A microRNA-1280/JAG2 network comprises a novel biological target in high-risk medulloblastoma

Abstract

Over-expression of PDGF receptors (PDGFRs) has been previously implicated in high-risk medulloblastoma (MB) pathogenesis. However, the exact biological functions of PDGFRα and PDGFRβ signaling in MB biology remain poorly understood. Here, we report the subgroup specific expression of PDGFRα and PDGFRβ and their associated biological pathways in MB tumors. c-MYC, a downstream target of PDGFRβ but not PDGFRα, is involved in PDGFRβ signaling associated with cell proliferation, cell death, and invasion. Concurrent inhibition of PDGFRβ and c-MYC blocks MB cell proliferation and migration synergistically. Integrated analysis of miRNA and miRNA targets regulated by both PDGFRβ and c-MYC reveals that increased expression of JAG2, a target of miR-1280, is associated with high metastatic dissemination at diagnosis and a poor outcome in MB patients. Our study may resolve the controversy on the role of PDGFRs in MB and unveils JAG2 as a key downstream effector of a PDGFRβ-driven signaling cascade and a potential therapeutic target.

Figure 1. The subgroup specific expression of PDGFRα and PDGFRβ in primary MB

(A) Boxplot showing PDGFRα expression in normal adult cerebellar samples and MB subgroups based on the Boston cohort (n = 199). (B) Relative expression of PDGFRα as a log2-ratio compared to a pool of normal cerebellar samples according to MB subgroups based on the Heidelberg cohort (n = 64). (C) Boxplot showing PDGFRβ expression in normal adult cerebellar samples and MB subgroups based on the Boston cohort. (D) Relative expression of PDGFRβ as a log2-ratio compared to a pool of normal cerebellar samples according to MB subgroups based on the Heidelberg cohort.

Figure 2. PDGFRs have distinct cellular functions and PDGFRβ regulates c-MYC

(A) Effect of siRNA-mediated knockdown of PDGFRα and PDGFRβ in MB cells on cell proliferation. After 48 h of transfection, the rates of cell proliferation were determined using MTS assay; (B) siRNA-mediated knockdown of PDGFRα and PDGFRβ in MB cells on cell death. PDGFRα and PDGFRβ in MB cells were knocked down using siRNA and the rate of cell death was determined by LDH at 48 h; (C) Daoy cell migration/invasion was assessed in the presence of a control antibody, PDGFR neutralizing antibodies (mouse IgG as control for anti-PDGFRα, goat IgG as control for anti-PDGFRβ) as described in the Materials and Methods. *p < 0.05, **p < 0.01 (paired Student's t-test, sample vs. control); (D) PDGFRα and PDGFRβ signaling differentially regulates the expression of c-MYC in MB cells. PDGFRα and PDGFRβ in MB cells were knocked down using siRNAs. After 48 h of siRNA transfection, cells were harvested as protein lysates for Western blotting analysis; (E) The relative levels of PDGFRs and c-MYC in response to the siRNA treatments were calculated from the gel images of (D).

Figure 3. Co-inhibition of PDGFRβ and c-MYC suppresses MB cell proliferation and migration

MB cells were transfected with gene-specific siRNAs for PDGFRβ and c-MYC and also with PDGFR and c-MYC specific inhibitors alone or in combination for 48 h. (A) Confirmation of specific gene knockdown by Western blotting analysis. β-actin was used as the loading control. (B) The effects of c-MYC inhibitor 10058-F4 on MB cell proliferation. (C) The effects of siRNAs and inhibitors on MB cell proliferation were determined using MTS. *p < 0.05, **p < 0.01 (paired t-test, sample vs. control). (D) The effects of co-inhibiting PDGFRβ and c-MYC on MB cell migration. Daoy cells were transfected with gene specific siRNAs for PDGFRβ and c-MYC and also with PDGFR and c-MYC specific inhibitors alone or in combination for 36 h. Treated cells were then detached and re-distributed in equal amounts in a 48-well plate before a linear wound was made. The image was captured immediately after that an artificial wound was made at 0^th h and also at 24^th h (Figures S3a, S3b). Quantified results were calculated from the images. Percentage wound closure shows the migration rate in PDGFRβ^KD, c-MYC^KD or PDGFRβ^KDc-MYC^KD cells when compared to control sample, *p < 0.05, **p < 0.01, ***p < 0.001 (paired t-test, sample vs. control).

