup analyses were performed according to variables including age, histologic subtype, WHO grade, IDH mutation status, 1p/19q codeletion status, and treatment-related information, where available. Figures were generated using standard statistical plotting workflows in R and GraphPad Prism.

2. Pan-cancer expression and survival analyses

MRC2 expression was compared between tumor and normal tissues across cancer types using TCGA and GTEx datasets. Differences were visualized using boxplots and summarized using radar plots. Cox proportional hazards regression analyses were performed to evaluate associations between MRC2 expression and overall survival (OS), disease-specific survival (DSS), and progression-free interval (PFI) across cancer types. Hazard ratios and corresponding significance levels were visualized using forest plots and heatmaps.

3. Prognostic validation and subgroup analyses in glioma cohorts

In the TCGA lower-grade glioma (TCGA-LGG) cohort, patients were stratified into high- and low-MRC2 expression groups. Kaplan–Meier survival analyses were performed to compare OS, DSS, and PFI between groups. Time-dependent receiver operating characteristic (ROC) curves were generated to assess predictive performance. Univariate and multivariate Cox regression analyses were conducted to evaluate the independent prognostic value of MRC2. External validation was performed using the CGGA glioma dataset, including Kaplan–Meier survival analysis, ROC analysis, and Cox regression. Subgroup analyses were conducted to evaluate MRC2 expression across clinicopathologic variables, including age, tumor grade, histological subtype, IDH mutation status, 1p/19q codeletion status, and treatment-related variables.

4. MRC1 expression in IDH-mutant versus IDH-wildtype gliomas

The association between MRC1 expression and IDH mutation status was evaluated using the GlioVis web platform and the TCGA GBMLGG dataset. Cases were grouped based on annotated IDH status into IDH-mutant and IDH-wildtype gliomas. MRC1 mRNA expression was queried within the selected dataset, and expression levels between the two groups were compared using the platform’s built-in statistical analysis function. Group-wise expression distributions were displayed as generated by the platform, and statistical significance was defined as P < 0.05.

5. Cell culture

Human glioma cell lines and normal human astrocytes were obtained from established cell repositories. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and maintained at 37 °C in a humidified incubator containing 5% CO₂ and approximately 95% relative humidity. U87 and U251 cells were selected for loss-of-function experiments because baseline screening across the tested glioma cell panel showed that these two cell lines expressed relatively higher MRC2 levels and were therefore suitable for knockdown-based functional analyses.

6. Lentiviral knockdown of MRC2

Short hairpin RNAs (shRNAs) targeting MRC2 and a non-targeting control were cloned into lentiviral vectors. Lentiviral particles were produced by co-transfecting HEK293T cells with transfer plasmids and packaging plasmids using a standard transfection reagent. Viral supernatants were collected at 48 h and 72 h after transfection, centrifuged at 2,100 × g for 5 min to remove packaging cells and cell debris, and then passed through a 0.45 µm filter before immediate use or storage at −80 °C¹⁹. Virus-containing supernatants were used immediately or aliquoted and stored at −80 °C. Repeated freeze–thaw cycles were avoided. Glioma cells were infected with lentiviral particles in the presence of polybrene. After infection, cells were selected with puromycin to establish stable knockdown cell lines. The sequences used were as follows: sh-MRC2-1: 5′-GAAATGAATGAGCAGCAAGAA-3′; sh-MRC2-2: 5′-CCGGTATTGCTATAAGGTGTT-3′; sh-NC: 5′-TTCTCCGAACGTGTCACGT-3′.

7. Validation of MRC2 knockdown by RT-qPCR and immunoblotting

Total RNA was extracted using TRIzol reagent, and cDNA was synthesized using a reverse-transcription kit according to the manufacturer’s instructions. Quantitative PCR was performed using SYBR Green chemistry under the following cycling conditions: initial denaturation at 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 30 s, with a dissociation curve analysis performed at the end of amplification. The primer sequences were as follows: MRC2 forward, 5′-GGCAAGGACAAGAAGTGCGTGT-3′; MRC2 reverse, 5′-CTTTGGTGACGTTGCTGCGCTT-3′; GAPDH forward, 5′-TCCACCCATGGCAAATTCC-3′; and GAPDH reverse, 5′-TCGCCCCACTTGATTTTGG-3′. Relative mRNA expression was calculated using the comparative C_t (2^-ΔΔCt) method with GAPDH as the internal control²⁰. Immunoblotting was performed in parallel to confirm MRC2 knockdown at the protein level. For RT-qPCR, three independent biological replicates were analyzed, and each sample was run in technical triplicate.

8. Cell proliferation assay

Cell proliferation was assessed using the CCK-8 assay. Cells were seeded into 96-well plates at an appropriate density and cultured for the indicated time periods. At each time point, CCK-8 reagent was added to each well and incubated according to the manufacturer’s instructions, after which absorbance at 450 nm was measured using a microplate reader. At least three independent biological replicates were performed.

