Targeting of TGFβ signature and its essential component CTGF by miR-18 correlates with improved survival in glioblastoma

Abstract

The miR-17∼92 cluster is thought to be an oncogene, yet its expression is low in glioblastoma multiforme (GBM) cell lines. This could allow unfettered expression of miR-17∼92 target genes such as connective tissue growth factor (CTGF; or CCN2), which is known to contribute to GBM pathogenesis. Indeed, microRNA-18a (but not other miR-17∼92 members) has a functional site in the CTGF 3' UTR, and its forced reexpression sharply reduces CTGF protein and mRNA levels. Interestingly, it also reduces the levels of CTGF primary transcript. The unexpected effects of miR-18a on CTGF transcription are mediated in part by direct targeting of Smad3 and ensuing weakening of TGFβ signaling. Having defined the TGFβ signature in GBM cells, we demonstrate a significant anti-correlation between miR-18 and TGFβ signaling in primary GBM samples from The Cancer Genome Atlas. Most importantly, high levels of miR-18 combined with low levels of the TGFβ metagene correlate with prolonged patient survival. Thus, low expression of the miR-17∼92 cluster, and specifically miR-18a, could significantly contribute to GBM pathogenesis.

FIGURE 1.

miR-18a is the miR-17∼92 component that regulates CTGF in glioblastoma. (A, top) Alignment of the CTGF 3′ UTR with miR-18a and miR-19 sequences, with predicted seed sequences indicated. (Bottom) Partial sequences of luciferase sensors with underlines indicating mutated nucleotides. (B) Dual luciferase assay performed in DLD-1 Dicer hypomorph cells cotransfected with indicated luciferase constructs and control, miR-18a, or miR-19b mimic. (C) Representative Western blot of CTGF and actin (loading control) from A172 human GBM cell line transfected with indicated microRNA mimics 48 h prior to harvesting. Numbers below the CTGF band indicate CTGF band intensity relative to that of the actin band. (D) qPCR for CTGF mRNA in A172 cells transfected with the indicated mimics for 24–48 h. (E) 3′-RACE products specific for GAPDH or CTGF 3′ UTR from HCT116 or A172 cells. (F) qPCR for CTGF from human GBM cell lines transfected with control or miR-18a mimic for 24 h. Error bars represent the standard deviation of three or more independent experiments; (**) P < 0.01 by Student’s t-test.

FIGURE 2.

miR-18a significantly inhibits CTGF transcription. (A) qPCR and quantitated Western blot analysis for CTGF in A172 cells transfected with indicated mimics for 24–48 h. (B) qPCR for indicated genes in A172 cells treated with Actinomycin D relative to untreated samples. (Vertical dashed lines) Approximate half-life. (C) qPCR for CTGF in A172 cells treated with Actinomycin D and simultaneously transfected with control or miR-18a mimic. (Vertical dashed lines) Approximate half-life. (D) qPCR for CTGF mRNA or hnRNA from A172 cells treated with Actinomycin D for 8 h. (E) qPCR for CTGF mRNA or hnRNA from A172 cells treated with vehicle or TGFβ1 for 24 h. (F) qPCR for CTGF mRNA or hnRNA from A172 cells transfected with control or miR-18a mimic for 24 h. Error bars represent the standard deviation of three or more independent experiments; (**) P < 0.01 by Student’s t-test.

FIGURE 3.

miR-18a regulates CTGF transcription in part via direct regulation of human Smad3. (A) Simplified diagram of Smad-dependent TGFβ signaling pathway. (B) Western blot analysis of A172 cells transfected with nontargeting siRNA or siRNA targeting TGFBR2 for 24 h, then treated with vehicle or 5 ng/mL TGFβ1 for 3 h. (C, top) Western blot of lysates from A172 cells transfected with nontargeting siRNA or siRNA targeting TGFBR2 for 24 h, then transfected with control or miR-18a mimic for 24 h. (Bottom) Quantitation of CTGF Western blot above; levels of CTGF protein are normalized to the control mimic in each cell type. The bracket indicates contribution of TGFβ signaling to miR-18a repression of CTGF. (D) Representative CTGF Western blot from A172 cells transfected with 20 nM indicated microRNA mimics for 48 h, followed by 8 h of stimulation with vehicle or 5 ng/mL TGFβ1. The numbers below the CTGF band indicate CTGF band intensity relative to that of the actin band. (E) Western blot analysis of A172 cells transfected with 20 nM control or miR-18a mimic for 24 h, then treated with vehicle or 5 ng/mL TGFβ1 for the indicated times. (F, left) Alignment of predicted miR-18 sites in Smad3 3′ UTR and miR-18a sequence. (Right) Distribution of miR-18 sites within Smad3 3′ UTR; (black boxes) 100-bp fragments used in luciferase sensor constructs. (G) Dual luciferase assay performed in DLD-1 Dicer hypomorph cells cotransfected with indicated luciferase sensors and miR-18a mimic. Values were normalized to luciferase activity from cells simultaneously cotransfected with indicated luciferase sensors and control mimic. Error bars represent the standard deviation of two independent experiments; (**) P < 0.01 WT versus Mut sequence by Student’s t-test.

FIGURE 4.

miR-18 expression inversely correlates with the TGFβ signature and influences survival in human GBM. (A) Correlation between TGFβ metagene and miR expression. (Blue) The miRs that are negatively or positively correlated at a false discovery rate of 0.1; (red) miR-18a and miR-18b. (B) Gene set enrichment analysis (GSA) demonstrating inverse correlation between TGFβ signature genes and miR-18 expression. The heatmap shows the expression of the 149 genes in columns with each tumor sample in rows. (Orange) High expression; (blue) low. For each gene, the gene score from the GSA is indicated in the plot above the heatmap, with negative scores indicating negative association with miR-18 expression. The expression of miR-18 for each sample is shown in the graph to the right of the heatmap. (C) Negative correlation between select TGFβ target genes and average miR-18 expression levels. The correlation coefficients are indicated; all P-values are <0.05. (D) Kaplan-Meier survival curves for GBM patients stratified by high and low expression of miR-18 and the TGFβ metagene (TGFbMeta). The P-value shown is from a comparison between miR-18(hi)/TGFbMeta(lo) versus the other groups. (E) Proposed model wherein miR-18a inhibits CTGF via direct inhibition of mRNA (and possibly inhibition of translation), as well as via Smad3-dependent CTGF transcription.