The Relationship of Mir-21, Mir-126 and Mir-205 to P-Glycoprotein, Mrp1 and Lrp/Mvp in Non-Small Cell Lung Cancer

Research Article

Austin J Cancer Clin Res 2015;2(5):1042.

The Relationship of Mir-21, Mir-126 and Mir-205 to P-Glycoprotein, Mrp1 and Lrp/Mvp in Non-Small Cell Lung Cancer

Zizkova V¹†, Skarda J¹,²†, Janikova M¹,²*, Luzna P³, Radova L², Kurfurstova D¹ and Kolar Z¹,²

¹Department of Clinical and Molecular Pathology and Laboratory of Molecular Pathology, Palacky University Olomouc and University Hospital Olomouc, Czech Republic

²Institute of Molecular and Translational Medicine, Palacky University Olomouc and University Hospital Olomouc, Czech Republic

³Department of Histology and Embryology, Palacky University Olomouc and University Hospital Olomouc, Czech Republic †Authors’ contributions: Veronika Zizkova and Jozef Skarda contributed equally to this work

*Corresponding author: Janikova M, Department of Clinical and Molecular Pathology and Laboratory of Molecular Pathology, Institute of Molecular and Translational Medicine, Palacky University Olomouc and University Hospital Olomouc, Hnevotinska 3, 775 15 Olomouc, Czech Republic.

Received: May 20, 2015; Accepted: June 15, 2015;Published: July 29, 2015

Abstract

Protein transporters P-gp, MRP1 and LRP/MVP participate in the emergence of multidrug resistance (MDR) in non-small cell lung cancer (NSCLC). Their expression is post-transcriptionally regulated by microRNAs (miRNAs). Dysregulation of miR-21, miR-126 and miR-205 is often found in NSCLC. The aim of this study was to determine whether the level of miRNAs is associated with expression of the above mentioned proteins involved in MDR and whether they can be used as prognostic and diagnostic markers. We analysed miR-21, miR-126 and miR-205 in various histological subtypes of NSCLC. Their expression was then correlated with clinico-pathological characteristics, such as progression free survival (PFS), overall survival (OS) and different histological subtypes of NSCLC and, with expression of P-gp, MRP1 and LRP/MVP. We found no significant relationship between miR-21 and miR-126 expression and clinico-pathological parameters. However, miR- 205 levels were significantly increased in squamous cell carcinomas (p<10-6) compared with other histological subtypes of NSCLC. Additionally, the level of miR-205 inversely correlated with P-gp expression in NSCLC patients (p=0.03). Results of this study suggest that miR-205 could be used as a diagnostic marker and its downregulation may indicate the emergence of P-gp mediated drug resistance in NSCLC patients.

Keywords: microRNA; NSCLC; P-gp; MRP1; LRP/MVP

Abbreviations

ADC: Adenocarcinoma; EGFR: Epidermal Growth Factor Receptor; FFPE: Formalin-fixed and Paraffin-embedded; HER3: v-erb-b2 Erythroblastic Leukemia Viral Oncogene Homolog 3; LCC: Large Cell Carcinoma; LRP/MVP: Lung Resistance-related Protein/major Vault Protein; MDR: Multidrug Resistance; miRNA: microRNAs; MRP1: Multidrug Resistance-associated Protein 1; NSCLC: Non-small Cell Lung Cancer; OS: Overall Survival; PFS: Progression Free Survival; P-gp: P-glycoprotein; PTEN: Phosphatase and Tensin Homologue; RNAi: RNA Interference; RT-PCR: Reverse Transcription Polymerase Chain Reaction; SCC: Squamous Cell Carcinoma; SCLC: Small Cell Lung Cancer; TMA: Tissue Microarrays; VEGF: Vascular Endothelial Growth Factor; ZEB: Zinc Finger E-box Binding Homeobox

Introduction

A major cause of cancer mortality worldwide is lung cancer and non-small cell lung cancer (NSCLC) represents approximately 80-85% of all cases. Many NSCLC patients do not respond to therapy due to the emergence of multidrug resistance (MDR). For treatment of advanced forms of NSCLC is frequently treated using radiotherapy, chemotherapy or biological therapies. In recent years, RNA interference (RNAi) has also been introduced [1-3]. RNAi is a molecular mechanism of gene silencing which inhibits gene expression at post-transcriptional level. In human it is govern by small (~22 nt) non-coding, endogenous, single-stranded RNAs, called microRNAs (miRNAs). Based on complementarity, these molecules bind to the target mRNA, which is either completely degraded or prevented from translation, without any split [3]. Currently, according to miRBase 21, 2588 mature human miRNAs are known to regulate many protein-coding genes [4-7]. MiRNAs play an important role in many biological processes, such as proliferation, apoptosis, development, differentiation, DNA damage response and other processes. They can also affect carcinogenesis, chemoresistance and radioresistance and they are involved in diverse regulatory pathways [8-12]. For these reasons, miRNAs often dysregulated in tumours could be classified as a class of oncogenes or tumour suppressor genes and, they might be used as markers for monitoring carcinogenesis [8, 13].

