miR-199a-5p和miR-495在肺癌的未折叠蛋白反应途径中靶向GRP78。

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

作者信息

Ahmadi Azam, Khansarinejad Behzad, Hosseinkhani Saman, Ghanei Mostafa, Mowla Seyed Javad

机构信息

Department of Molecular Genetics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran.

Infectious Diseases Research Center (IDRC), Department of Microbiology and Immunology, Arak University of Medical Sciences, Arak, Iran.

出版信息

Gene. 2017 Jul 15;620:15-22. doi: 10.1016/j.gene.2017.03.032. Epub 2017 Mar 28.

DOI:10.1016/j.gene.2017.03.032

PMID:28363780

Abstract

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.

摘要

引言

非小细胞肺癌（NSCLC）是肺癌最常见的亚型。与NSCLC相关的信号转导通路之一是未折叠蛋白反应（UPR），其主要由GRP78（HSPA5，基因ID：3309）调控。本研究的目的是利用生物信息学工具预测影响GRP78表达的微小RNA（miRNA），通过实验验证这些miRNA与GRP78的相互作用，并评估GRP78及其预测的miRNA在临床样本中的表达相关性。

材料与方法

使用各种软件预测同时靶向UPR中GRP78的所有上游和下游成分以及PI3K/AKT、MAPK、ErbB和钙信号通路主要成分的miRNA。为进行实验分析，获取了36对福尔马林固定石蜡包埋（FFPE）的肺肿瘤和非肿瘤组织样本。此外，使用A549和QU-DB肺癌细胞系来测定GRP78及其预测的靶向miRNA的表达。我们还采用荧光素酶测定法评估候选miRNA与GRP78的3'-UTR之间的相互作用。

结果

基于包括miRNA与靶转录本的热力学结合特征、识别位点数量以及GRP78的3'-UTR内结合位点的保守性等多个标准，选择了hsa-miR-495和hsa-miR-199-5p。RT-qPCR数据显示，肿瘤样本中GRP78显著上调（3.87倍，P = 0.002），而miR-199a-5p（0.13倍，P = 0.0001）和miR-495（0.085倍，P = 0.0001）显著下调。荧光素酶测定法证实了hsa-miR-199a-5p和hsa-miR-495与GRP78转录本的3'-UTR之间存在相互作用。此外，上述miRNA的过表达和竞争性抑制显著改变了GRP78的表达以及剪接的XBP1水平。