MiR-328 promotes glioma cell invasion via SFRP1-dependent Wnt-signaling activation

Abstract

Background Diffusely infiltrative growth of human astrocytic gliomas is one of the major obstacles to successful tumor therapy. Thorough insights into the molecules and pathways signaling glioma cell invasion thus appear of major relevance for the development of targeted and individualized therapies. By miRNA expression profiling of microdissected human tumor biopsy specimens we identified miR-328 as one of the main miRNAs upregulated in invading glioma cells in vivo and further investigated its role in glioma pathogenesis. Methods We employed miRNA mimics and inhibitors to functionally characterize miR-328, 3' untranslated region luciferase assays, and T-cell factor/lymphoid enhancer factor reporter assays to pinpoint miR-328 targets and signaling pathways, and analyzed miR-328 expression in a large panel of gliomas. Results First, we corroborated the invasion-promoting role of miR-328 in A172 and TP365MG glioma cells. Secreted Frizzled-related protein 1 (SFRP1), an inhibitor of Wnt signaling, was then pinpointed as a direct miR-328 target. SFRP1 expression is of prognostic relevance in gliomas with reduced expression, being associated with significantly lower overall patient survival in both the Repository of Molecular Brain Neoplasia Data (REMBRANDT) and The Cancer Genome Atlas. Of note, miR-328 regulated both SFRP1 protein expression levels and Wnt signaling pathway activity. Finally, in human glioma tissues miR-328 appeared to account for the downregulation of SFRP1 preferentially in lower-grade astrocytic gliomas and was inversely related to SFRP1 promoter hypermethylation. Conclusion Taken together, we report on a novel molecular miR-328-dependent mechanism that via SFRP1 inhibition and Wnt activation contributes to the infiltrative glioma phenotype at already early stages of glioma progression, with unfavorable prognostic implications for the final outcome of the disease.

Keywords: astrocytoma; brain tumor; epigenetic; glioblastoma; miRNA.

Fig. 1.

MiR-328 expression is upregulated in infiltrating glioma cells in vivo. (A) MiR-328 expression in microdissected tumor areas from the infiltration zone (INF) and more central cell-rich tumor regions (CEN) of 7 malignant gliomas was analyzed by quantitative PCR assays based on Micro Fluidic Cards and additionally compared with miR-328 expression in 7 commercially available nonneoplastic brain (NB) samples. (B) Results were validated for microdissected INFs and CENs by singleplex microRNA assays (miR-328 expression normalized to RNU48) (t-test with pairwise comparison for INF vs CEN, unpaired t-test for INF vs NB. * P < .05, *** P < .001).

Fig. 2.

MiR-328 promotes glioma invasion and proliferation in vitro. (A and B) MiR-328 downregulation leads to significantly decreased invasion of the glioma cell lines A172 and TP365MG in modified Boyden chamber assay experiments. In TP365MG, miR-328 upregulation resulted in significantly increased invasion. (C and D) Upregulation of miR-328 expression significantly increased cell proliferation of A172 and TP365MG glioma cells. (E and F) Apoptosis remained unaffected by miR-328 up- or downregulation in A172 and TP365MG cells (anti-control [CTRL]: transfected with nontargeting miRNA inhibitor; anti-328: transfected with miR-328 inhibitor; pre-CTRL: transfected with nontargeting miRNA precursor; pre-328: transfected with miR-328 precursor; t-test. * P < .05).

Fig. 3.

SFRP1 and Wnt signaling is a target of miR-328 in glioma cell lines. (A) A SFRP1 3′ UTR luciferase reporter gene assay containing a wild-type SFRP1 3′ UTR fragment or a mutated SFRP1 3′ UTR fragment was used to analyze the effect of miR-328 up- and downregulation in T98G glioma cells. The SFRP1 3′ UTR sequence corresponding to the miR-328 seed sequence is indicated in bold letters, and the 7 base pairs missing in the mutated SFRP1 3′ UTR are underlined. With the luciferase reporter gene assay containing the wild-type SFRP1 3′ UTR, downregulation of miR-328 expression resulted in higher luciferase activity, while miR-328 upregulation decreased luciferase activity. There is no significant effect of miR-328 on luciferase activity if the potential miR-328 binding site is mutated. (B) The expression of endogenous SFRP1 protein was analyzed by western blotting after transfection with either miR-328 inhibitor (anti-328) or miR-328 precursor (pre-328) in A172 and TP365MG glioma cells (shown is a representative western blot). Note that transfection of anti-328 resulted in markedly higher SFRP1 expression, while transfection with pre-328 nearly completely abolishes SFRP1 expression in both cell lines. (C) TCF/LEF reporter assay was applied to assess Wnt signaling activity in T98G cells in relation to miR-328 expression. Transfection with anti-328 significantly decreased Wnt signaling pathway activation, while transfection with pre-328 increased Wnt pathway activation (anti-control [CTRL]: transfection with nontargeting miRNA inhibitor; anti-328: transfection with miR-328 inhibitor; pre-CTRL: transfection with nontargeting miRNA precursor; pre-328: transfection with miR-328 precursor; t-test. * P < .05).

