Abstract
Objectives: Our previous findings demonstrate that channel-kinase transient receptor potential (TRP) ion channel subfamily M, member 7 (TRPM7) is critical in regulating human glioma cell migration and invasion. Since microRNAs (miRNAs) participate in complex regulatory networks that may affect almost every cellular and molecular process during glioma formation and progression, we explored the role of miRNAs in human glioma progression by comparing miRNA expression profiles due to differentially expressed TRPM7.Methods: First, we performed miRNA microarray analysis to determine TRPM7's miRNA targets upon TRPM7 silencing in A172 cells and validated the miRNA microarray data using A172, U87MG, U373MG, and SNB19 cell lines by stem-loop RT-qPCRs. We next determined whether TRPM7 regulates glioma cell proliferation and migration/invasion through different functional domains by overexpressing wild-type human TRPM7 (wtTRPM7), two mutants with TRPM7's α-kinase domain deleted (Δkinase-DK), or a point mutation in the ATP binding site of the α-kinase domain (K1648R-KR). In addition, we determined the roles of miR-28-5p in glioma cell proliferation and invasion by overexpressing or under expressing miR-28-5p in vitro. Lastly, we determined whether a Ras-related small GTP-binding protein (Rap1b) is a target of miR-28-5p in glioma tumorigenesis.Results: The miRNA microarray data revealed a list of 16 downregulated and 10 upregulated miRNAs whose transcripts are significantly changed by TRPM7 knock-down. Cell invasion was significantly reduced in two TRPM7 mutants with inactive kinase domain, Δkinase, and K1648R transfected glioma cells. miR-28-5p overexpression suppressed glioma cells' proliferation and invasion, and miR-28-5p under expression led to a significant increase in glioma cell proliferation and migration/invasion compared to that of the controls. miR-28-5p suppressed glioma cell proliferation and migration by targeting Rap1b. Co-transfection of siRap1b with miR28-5p inhibitor reduced the glioma cell proliferation and invasion, caused by the latter.Conclusions: These results indicate that TRPM7's channel activity is required for glioma cell growth while the kinase domain is required for cell migration/invasion. TRPM7 regulates miR-28-5p expression, which suppresses cell proliferation and invasion in glioma cells by targeting Rap1b signaling.
Highlights
High grade malignant gliomas, called glioblastoma multiforme (GBM), the most common and aggressive primary brain tumor, are devastating, uniformly fatal tumors for which no effective therapies currently exist
We further investigated whether or not changes in glioma cell proliferation and migration might be caused by channel domain-mediated and/or kinase domain-mediated TRPM7 activation
Cell proliferation was not significantly changed when cells were transfected with kinase or K1648R compared to those transfected with wt TRPM7, which indicates that TRPM7 channel, rather than kinase activity, is required for A172 cell growth (Figure 1B)
Summary
Called glioblastoma multiforme (GBM), the most common and aggressive primary brain tumor, are devastating, uniformly fatal tumors for which no effective therapies currently exist. MiRNAs, one of the epigenetic effectors, reversibly regulate transcription through binding to complementary sequences of mRNA and silencing its translation into proteins. MiRNAs are small noncoding RNAs, generally 19– 22 nucleotides in length, that modulate protein-coding genes by binding to the 3’-untraslated region (UTR) of the target mRNA, disrupting transcription [2,3,4]. Many studies have shown that miRNA are expressed in a variety of human tumors and exert dramatic functions in human tumorigenesis and metastasis [5]. Since miRNAs have significant roles in human tumorigenesis and metastasis [5], identification of aberrantly expressed miRNAs is a crucial initial step in illustrating miRNA-mediated tumorigenic pathways
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