MiR-135a inhibits migration and invasion and regulates EMT-related marker genes by targeting KLF8 in lung cancer cells

https://doi.org/10.1016/j.bbrc.2015.07.145Get rights and content

Highlights

  • MiR-135a regulated key EMT markers.

  • MiR-135a inhibits the migration and invasion of NSCLC cells.

  • MiR-135a targets KLF8 and decreases its expression in NSCLC cells.

  • KLF8 is involved in miR-135a-regulated migration and invasion of NSCLC cells.

  • KLF8 is involved in TGF-β1-induced EMT in A549 cells.

Abstract

Epithelial–mesenchymal transition (EMT) has been shown to be related to the pathogenesis of various diseases. Recently, microRNAs (miRNA) have been recognized as a new class of genes involved in human tumorigenesis. In this study, we found that the expression levels of miR-135a were dramatically decreased in NSCLC cell lines and clinical NSCLC tissue samples. Then, we demonstrated that miR-135a significantly suppressed the migration and invasion of lung cancer cells in vitro, suggesting that miR-135a may be a novel tumor suppressor. Further studies revealed that the transcription factor KLF8 was a target gene of miR-135a in NSCLC cells, as miR-135a bound directly to the 3′-untranslated region (3′-UTR) of KLF8, thus reducing both the expression of KLF8 at the mRNA and protein levels. In addition, the EMT marker E-cadherin or vimentin was also down-regulated or up-regulated on miR-135a treatment. Moreover, silencing KLF8 was able to inhibit the migration and invasion of lung cancer cells. In conclusion, these findings indicate that miR-135a suppresses the migration and invasion of NSCLC cells through targeting KLF8, which is involved in the EMT process. This finding provides new insight into the mechanism of NSCLC progression.

Introduction

Lung cancer is the most common cancer worldwide in terms of both incidence and mortality, with very poor prognosis and high possibilities of tumor invasion and migration. Although invasion and migration have been acknowledged as the most lethal attributes of solid tumors, the molecular mechanism underlying them is still undetermined.

Epithelial–mesenchymal transition (EMT) is one of the major molecular mechanisms involved during oncogenesis to promote cancer progress [1]. EMT is characterized by the breakdown of cell junctions and loss of epithelial phenotypes and leads to depolarization of cells, thus contributing to the progression of cancer [2]. Besides gaining mesenchymal markers, EMT endows cancer cells with a higher ability of migration, invasion and metastasis. Recently, several studies have demonstrated that EMT was correlated with carcinogenesis, metastasis and poor prognosis in various human cancers, including lung cancer [3].

MicroRNAs (miRNAs) are small, non-coding RNAs that modulate gene expression post-transcriptionally. These small RNAs modulate protein expression post-transcriptionally by interacting with complementary sites within the 3′-UTR of a target mRNA. MiRNAs have been recognized as a new class of genes involved in human tumorigenesis [4], [5] and have recently been shown to be diagnostic, prognostic and therapeutic biomarkers in lung cancer [5], [6]. MiRNAs have recently been described as crucial regulators of the EMT and metastasis. It has been reported that miR-148 suppresses EMT by target ROCK1 in NSCLC cells [7]. MiR-132 suppresses the migration and invasion of NSCLC cells through targeting ZEB2 involving the EMT [8]. In the last decade, many studies have shown possible links between miR-135a and tumor biological behavior. In classic Hodgkin's lymphoma and gastric cancer, miR-135a can function as a tumor suppressor through the targeting of JAK2 to repress STAT3 activation, reduce cyclin D1 and Bcl-xL expression, and inhibit tumor cell proliferation [9], [10]. Enforced miR-135a expression sensitized lung cancer cells to cisplatin-induced apoptosis by targeting MCL1, and paclitaxel resistance of non-small cell lung carcinoma cell is associated with the up-regulation of miR-135a [11], [12].

Krüppel-like factor 8 (KLF8) belongs to the Krüppel-like C2H2 zinc-finger transcription factor family of proteins [13]. Several KLF family members, including KLF4, KLF5 and KLF6, have been identified as either oncogenes or tumor suppressors [14], [15]. KLF8 is a relatively new member of this family and is emerging as a crucial regulator of cancer initiation and progression. It has been reported that KLF8 is involves in TGF-beta-induced EMT and promotes invasion and migration in gastric cancer cells [16]. Other studies have linked KLF8 with EMT [17], [18].

The aim of this study was to investigate the role of miR-135a in of NSCLC. We found that miR-135a is down-regulated in metastatic lung cancer cell lines and clinical tissue samples, suggesting that miR-135a might act as a tumor suppressor. We explored the possibility that the EMT regulator KLF8 is one of the direct target genes of miR-135a. MiR-135a is able to inhibit EMT and metastasis of NSCLC cells through paralyzing the function of KLF8.

Section snippets

Cell lines and clinical specimens

Six human lung cancer cell lines (A549, H1299, H358, SPC-A-1sci, SPC-A-1 and H460) and normal lung cell (BEAS2B) were purchased from the Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences (Shanghai, China). Lung cancer cell lines were routinely maintained in DMEM medium (HyClone) supplemented with 10% fetal bovine serum (Biowest, South America Origin), 100 U/ml penicillin sodium, and 100 mg/ml streptomycin sulfate at 37 °C in a humidified air atmosphere containing 5%

MiR-135a is frequently down-regulated in lung cancer cells and tissue specimens

We first investigated the expression of miR-135a in six human NSCLC cell lines by qRT-PCR. The results showed that miR-135a was significantly down-regulated in NSCLC cell lines compared with normal lung cell BEAS-2B (Fig. 1A). Further, we detected miR-135a expression in 40 paired clinical primary lung cancer tissue and metastatic lymph node cancer tissue samples (Fig. 1B). Compared to their primary counterparts, the lymph metastatic tissue samples harbored significantly lower miR-135a levels.

Discussion

Tumor invasion and metastasis is a complicated process involving various factors and multiple steps. One of the well-defined processes that occurred during the invasion and distant metastasis of primary epithelial tumors is called the EMT, which is commonly observed in various types of malignant tumors.

In this study, we found that miR-135a was down-regulated in NSCLC cell lines and tissue specimens. Thus, we supposed that miR-135a may be a novel tumor suppressor miRNA and its down-regulation

Conflict of interest

The authors have no conflict of interest.

Acknowledgments

None.

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