MicroRNA 133B targets pro-survival molecules MCL-1 and BCL2L2 in lung cancer

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Abstract

Lung cancer is the most frequent cause of cancer-related death in this country for men and women. MicroRNAs (miRNAs) are a family of small non-coding RNAs (approximately 21–25 nt long) capable of targeting genes for either degradation of mRNA or inhibition of translation. We identified aberrant expression of 41 miRNAs in lung tumor versus uninvolved tissue. MiR-133B had the lowest expression of miRNA in lung tumor tissue (28-fold reduction) compared to adjacent uninvolved tissue. We identified two members of the BCL-2 family of pro-survival molecules (MCL-1 and BCL2L2 (BCLw)) as predicted targets of miR-133B. Selective over-expression of miR-133B in adenocarcinoma (H2009) cell lines resulted in reduced expression of both MCL-1 and BCL2L2. We then confirmed that miR-133B directly targets the 3′UTRs of both MCL-1 and BCL2L2. Lastly, over-expression of miR-133B induced apoptosis following gemcitabine exposure in these tumor cells. To our knowledge, this represents the first observation of decreased expression of miR-133B in lung cancer and that it functionally targets members of the BCL-2 family.

Introduction

Lung cancer is the leading cause of cancer-related deaths in the United States among both men and women [1]. Analysis of the lung cancer genome and proteome has demonstrated that focusing on molecular heterogeneity within lung cancers may be a viable approach to identify and develop novel therapeutics. Aberrant expression of miRNAs in malignancy and their frequent location in fragile chromosomal regions suggests their importance to the pathogenesis of disease [2]. MicroRNAs (miRNAs or miRs) represent a family of small non-coding RNAs (approximately 21–25 nt long) expressed in many organisms including animals, plants, and viruses [3]. MiRNAs are integral to gene regulation, apoptosis, hematopoietic development, and the maintenance of cell differentiation [3], [4]. Researchers have identified abnormal expression of miRNAs in several types of malignancies including lung cancer [5], [6], [7], [8], [9], [10]. MiRNAs have the capacity to target multiple biological functions essential to tumor progression. For example, miR-221 and -222 are upregulated in TNF-α related apoptosis-inducing ligand lung cancer cell lines. Silencing of these miRNAs sensitized resistant cell lines to TRAIL agents [11]. In human lung cancers, particularly small cell carcinoma, miR-17–92 is also over-expressed and in vitro introduction enhanced cell proliferation [12]. Selective silencing of both miR-17–5p and miR-20a induced apoptosis selectively in lung cancer cells over-expressing miR-17–92[13].

MiR-133A and B are currently regarded as are muscle-specific miRNAs [14]. MiR-133A shares a transcriptional unit with miR-1[15]. Through targeting of critical genes involved in cardiac development (Rho-A, Ccd42) and genes involved in cardiac channel expression (HCN2 and HCN4), miR1/133A/133B are implicated in the regulation of cardiac myogenesis and development and cardiac ion channel expression [16], [17]. In addition, both miR-1 and 133 appear to alter cardiomyocyte apoptosis through targeting of HSP60 and 70 [14]. Few if any studies have investigated a potential role for these miRNAs in non-cardiac disease. Recently, Nasser et al. demonstrated that miR-1 was decreased in lung cancer and that over-expression of miR-1 both in vitro and in vivo resulted in reduced tumor growth, migration and increased sensitivity to doxorubicin [18]. Herein, we demonstrate that miR-133B expression is reduced in human non-small cell lung cancer (NSCLC). In addition, miR-133B functionally targets the pro-survival molecules (myeloid cell leukemia 1) MCL-1 and B-cell CLL/lymphoma 2 like 2 (BCL2L2 or BCL-W) and induces apoptosis NSCLC in the setting of chemotherapeutic agents.

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Materials and methods

Cell culture and reagents. H23, H2172, H522, H2009, A549 (adenocarcinoma), H226, H1703 (squamous cell) (American Type Culture Collection, Manassas, VA) and primary normal human bronchial epithelial cells (NHBE) (ScienCell, Research Laboratories, Carlsbad, California) were maintained in 37 °C humidified CO2 incubator and grown in appropriate media. Gemcitabine (25 nM) was used for drug treatment experiments (Eli Lilley, Indianapolis, IN).

Western blotting. Cell and tissue lysates were prepared

Differential expression of miR-133B in human NSCLC

In this study, we examined differentially expressed miRNAs in a cohort of 8 cases of matched adenocarcinomas and adjacent uninvolved lung tissue and 4 additional non-paired adenocarcinomas. We used a high throughput qRT-PCR assay [19]. p-Values were obtained and the significance level was determined by controlling the mean number of false positives [20], [21]. Heat-maps of the expression values with hierarchical clustering were generated to aid in visualization. Out of 198 detected miRNA, we

Acknowledgments

This work was supported by National Institutes of Health Grants #HL077717 (S.P.N.) and Chest/LUNGevity Foundation Grant (S.P.N.).

The authors have no conflicts of interest to declare.

The authors would like to thank Drs. Carlo Croce and Melissa Piper for their assistance in the preparation of this manuscript.

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