miR-513a-3p sensitizes human lung adenocarcinoma cells to chemotherapy by targeting GSTP1

Abstract

Cisplatin is a classic chemotherapy agent used for treating human non-small cell lung cancer (NSCLC). However, cisplatin resistance is a challenge against successful clinical use. Glutathione S-transferase P1 (GSTP1) has been reported to contribute to cisplatin resistance in many studies. MicroRNAs (miRNAs) are short non-coding RNAs that are 21–25 nucleotides in length. They play a role in post-transcriptional gene regulation by inducing repression and/or mRNA degradation. Recent studies have shown that miRNAs are responsible for cisplatin resistance. This study aims to determine whether deregulated miRNAs can sensitize human lung adenocarcinoma cells to cisplatin by targeting GSTP1. Real-time RT-PCR revealed that GSTP1 mRNA expression was 2.7 ± 0.38 folds (p = 0.039) upregulated in A549/CDDP cells, compared with the parental A549 cells, while miR-513a-3p expression was 0.34 ± 0.03 folds (p = 0.023) downregulated. Luciferase activity assay proved that GSTP1 was a target gene of miR-513a-3p, which was confirmed by Western blot analysis. Furthermore, CCK-8 assay showed that overexpression of miR-513a-3p could enhance cisplatin-induced apoptosis in human lung adenocarcinoma cell lines, A549/CDDP and SPC-A-1. In conclusion, our data demonstrated that miR-513a-3p can sensitize human lung adenocarcinoma cells to cisplatin by targeting GSTP1.

Introduction

Lung cancer is the leading cancer site in males, comprising 17% of total new cancer cases and 23% of total cancer deaths; while mortality for females in developing countries is as high as that of cervical cancer, accounting for 11% of total female cancer deaths [1]. Histologically, lung cancer can be classified into non-small-cell lung cancer (NSCLC) and small-cell lung cancer. Non-small-cell lung cancer, including adenocarcinoma, squamous cell carcinoma, large cell carcinoma, and bronchioloalveolar carcinoma, accounts for nearly 85% of all lung cancer cases [2]. About 50% of patients with lung cancer have advanced stage disease at the time of diagnosis [3].

Systemic therapy remains the main treatment strategy for advanced stage NSCLC. Combinational chemotherapy with a platinum-based regimen has emerged as standard therapy for patients with advanced stage disease [4]. Cisplatin [cis-diamminedichloroplatinum (II), DDP], an electrophilic platinum coordinate compound which causes DNA damage by forming platinum-DNA coordination complexes [5], has been frequently used as a chemotherapeutic agent against lung adenocarcinoma.

However, resistance to cisplatin-induced cellular toxicity is one of the obstacles against successful chemotherapy in clinical use [6]. Until now, the molecular mechanisms of cisplatin resistance have not been fully understood and may be include: (1) decreased accumulation of cisplatin, (2) increased detoxification systems, such as GSH, GSTP1, and metallothionein, and (3) decreased DNA damage or increased repair [7]. Many genes have been reported to contribute to cisplatin resistance, such as BCL-2 [8], [9], [10], excision repair cross-complementation group 1 (ERCC1) [11], [12], [13], resistance-related protein (LRP) [8], survivin [14], [15], multidrug resistance protein 1 (MDR1) [16], [17], multidrug resistance associated protein 1(MRP) [8], [18], and Glutathione S-transferase P1 (GSTP1) [5], [19], [20], [21]. In cells, numerous factors regulate the functions of those genes. Among them, microRNAs (miRNAs) play an important role.

MiRNAs are short non-coding RNA molecules that are 21–25 nucleotides long. Mature microRNAs are able to modulate gene expression via direct or indirect interaction with one or more mRNA targets. In mammalian cells, miRNAs regulate gene expression by binding to the 3′-UTR of target mRNAs, leading to translational repression or degradation of the mRNAs [22]. For instance, miR-16 [9], miR-181b [10], miR-497 [23], [24], miR-302b [24], miR-200bc/429 cluster [25], and miR-885-3p [26] could regulate BCL-2 expression. MiR-138 [27] could repress ERCC1expression. MiR-708 [28], miR-218 [29], and miR-542-3p [30] could downregulate survivin expression. These miRNAs could play a crucial role in drug resistance by targeting their respective genes.

In this study, we demonstrated that miR-513a-3p was downregulated in cisplatin resistance human lung adenocarcinoma cell line A549/CDDP, compared with its parental cell line A549. While GSTP1 mRNA and protein expression level were upregulated in A549/CDDP cells, we showed that exogenous miR-513a-3p could sensitize human lung adenocarcinoma cell lines (A549/CDDP and SPC-A-1) to cisplatin by repressing GSTP1 expression.