Fig. 4.

MiR-328 modifies invasion and proliferation of glioma cells by regulating SFRP1. (A–C) Effects on glioma cell invasion: cotransfection of a miR-328 precursor (pre-328) and an empty control vector increases invasion of TP365MG (white bars) and slightly also of A172 glioma cells (gray bars) (A). When cotransfecting an SFRP1 expression vector instead of the empty control vector, the stimulating effect of miR-328 overexpression on tumor cell invasion is decreased or completely abolished (B). When cotransfecting C140Y mutated SFRP1, the stimulating effect of miR-328 overexpression on tumor cell invasion remains unaffected (C). Same applies to proliferation using BrdU incorporation assays (D–F): mir-328 effect in presence of the empty vector (D), rescue of the miRNA-328 effect through presence of the SFRP1 expression vector (E), and no rescue through coexpression of mutated SFRP1 (F). * P < .05; ** P < .01.

Fig. 5.

SFRP1 expression in the tumor center vs the tumor periphery. (A) Prostate adenocarcinoma as positive control of SFRP1 immunoreactivity. (B) Negative control of a malignant glioma tissue with normal rabbit immunoglobulin G as the primary antibody. (C–H) Examples of matched pairs of tumor center and periphery from 3 malignant glioma patients. Note that SFRP1 staining in the tumor periphery/infiltration zone (D, F, and H) is generally low. In sample pairs C–F, SFRP1 is decreased in the tumor rim (D, F) relative to the tumor center (C, E), most likely due to miR-328 upregulation. In sample pair G and H, SFRP1 is low also in the tumor center (G), which may be explained by alternative mechanisms of SFRP1 downregulation, such as aberrant promoter methylation.

Fig. 6.

Synopsis of miR-328 expression with SFRP1 expression and promoter methylation in gliomas. (A) MiR-328 expression is highest in WHO grade II astrocytomas and decreases with malignant progression to secondary glioblastoma. Primary glioblastomas exhibit median miR-328 expression at about the level observed in nonneoplastic brain tissue. (B) SFRP1 promoter hypermethylation exhibits an inverse pattern to miR-328 expression, with the by far highest methylation frequency in primary glioblastomas. Data shown in B are from Götze et al. (C) SFRP1 mRNA expression levels do not significantly differ among WHO grades. However, median SFRP1 expression levels for each individual WHO grade are decreased relative to nonneoplastic brain tissue. (D) Synopsis of the molecular changes illustrated in A–C indicating that miR-328 overexpression and SFRP1 promoter hypermethylation are mutually exclusive, depending on tumor type and grade, and may alternatively explain the uniformly low SFRP1 transcript levels in gliomas. (E) Concomitant analysis of miR-328 expression and SFRP1 hypermethylation in 38 gliomas (left) and 19 pGBIVs (right) revealed significantly higher miR-328 expression in tumors with unmethylated SFRP1 promoter (u.) compared with tumors with SFRP1 promoter methylation (m.) (AII: diffuse astrocytoma, WHO grade II; AAIII: anaplastic astrocytoma, WHO grade III; sGBIV: secondary glioblastoma, WHO grade IV; pGBIV: primary glioblastoma, WHO grade IV; * P < .05; ** P < .01; *** P < .001; • marks mild outlier, ▴ marks extreme outliers; dotted line in A, C, and E marks expression levels in nonneoplastic brain tissue).

Fig. 7.

Kaplan–Meier survival analysis in glioma patients stratified for SFRP1 and miR-328 expression. (A) The REMBRANDT data set, containing astrocytomas, oligodendrogliomas, and mixed oligoastrocytic tumors of different WHO grades, reveals statistically significant overall survival differences among patients stratified for SFRP1 mRNA expression. (B) Analysis within TCGA shows statistically significant overall survival differences depending on SFRP1 transcript levels also in the group of WHO grade IV glioblastoma patients. (C) In the own cohort of 114 glioblastoma patients, there is a (nonsignificant) trend toward longer overall survival in the small subset of patients with high SFRP1 protein expression. (D) MiR-328 expression does not significantly correlate with overall survival in the group of WHO grade IV glioblastoma patients (log-rank tests).