miR-124-3p and miR-194-5p regulation of the PI3K/AKT pathway via ROR2 in medulloblastoma progression

Abstract

Medulloblastoma (MB), a prevalent pediatric central nervous system tumor, is influenced by microRNAs (miRNAs) that impact tumor initiation and progression. However, the specific involvement of miRNAs in MB tumorigenesis remains unclear. Using single-cell RNA sequencing, we identified ROR2 expression in normal human fetal cerebellum. Subsequent analyses, including immunofluorescence, quantitative real-time PCR (qRT-PCR), and Western blot, assessed ROR2 expression in MB tissues and cell lines. We investigated miR-124-3p and miR-194-5p and their regulatory role in ROR2 expression through the dual-luciferase reporter, qRT-PCR, and western blot assays. Mechanistic insights were gained through functional assays exploring the impact of miR-124-3p, miR-194-5p, and ROR2 on MB growth in vitro and in vivo. We observed significantly reduced miR-124-3p and miR-194-5p expression and elevated ROR2 expression in MB tissues and cell lines. High ROR2 expression inversely correlated with overall survival in WNT and SHH subgroups of MB patients. Functionally, overexpressing miR-124-3p and miR-194-5p and inhibiting ROR2 suppressed in vitro malignant transformation and in vivo tumorigenicity. Mechanistically, miR-124-3p and miR-194-5p synergistically regulated the ROR2/PI3K/Akt pathway, influencing MB progression. Our findings indicate that miR-124-3p and miR-194-5p function as tumor suppressors, inhibiting MB progression via the ROR2/PI3K/Akt axis, suggesting a key mechanism and therapeutic targets for MB patients.

Fig. 1. Elevated ROR2 expression in MB correlates with an unfavorable prognosis.

A Uniform Manifold Approximation and Projection (UMAP) visualization and marker-based annotation of cells from the human fetal cerebellum colored by cell type (left panel). Plots in the right panel were colored by the normalized expression of cell-type-specific ROR2 in the human fetal cerebellum. B Expression analysis for ROR2 in MB and control groups using the R2 platform. Control cerebellum, Roth et al., 2008 (n = 9, GSE3526); MB Group1, Pfister et al., 2015 (n = 73, GSE49243); MB Group2, Pfister et al., 2017 (n = 223); MB Group 3, Delattre et al., 2012 (n = 57); MB Group 4, Kool et al., 2009 (n = 62, GSE10327). C, D Expression analysis for ROR2 in WNT, SHH, G3, and G4 groups using the R2 platform. Swartling et al., 2021 (n = 1641, GSE124814). E Survival analysis for ROR2 in WNT and SHH subgroup using the R2 platform. Cavalli et al., 2017 (n = 763, GSE85217). F IF analysis of ROR2 in MB subgroups and control tissues. G Western blot analysis of ROR2 protein in MB and control tissues. H qRT-PCR analysis of relative ROR2 mRNA expression from MB and control tissues. I qRT-PCR analysis of relative ROR2 mRNA expression in MB cell lines and NHA (normal human astrocytes). Data are shown as mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 2. Inhibition of ROR2 suppresses proliferation and induces apoptosis via the PI3K/AKT pathway in MB cells.

A qRT-PCR analysis of relative ROR2 mRNA expression in MB cell lines transfected with siRNAs. B Western blot analysis of ROR2, Akt and p-Akt proteins in MB cell lines. C, D CCK-8 and colony formation analysis of cell proliferation ability in MB cell lines transfected with siROR2 or NC. E Apoptosis analysis of early and late apoptosis in MB cell lines transfected with siROR2 or NC. F Western blot analysis of standard apoptosis-related proteins in MB cell lines transfected with siROR2 or NC. Data are shown as mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 3. ROR2 enhances migration and invasion of MB cells through epithelial–mesenchymal transition (EMT).

A Wound healing assay in DAOY and D283 cells transfected with siROR2 or NC. B, C Transwell migration and invasion assays in MB cell lines transfected with siROR2 or NC. D Western blot analysis of standard EMT-related proteins in MB cell lines transfected with siROR2 or NC. Data were showed as mean ± SEM; *P < 0.05, **P < 0.01.

Fig. 4. miR-124-3p and miR-194-5p dually regulate ROR2 in MB cells.

A Expression analysis for miR-124-3p/miR-194-5p in MB and control groups using the GEO2R platform. Ferretti et al., 2021 (n = 48, GSE12303). B qRT-PCR analysis of relative miR-124-3p/miR-194-5p expression from MB and control tissues. C qRT-PCR analysis of relative miR-124-3p/miR-194-5p expression in MB cell lines and normal cerebellum. D The potential prediction binding sites between the target gene ROR2 and miR-124-3p/miR-194-5p and the schematic illustration of ROR2-WT/ROR2-MUT dual-luciferase reporter vectors. E Dual-luciferase reporter analysis of luciferase activities in cells after co-transfection with ROR2-WT, ROR2-MUT and miR-124-3p/miR-194-5p mimics or mimics NC, respectively. F, G Western blot and qRT-PCR analysis of ROR2 protein and mRNA in MB cell lines transfected with miR-124-3p mimics, miR-194-5p mimics, mimics NC, miR-124-3p inhibitor, miR-194-5p inhibitor and inhibitor NC, respectively. Data are shown as mean ± SEM; n.s. indicated no significance. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 5. Elevated expression of miR-124-3p and miR-194-5p suppresses proliferation and migration in MB cells.

A CCK-8 analysis of cell proliferation ability in MB cell lines transfected with miR-124-3p mimics, miR-194-5p mimics, mimics NC, miR-124-3p inhibitor, miR-194-5p inhibitor and inhibitor NC, respectively. B Wound-healing assay in DAOY and D283 cells transfected with miR-124-3p mimics, miR-194-5p mimics, mimics NC, miR-124-3p inhibitor, miR-194-5p inhibitor and inhibitor NC, respectively. Data are shown as mean ± SEM; *P < 0.05, **P < 0.01.

Fig. 6. Overexpression of miR-124-3p/miR-194-5p and inhibition of ROR2 suppress the growth of MB in vivo.

A qRT-PCR analysis of relative miR-124-3p, miR-194-5p and ROR2 expression in DAOY cells stably transfected with miR-124-3p agomir, miR-194-5p agomir, shROR2, and shNC. B Western blot analysis of ROR2 protein in DAOY cells stably transfected with miR-124-3p agomir, miR-194-5p agomir, shROR2, and shNC. C Proliferation imaging of DAOY cells stably transfected with miR-124-3p agomir, miR-194-5p agomir, shROR2, and shNC after 48 h culture. D Image of dissected subcutaneous tumors (left), tumor growth curves and tumor weight analysis (right) from miR-124-3p agomir, miR-194-5p agomir, shROR2 and control group. A ruler was used to indicate the size of the tumors. E IF analysis of ROR2 in shROR2, miR-124-3p agomir, miR-194-5p agomir, and control xenograft tumor groups. Data are shown as mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001.