Figure 4. PDGFRβ and c-MYC differentially regulate miRNA expression in MB cells

(A) Heat map represents the differentially regulated miRNAs by Control (Mock), PDGFRα^KD, PDGFRβ^KD, c-MYC^KD or PDGFRβ^KDc-MYC^KD cells. The normalized log ratio values were used for the analysis. The clustering was performed on all samples, and the highly differentially regulated 39 miRNAs were selected. Each row represents a miRNA and each column represents a sample. The color scale illustrates the relative expression levels of miRNAs. Green color represents an expression level below the reference channel, and red color represents the expression higher than the reference. (B) The expression levels of miRNAs regulated by PDGFRα, PDGFRβ, and c-MYC in MB cell lines. MB cells were transfected with gene specific siRNAs for PDGFRα, PDGFRβ, and c-MYC alone or in combination of PDGFRβ and c-MYC for 24 h. p < 0.05 was considered to be statistically significant. (C) miR-1280 inhibitor suppresses miR-1280 expression in Daoy cells. PDGFRβ^KD Daoy cells were transfected with control or increasing concentrations of miR-1280 specific inhibitor using Lipofectamineltx in opti-MEM reduced serum medium following the manufacturer's instruction for 4 h. The cells were then fed with equal volume of MEM medium with 10% FBS. After 24 h, total RNA isolated was subjected to TaqMan microRNA assay to verify for specific inhibition of miRNAs. Data are presented as mean values (n = 3) ± standard deviation. Differences between 2 groups were analyzed using Student's t-test. p < 0.05 was considered to be statistically significant. (D) miR-1260 inhibitor suppresses miR-1260 expression in Daoy cells. (E) The effects of miR-1280 and miR-1260 on MB cell proliferation. PDGFRβ^KD Daoy cells were treated with control or miR-1280 inhibitor or miR-1260 inhibitor. The effects on cell proliferation were determined by MTS assay. (F) The effects of miR-1280 and miR-1260 on PDGFRβ^KD Daoy cell migration. PDGFRβ^KD Daoy cells were treated with control or miR-1280 inhibitor or miR-1260 inhibitor. The treated cells were then detached and re-distributed in equal amounts in a 48-well plate before a linear wound was made. The image was captured immediately after an artificial wound was made at 0^th h and also at 24^th h (Figure S4a). Quantified results were calculated from the images. The treated samples were compared to control sample, *p < 0.05, **p < 0.01, ***p < 0.001 (paired t-test, sample vs. control).

Figure 5. Identification of JAG2 as a potential target of MB

(A) The expression of JAG2 in MB cells is regulated by PDGFRβ and c-MYC. MB cells were transfected with gene specific siRNAs against PDGFRβ and/or c-MYC for 48 h. Protein lysates extracted from treated samples were used for the expression levels of JAG2 by Western blotting. β-actin was used as a loading control. (B) The expression levels of JAG2 are regulated by a miR-1280 inhibitor. PDGFRβ^KD Daoy cells were transfected with increasing concentrations of a miR-1280 specific inhibitor. After 48 h, protein lysates extracted were subjected to Western blotting analysis to check for change in expression of JAG2 protein. β-actin was used as a loading control. (C) Confirmation of specific knockdown JAG2 by Western blotting analysis. β-actin was used as the loading control. (D) The effects of JAG2 siRNA on MB cell proliferation. MB cells were transfected with control or JAG2 specific siRNAs for 48 h. Cell proliferation was determined using MTS. (E) The effects of JAG2 on MB cell migration. PDGFR^KD Daoy cells were treated with control or JAG2 siRNAs for 36 h and then detached and re-distributed in equal amounts in a 48-well plate before a linear wound was made. The image was captured immediately after an artificial wound was made at 0^th h and also at 24^th h (Supplemental Figure S4B). Quantified results were calculated from the images. The significance of JAG2 siRNAs on MB cell migration were analyzed using paired t-test, sample vs. control (*p < 0.05, **p < 0.01). (F) The expression of JAG2 in MB tissues of different stages of metastasis (45 patients at M0 stage, 5 patients at M1 stage, 4 patients at M2 stage, and 9 patients at M3 stage); (G) Kaplan-Meier plots of overall survival (OS) time according to JAG2 levels in MB patients.

Figure 6. Schematic representation of pathways promoting MB progression by PDGFRβ and c-MYC, and the axis connecting PDGFRβ to JAG2, through c-MYC and miR-1280