Cancer Letters

Cancer Letters

Volume 335, Issue 2, 28 July 2013, Pages 487-494
Cancer Letters

MicroRNA-365 regulates NKX2-1, a key mediator of lung cancer

https://doi.org/10.1016/j.canlet.2013.03.006Get rights and content

Highlights

  • We found microRNA-365 is an important regulator of NKX2-1 in lung oncogenesis.

  • miRNA-365 expression levels were low, NKX2-1 high, in human lung cancers.

  • We identified the miR-365 response element in the NKX3-1 3’UTR.

  • miR-365 decreased NKX2-1 and reduced proliferation of lung cancer cells.

Abstract

MicroRNAs constitute a class of small noncoding RNAs that play roles in tumorigenesis. We found that NKX2-1 protein levels were generally high in the lung cancer tissues whereas miRNA-365 expression levels were downregulated. Ectopic miR-365 expression decreased NKX2-1 expression in lung cancer cell lines. Transfection of a miR-365 mimic led to reduced proliferation of lung cancer cells; conversely, a miR-365 inhibitor slightly increased cell proliferation. The NKX2-1 overexpression significantly increased the cell proliferation by overcoming the suppressive effect of miR-365. Our data suggest that miR-365 is an important regulator of NKX2-1 and can be a target for lung cancer therapies.

Introduction

Lung cancer is one of the leading causes of cancer-related deaths worldwide [36]. Lung cancer was responsible for almost 27.4% of all cancer deaths in the United States in 2011 [43]. Lung cancer has two major histological types; small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). NSCLC, which accounts for 80% of lung cancers, consists of squamous cell carcinoma, large cell carcinomas and adenocarcinoma [7], [10]. Many previous studies have suggested that genetic abnormalities play roles in lung cancer.

In addition, recent work has focused on identifying recurrent genetic alterations associated with the development and progression of malignancy. Previous studies in NSCLC revealed frequent focal DNA amplifications at chromosome 14q13.3 [22]. This area contains three genes with potential roles in lung cancer, including NK2 homeobox 1 (NKX2-1) [22], [24], [37], [47], [51]. Adjacent to NKX2-1 is the related transcription factor NKX2-8. The function of NKX2-8 in lung cancer is less clear; genetic deletion of NKX2-8 leads to proliferation in lung progenitor cells and widespread dysplasia in the large airways of mice [49]. However, overexpression of this protein seems to enhance the tumorigenicity of malignant cell lines [22]. Another transcription factor found in this region is paired-box 9 (PAX-9), which may synergize with either NKX2-1 or NKX2-8 to promote growth in premalignant lung epithelial cells [22].

NKX2-1, also known as thyroid transcription factor-1 (TTF-1) and thyroid-specific enhancer binding protein (T/EBP), is a member of the homeodomain-containing transcription factor family; it activates the expression of selected genes in the thyroid, lung and parts of the brain. Within the lung, NKX2-1 expression is confined mainly to the peripheral airways and small bronchioles [4], [5], [55]. The regulation of NKX2-1 expression in normal lung tissue and pulmonary adenocarcinoma is poorly understood. The oncogenic and suppressive function of NKX2-1 in the same tumor type substantiates its role as a dual function lineage factor. NKX2-1 controls the transcription of genes specifically expressed in the lung, such as those encoding surfactant protein A (SP-A) [8], [26], [33] and uteroglobin-related protein 1 (uGRP1) [35].

MicroRNAs (miRNAs) are noncoding 21–23 nucleotide small RNAs that usually regulate posttranscriptional processes via sequence-specific interactions with the 3′ untranslated regions (UTRs) of cognate messenger RNA (mRNA) targets in animals [1], [38]. miRNA genes are transcribed by RNA polymerase II, which produces long primary transcripts (pri-miRNAs). Nuclear RNase III Drosha cleaves the pri-miRNA, releasing a small hairpin-shaped precursor miRNA (pre-miRNA). Following nuclear export, the pre-miRNA is processed by another RNase III enzyme Dicer, generating a small RNA duplex. One strand of the duplex is loaded onto the Argonaute (Ago) protein to produce the RNA-induced silencing complex (RISC). Nucleotides at positions 2–8 in miRNA are important for base-pairing with the target mRNA and are referred to as seed sequences [23], [25]. In this study, we investigated NKX2-1 protein expression levels and its possible regulatory miRNAs in the tissues from lung cancer patients. In a searching effort for miRNAs that directly regulate NKX2-1 expression, we found a candidate miRNA, miR-365. Ectopic miR-365 expression reduced both the mRNA and protein expression levels of NKX2-1. miR-365 decreased the proliferation of lung cancer cells whereas NKX2-1 rescued the suppressive effect of miR-365. Our data suggests that miR-365 suppresses the expression and therefore the oncogenic functions of NKX2-1 in lung cancer.

Section snippets

Fresh clinical tissue specimens

Fresh human lung tumor (T) and adjacent normal (N) tissue specimens were obtained from lung patients who underwent tumor resection for lung cancer treatment between 2010 and 2011 at the Kyungpook National University Hospital. Informed consent was obtained from all patients before the collection of lung specimens, and the study was approved by the Ethics Committee of the Kyungpook National University Hospital. The patients’ clinical information is summarized in Table 1. Chopped tissue samples

NKX2-1 transcript and protein levels in various human organs and lung cancer patient samples

Previous studies have reported that NKX2-1 controls the expression of several important lung- and thyroid-specific genes. In lung, NKX2-1 is essential for the expression of the SP-A [8], [26], SP-B [6], [30], SP-C [21], [27], Clara cell secretory protein (CCSP) [50], [58] and uGRP1 [35] genes. In thyroid, NKX2-1 is known to control the expression of the thyroglobulin [12], thyroperoxidase [14] and thyrotropin receptor [42] genes. To test whether NKX2-1 is expressed in specific organs, we

Discussion

Here, we describe a miRNA that directly regulates NKX2-1, a gene that is critical in the development of lung cancer. Previous studies have drawn attention to the role of acquired, recurrent chromosomal alterations in common epithelial malignancies, such as adenocarcinomas of the lung, breast and prostate [41], [44], [51]. These events, which include amplifications, deletions, gene rearrangements and other mutations, are implicated as causal events in tumor origin and progression and often carry

Conflict of interest statement

The authors have no conflict of interests.

Acknowledgments

We thank Dr. Takashi Takahashi for his generous gift of the expression construct of full-length human NKX2-1 cDNA in pCMV-puro (pCMVpuro-NKX2-1) and the control vector. This work was supported by the Proteogenomic Research Program (Grant No. 2012036669), and Grant No. 2012M3C5A1053342 & 2012K001536 through the National Research Foundation of Korea (NRF) grant funded by MEST.

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