9. Colony formation assay

Cells were seeded at low density into six-well plates and cultured for 10–14 days. Colonies were fixed with 4% paraformaldehyde for 15 min at room temperature and stained with 0.1% crystal violet for 15 min. Colonies were washed with phosphate-buffered saline (PBS), air-dried, and counted manually or using image analysis software²¹. At least three independent biological replicates were performed.

10. Cell migration and invasion assays

Cell migration and invasion were evaluated using porous-membrane insert assays²². For invasion assays, the upper inserts were pre-coated with extracellular matrix gel; for migration assays, uncoated inserts were used. Cells suspended in serum-free medium were added to the upper chamber, and medium containing serum was placed in the lower chamber as a chemoattractant. After incubation, nonmigrated cells on the upper membrane surface were gently removed with a cotton swab. Cells that had migrated or invaded the lower surface were fixed with 4% paraformaldehyde for 15 min, stained with 0.1% crystal violet for 15 min, rinsed with cold phosphate-buffered saline, air-dried, and imaged under an inverted microscope at ×200 magnification. Cells were counted in five randomly selected microscopic fields per insert. At least three independent biological replicates were performed.

11. Western blot analysis

Cells were lysed in RIPA buffer supplemented with protease inhibitors, and protein concentration was determined using a BCA assay. Equal amounts of total protein were separated by SDS-PAGE and transferred to PVDF membranes. Membranes were blocked in 5% non-fat milk for 1 h at room temperature and incubated with primary antibodies overnight at 4 °C. The following primary antibodies and working dilutions were used for western blotting: anti-MRC2 (1:5,000), anti-CDK4 (1:1,000), anti-CDK6 (1:2,000), anti-β-catenin (1:5,000), anti-E-cadherin (1:10,000), anti-N-cadherin (1:3,000), and anti-β-actin (1:5,000). After washing, membranes were incubated with horseradish peroxidase-conjugated goat anti-mouse IgG or goat anti-rabbit IgG secondary antibodies (1:5,000) for 1 h at room temperature. Signals were visualized using chemiluminescence, and band intensities were quantified and normalized to β-actin. At least three independent biological replicates were performed.

12. Statistical analysis

Statistical analyses were performed using R and GraphPad Prism. Survival curves were compared using the log-rank test. Cox proportional hazards regression models were used for univariate and multivariate analyses. For cell-based experiments, data are presented as mean ± SD. Before parametric testing, normality was assessed using the Shapiro–Wilk test. For normally distributed data, differences between two groups were evaluated using Student’s t-test, and comparisons among multiple groups were performed using one-way ANOVA. If normality assumptions were not satisfied, the corresponding nonparametric tests were used. P values were adjusted for multiple testing using the Benjamini–Hochberg false discovery rate procedure^23,24. A two-sided P < 0.05 was considered statistically significant.

Results

Pan-cancer expression and prognostic landscape of MRC2
Pan-cancer analyses were first performed to characterize the expression pattern and prognostic relevance of MRC2 across human malignancies. Comparisons based on TCGA and GTEx datasets showed that MRC2 expression differed significantly between tumor and normal tissues in multiple cancer types (Figure 2A, 2B). A radar plot summary further illustrated the broad variation in MRC2 expression across cancer...

