miR-199a-5p and miR-495 target GRP78 within UPR pathway of lung cancer

Introduction: Non-small cell lung cancer (NSCLC) is the most common subtype of lung cancer. One of the signal transduction pathways related to NSCLC is Unfolded Protein Response (UPR), which is mainly regulated by GRP78 (HSPA5, Gene ID: 3309). The aim of this study was to employ bioinformatics tools to predict microRNAs (miRNAs) affecting GRP78 expression, experimentally validate interaction of these miRNAs with GRP78 and also evaluating the expression correlation of GRP78 and its predicted miRNAs in clinical samples. Materials and methods: Various software were used to predict miRNAs that simultaneously target all upstream and downstream components of GRP78 in the UPR, as well as the main components of PI3K/AKT, MAPK, ErbB and calcium pathways. For experimental analysis, 36 pairs of Formalin-Fixed Paraffin-Embedded (FFPE) lung tumor and non-tumor tissue samples were obtained. Additionally, A549 and QU-DB lung cancer cell lines were used for expression determination of GRP78 and its predicted targeting miRNAs. We also employed a luciferase assay to evaluate interactions between candidate miRNAs with the 3'-UTR of GRP78. Results: hsa-miR-495 and hsa-miR-199-5p were chosen based on several criteria including thermodynamic binding features of miRNAs to the target transcripts, number of recognition sites, and conservation of binding sites within the 3'-UTR of GRP78. RT-qPCR data revealed a significant up-regulation of GRP78 (3.87 times, P = 0.002) and down-regulation of miR-199a-5p (0.13 times, P = 0.0001) and miR-495 (0.085 times, P = 0.0001) in tumor samples. Luciferase assay confirmed an interaction of hsa-miR-199a-5p and hsa-miR-495 with the 3'-UTR of GRP78 transcript. In addition, over-expression and competitive inhibition of the aforementioned miRNAs, significantly altered the expression of GRP78 and spliced XBP1 level. Conclusion: Our data revealed a significant up-regulation of GRP78 and a concomitant down-regulation of miR-495 and miR-199a-5p in NSCLC. Accordingly, our data suggest a causative role for miR-199-5p and miR-495 in tumorgenesis of lung and probably other cancer types. (C) 2017 Elsevier B.V. All rights reserved.