The human microRNA-21 gene (hsa-miR-21), located on chromosome 17q23.2, has been shown to be dysregulated in e.g. breast, colon, pancreatic, stomach, prostate, ovarian and lung cancer [14-16]. Overexpression of miR-21 in lung cancer in never-smokers is probably connected with activated epidermal growth factor receptor (EGFR) signalling [17]. miR-21 supports cell proliferation because it inhibits the tumour suppressor gene Phosphatase and tensin homologue (PTEN). This leads to constitutively active signalling through the PI3K/Akt pathway followed by K-Ras signalling which supports survival and proliferation of tumour cells [18-20]. miR-21 also participates in other processes, such as differentiation, cell cycle progression, apoptosis, tumour invasion and DNA-damage repair processes [21-23]. There is also evidence that the Akt2-dependent pathway activated by hypoxia can support tumour resistance via miR- 21 induction. Chemoresistance accompanied by miR-21 upregulation has been found in breast and ovarian carcinomas, pancreatic cancer, prostate cancer and glioblastomas [24-28].

The expression of human microRNA-126 gene (hsa-miR-126), located on chromosome 9q34.3, has been shown to be dysregulated in hepatocarcinomas, breast, colorectal, cervical and lung cancer [29-31,14,15]. miR-126 is the regulator of Vascular endothelial growth factor A (VEGF-A) and therefore, it has an important role in angiogenesis [29,32,33]. It is also involved in proliferation, differentiation and metastasis [31,34,35]. On the other hand, downregulation of miR-126 decreases the cytotoxic effect of gefitinib in adenocarcinomas cell lines. This can lead to the emergence of resistance to gefitinib [36].

The human microRNA-205 gene (hsa-miR-205), located on chromosome 1q32.2, has been shown to be upregulated in head and neck cancer, bladder cancer and in squamous cell carcinomas [37,38]. Likewise miR-21, miR-205 inhibits tumour suppressor gene PTEN and also regulates Zinc finger E-box binding Homeobox 1 and 2 (ZEB1 and ZEB2) regulating tumour invasion [18,39]. miR- 205 regulation of v-erb-b2 erythroblastic leukemia viral oncogene homolog 3 (HER3) receptor activating Akt can sensitize breast cancer to treatment using the tyrosine-kinase inhibitors gefitinib and lapatinib [40]. The pro-apoptotic effect of chemotherapeutic agents was also observed in prostate cancer cells with induced expression of miR-205 [41].

The following study on miR-21, miR-126 and miR-205, whose genes are located in regions frequently amplified (hsa-miR-21 and hsa-miR-205) or deleted (hsa-miR-126) in lung cancer was based on the work of Yanaihara et al. [14]. We correlated levels of these miRNAs with clinico-pathological status of NSCLC patients and with expression of known transporter proteins involved in MDR, such as P-glycoprotein (P-gp), Multidrug resistance-associated protein 1 (MRP1) and Lung resistance-related protein/Major vault protein (LRP/MVP). These proteins are able to efflux anti-cancer drugs from the cells which is one of the main mechanisms of MDR [2].

Materials and Methods

Clinical assessment and patients

Formalin-fixed and paraffin-embedded (FFPE) surgical tissue samples of lung cancer from years 1996-2000 were obtained from the archives of the Department of Clinical and Molecular Pathology, Faculty of Medicine and Dentistry, Palacky University and University Hospital, Olomouc. The group of patients comprised of 65 patients with lung cancer after approval by the ethics committee of University Hospital and Faculty of Medicine and Dentistry, Palacky University Olomouc. The cohort consistent of 65 patients: 56 male and 9 female and age range 33 to 78 years. 31 tumours were classified as adenocarcinomas (ADCs), 26 as squamous cell carcinomas (SCCs), 6 as large cell carcinomas (LCCs) and 2 as small cell lung cancer (SCLC) patients. 19 patients were in stage I/II and 34 patients in stage III/IV. For the rest of the patients, the stage was unknown. The characteristics of patients are shown in Table 1. 21 patients had received chemotherapy. 18 of them underwent platinum based chemotherapy regime and the rest of patients were treated with different chemotherapeutics, such as fluorouracil, doxorubicin or taxanes. The clinico-pathological parameters progression free survival (PFS) and overall survival (OS) were monitored over 15-years.