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The insulin-like growth factor (IGF)-1 signaling is relevant in regulating cell growth and cytokine secretions by glioblastomas. MicroRNAs determine the cell fate in glioblastomas. However, relationships between IGF-1 signaling and miRNAs in glioblastoma pathogenesis are still unclear. Our aim was to validate the IGF-1-mediated mRNA/miRNA regulatory network in glioblastomas. Using in silico analyses of mRNA array and RNA sequencing data from The Cancer Genome Atlas (TCGA), we identified 32 core enrichment genes that were highly associated with IGF-1-promoted cytokine-cytokine receptor interactions. To investigate the IGF-1-downregulated miRNA signature, microarray-based approaches with IGF-1-treated U87-MG cells and array data in TCGA were used. Four miRNAs, including microRNA (miR)-9-5p, miR-9-3p, miR-181d, and miR-130b, exhibited an inverse correlation with IGF-1 levels. The miR-181d, that targeted the most IGF-1-related cytokine genes, was significantly reduced in IGF-1-treated glioma cells. Statistical models incorporating both high-IGF-1 and low-miR-181d statuses better predicted poor patient survival, and can be used as an independent prognostic factor in glioblastomas. The C-C chemokine receptor type 1 (CCR1) and interleukin (IL)-1b demonstrated inverse correlations with miR-181d levels and associations with patient survival. miR-181d significantly attenuated IGF-1-upregulated CCR1 and IL-1b gene expressions. These findings demonstrate a distinct role for IGF-1 signaling in glioma progression via miR-181d/cytokine networks.

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Temozolomide (TMZ), an alkylating agent of the imidazotetrazine series, is a first-line chemotherapeutic drug used in the clinical therapy of glioblastoma multiforme, the most common and high-grade primary glioma in adults. Micro (mi)RNAs, which are small noncoding RNAs, post-transcriptionally regulate gene expressions and are involved in gliomagenesis. However, no studies have reported relationships between TMZ and miRNA gene regulation. We investigated TMZ-mediated miRNA profiles and its molecular mechanisms underlying the induction of glioma cell death. By performing miRNA microarray and bioinformatics analyses, we observed that expression of 248 miRNAs was altered, including five significantly upregulated and 17 significantly downregulated miRNAs, in TMZ-treated U87MG cells. miR-128 expression levels were lower in different glioma cells and strongly associated with poor survival. TMZ treatment significantly upregulated miR-128 expression. TMZ significantly enhanced miR-128-1 promoter activity and transcriptionally regulated miR-128 levels through c-Jun N-terminal kinase 2/c-Jun pathways. The overexpression and knockdown of miR-128 expression significantly affected TMZ-mediated cell viability and apoptosis-related protein expression. Furthermore, the overexpression of miR-128 alone enhanced apoptotic death of glioma cells through caspase-3/9 activation, poly(ADP ribose) polymerase degradation, reactive oxygen species generation, mitochondrial membrane potential loss, and non-protective autophagy formation. Finally, we identified that key members in mammalian target of rapamycin (mTOR) signaling including mTOR, rapamycin-insensitive companion of mTOR, insulin-like growth factor 1, and PIK3R1, but not PDK1, were direct target genes of miR-128. TMZ inhibited mTOR signaling through miR-128 regulation. These results indicate that miR-128-inhibited mTOR signaling is involved in TMZ-mediated cytotoxicity. Our findings may provide a better understanding of cytotoxic mechanisms of TMZ involved in glioblastoma development.

Temozolomide (TMZ) is a first-line alkylating agent for glioblastoma multiforme (GBM). Clarifying the mechanisms inducing TMZ insensitivity may be helpful in improving its therapeutic effectiveness against GBM. Insulin-like growth factor (IGF)-1 signaling and micro (mi)RNAs are relevant in mediating GBM progression. However, their roles in desensitizing GBM cells to TMZ are still unclear. We aimed to identify IGF-1-mediated miRNA regulatory networks that elicit TMZ insensitivity for GBM. IGF-1 treatment attenuated TMZ cytotoxicity via WNT/β-catenin signaling, but did not influence glioma cell growth. By miRNA array analyses, 93 upregulated and 148 downregulated miRNAs were identified in IGF-1-treated glioma cells. miR-513a-5p from the miR-513a-2 gene locus was upregulated by IGF-1-mediated phosphoinositide 3-kinase (PI3K) signaling. Its elevated levels were also observed in gliomas versus normal cells, in array data of The Cancer Genome Atlas (TCGA), and the GSE61710, GSE37366, and GSE41032 datasets. In addition, lower levels of neural precursor cell-expressed developmentally downregulated 4-like (NEDD4L), an E3 ubiquitin protein ligase that inhibits WNT signaling, were found in gliomas by analyzing cells, arrays, and RNA sequencing data of TCGA glioma patients. Furthermore, a negative correlation was identified between miR-513a-5p and NEDD4L in glioma. NEDD4L was also validated as a direct target gene of miR-513a-5p, and it was reduced by IGF-1 treatment. Overexpression of NEDD4L inhibited glioma cell viability and reversed IGF-1-repressed TMZ cytotoxicity. In contrast, miR-513a-5p significantly affected NEDD4L-inhibited WNT signaling and reduced TMZ cytotoxicity. These findings demonstrate a distinct role of IGF-1 signaling through miR-513a-5p-inhibited NEDD4L networks in influencing GBM's drug sensitivity to TMZ.

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