ABSTRACT
Previously, we have revealed that the miR-130 family (miR-130b, miR-301a, and miR-301b) functions as an oncomiR in bladder cancer. The pharmacological inhibition of the miR-130 family molecules by the seed-targeting strategy with an 8-mer tiny locked nucleic acid (LNA) inhibits the growth, migration, and invasion of bladder cancer cells by repressing stress fiber formation. Here, we searched for a functionally advanced target sequence with LNA for the miR-130 family with low cytotoxicity and found LNA #9 (A(L)^i^i^A(L)^T(L)^T(L)^G(L)^5(L)^A(L)^5(L)^T(L)^G) as a candidate LNA. LNA #9 inhibited cell growth in vitro and in an in vivo orthotopic bladder cancer model. Proteome-wide tyrosine phosphorylation analysis suggested that the miR-130 family upregulates a wide range of receptor tyrosine kinases (RTKs) signaling via the expression of phosphorylated Src (pSrcTyr416). SILAC-based proteome analysis and a luciferase assay identified protein tyrosine phosphatase non-receptor type 1 (PTPN1), which is implicated as a negative regulator of multiple signaling pathways downstream of RTKs as a target gene of the miR-130 family. The miR-130-targeted LNA increased and decreased PTPN1 and pSrcTyr416 expressions, respectively. PTPN1 knockdown led to increased tumor properties (cell growth, invasion, and migration) and increased pSrcTyr416 expression in bladder cancer cells, suggesting that the miR-130 family upregulates multiple RTK signaling by targeting PTPN1 and subsequent Src activation in bladder cancer. Thus, our newly designed miR-130 family targeting LNA could be a promising nucleic acid therapeutic agent for bladder cancer.
Subject(s)
Antineoplastic Agents/therapeutic use , MicroRNAs/antagonists & inhibitors , Neoplasm Proteins/physiology , Oligonucleotides/therapeutic use , Protein Tyrosine Phosphatase, Non-Receptor Type 1/physiology , RNA, Neoplasm/antagonists & inhibitors , Urinary Bladder Neoplasms/drug therapy , Animals , Carcinoma, Transitional Cell/drug therapy , Carcinoma, Transitional Cell/genetics , Carcinoma, Transitional Cell/metabolism , Cell Line, Tumor , Drug Screening Assays, Antitumor , Female , Gene Expression Regulation, Neoplastic , Genes, Reporter , Humans , Mice , MicroRNAs/genetics , RNA, Neoplasm/genetics , Receptor Protein-Tyrosine Kinases/biosynthesis , Receptor Protein-Tyrosine Kinases/genetics , Recombinant Proteins/metabolism , Up-Regulation , Urinary Bladder Neoplasms/genetics , Urinary Bladder Neoplasms/metabolism , Xenograft Model Antitumor AssaysABSTRACT
Non-small cell lung cancer (NSCLC) is the most frequent cause of cancer-related death worldwide. Although many molecular-targeted drugs for NSCLC have been developed in recent years, the 5-year survival rate of patients with NSCLC remains low. Therefore, an improved understanding of the molecular mechanisms underlying the biology of NSCLC is essential for developing novel therapeutic strategies for the treatment of NSCLC. In this study, we examined the role of miR-130b in NSCLC. Our results showed that high expression of miR-130b in clinical specimens was significantly associated with poor overall survival in patients with NSCLC. Moreover, miR-130b expression was significantly increased in NSCLC clinical specimens from patients with vascular and lymphatic invasion. Consistent with this, overexpression of miR-130b promoted invasion and matrix metalloproteinase-2 (MMP-2) activity in A549 cells. Argonaute2 immunoprecipitation and gene array analysis identified tissue inhibitor of metalloproteinase-2 (TIMP-2) as a target of miR-130b. Invasion activity promoted by miR-130b was attenuated by TIMP-2 overexpression in A549 cells. Furthermore, TIMP-2 concentrations in serum were inversely correlated with relative miR-130b expression in tumor tissues from the same patients with NSCLC. Overall, miR-130b was found to act as an oncomiR, promoting metastasis by downregulating TIMP-2 and invasion activities in NSCLC cells.
Subject(s)
Biomarkers, Tumor/metabolism , Carcinoma, Non-Small-Cell Lung/pathology , Gene Expression Regulation, Neoplastic , Lung Neoplasms/pathology , MicroRNAs/genetics , Tissue Inhibitor of Metalloproteinase-2/metabolism , Adult , Aged , Aged, 80 and over , Apoptosis , Biomarkers, Tumor/genetics , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/metabolism , Cell Movement , Cell Proliferation , Epithelial-Mesenchymal Transition , Female , Follow-Up Studies , Humans , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , Male , Middle Aged , Neoplasm Invasiveness , Prognosis , Signal Transduction , Survival Rate , Tissue Inhibitor of Metalloproteinase-2/genetics , Tumor Cells, CulturedABSTRACT
Bladder cancer causes an estimated 150,000 deaths per year worldwide. Although 15% of the recurrent bladder cancer becomes an invasive type, currently used targeted therapy for malignant bladder cancer is still not efficient. We focused on the miR-130 family (miR-130b, miR-301a, and miR-301b) that was significantly upregulated in bladder cancer specimens than that of the normal urothelial specimens. We analyzed the functional significance of miR-130 family using a 5637 bladder cancer cell line and revealed that miR-130 family of inhibitors suppressed cell migration and invasion by downregulating focal adhesion kinase (FAK) and Akt phosphorylation. Mechanistic analyses indicate that the miR-130 family directly targets phosphatase and tensin homolog deleted from chromosome 10 (PTEN), resulting in the upregulation of FAK and Akt phosphorylation. In clinical bladder cancer specimens, downregulation of PTEN was found to be closely correlated with miR-130 family expression levels. Overall, the miR-130 family has a crucial role in malignant progression of bladder cancer and thus the miR-130 family could be a promising therapeutic target for invasive bladder cancer.