Discussion

Standard-of-care therapy for glioma relies on maximal safe resection followed by radiotherapy and chemotherapy, most commonly temozolomide⁴. Nevertheless, diffuse infiltration frequently limits complete surgical removal and contributes to inevitable relapse, particularly in high-grade disease^3,25. Several molecular features—including MGMT promoter methylation, IDH mutation, 1p/19q co-deletion, and EGFR alterations—are used clin...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
anti-CDK4 antibody	Proteintech Group, Inc., USA	11026-1-AP	Primary antibody for western blotting. RRID:AB_2078702.
anti-CDK6 antibody	Proteintech Group, Inc., USA	66278-1-Ig	Primary antibody for western blotting. RRID:AB_2881661.
anti-E-cadherin antibody	Proteintech Group, Inc., USA	20874-1-AP	Primary antibody for western blotting. RRID:AB_10697811.
anti-MRC2 antibody	Proteintech Group, Inc., USA	86028-1-RR	Primary antibody for western blotting. RRID:AB_2879692.
anti-N-cadherin antibody	Proteintech Group, Inc., USA	22018-1-AP	Primary antibody for western blotting. RRID:AB_2813891.
anti-β-actin antibody	Proteintech Group, Inc., USA	66009-1-Ig	Loading control antibody for western blotting. RRID:AB_2687938.
anti-β-catenin antibody	Proteintech Group, Inc., USA	51067-2-AP	Primary antibody for western blotting. RRID:AB_2086128.
BCA protein assay kit	Beyotime Biotechnology, China	P0012	BCA protein assay kit for protein concentration determination.
Cell Counting Kit-8 (CCK-8)	Dojindo Laboratories, Japan	CK04-500T	Cell viability/proliferation assay.
Chemiluminescence imaging system	Uvitec Limited, France	Alliance Q9 Advanced Manual	Image acquisition system for western blot detection.
CO2 incubator	Generic laboratory equipment	Not specified	Humidified CO2 incubator used for cell culture at 37 °C and 5% CO2.
Crystal violet solution (0.1%)	Beyotime Biotechnology, China	C0121-100 mL	Crystal violet staining solution used at a 0.1% working concentration.
DMEM, high glucose	Gibco, Thermo Fisher Scientific, USA	C11995500BT	Basal medium for glioma cell culture.
Fetal bovine serum (FBS)	ExCell Bio, China	FSP500	Serum supplement for cell culture.
GlioVis	gliovis.bioinfo.cnio.es	Not applicable	Web-based platform for visualization and analysis of brain tumor expression datasets, particularly gliomas.
GraphPad Prism	GraphPad Software, USA	Version 10.4.0	Statistical analysis and graphing software. RRID:SCR_002798.
HEK293T cells	Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China	GNHu43	Packaging cell line used for lentiviral production. RRID:CVCL_0063.
HRP-conjugated Goat Anti-Mouse IgG (H+L)	Proteintech Group, Inc., USA	SA00001-1	Secondary antibody for western blotting. RRID:AB_2722565.
HRP-conjugated Goat Anti-Rabbit IgG (H+L)	Proteintech Group, Inc., USA	SA00001-2	Secondary antibody for western blotting. RRID:AB_2722564.
Human astrocytes	Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China	Not publicly available	Normal human astrocytes used as the non-tumor control; catalog number not publicly available.
Inverted microscope	Generic laboratory equipment	Not specified	Inverted microscope used for image acquisition in migration and invasion assays.
Matrigel matrix	Corning, USA	354234	Basement membrane matrix used to coat inserts for invasion assays.
Microplate reader	Generic laboratory equipment	Not specified	Microplate reader used for absorbance measurement at 450 nm in CCK-8 assays.
PAGE Gel Preparation Kit, 10%	EpiZyme, China	PG212	SDS-PAGE gel preparation kit.
PAGE Gel Preparation Kit, 7.5%	EpiZyme, China	PG111	SDS-PAGE gel preparation kit.
Paraformaldehyde solution (4%)	Beyotime Biotechnology, China	P0099-100 mL	4% paraformaldehyde fixative for colony formation and migration/invasion assays.
Penicillin-Streptomycin solution	Gibco, Thermo Fisher Scientific, USA	15140163	Antibiotic supplement for cell culture.
Polybrene	Beyotime Biotechnology, China	C0351-1 mL	Hexadimethrine bromide solution (10 mg/mL) used to facilitate lentiviral transduction.
Prestained protein marker	Elabscience, China	E-IR-R331	Protein molecular weight marker.
PrimeScript RT Reagent Kit	Takara Bio Inc., Japan	RR047A	Reverse transcription kit for cDNA synthesis.
Puromycin	Beyotime Biotechnology, China	ST551-10 mg	Puromycin dihydrochloride used for selection of stable knockdown cells.
PVDF membrane	Beyotime Biotechnology, China	FFP20	Hydrophilic PVDF membrane (0.45 µm) for western blot transfer.
R	R Foundation for Statistical Computing, Austria	Version 4.5.1	Statistical computing environment used for bioinformatic analyses. RRID:SCR_001905.
RIPA lysis buffer	Beyotime Biotechnology, China	P0013B	RIPA lysis buffer (strong) for protein extraction.
SDS-PAGE loading buffer (5×)	Beyotime Biotechnology, China	P0015L	Protein sample loading buffer.
SYBR Premix Ex Taq II	Takara Bio Inc., Japan	RR066A	qPCR master mix.
Transwell inserts, 8.0-µm pore size	Corning, USA	3422	Cell culture inserts with an 8.0 µm pore-size polycarbonate membrane; standard 24-well format for migration and invasion assays.
TRIzol reagent	Invitrogen, Thermo Fisher Scientific, USA	10296010CN	RNA extraction reagent.
U251 glioma cells	National Collection of Authenticated Cell Cultures, China	SCSP-559	Human glioma cell line used for knockdown and functional assays. RRID:CVCL_0021.
U87 glioma cells	National Collection of Authenticated Cell Cultures, China	SCSP-5432	Human glioma cell line used for knockdown and functional assays. RRID:CVCL_0022.