Background
Lung cancer is the most malignant tumor and the leading cause of cancer deaths worldwide, with 1.8 million new cases in 2012 [
1]. In Estonia, the incidence rate for lung cancer per 100,000 was 71 for men and 14 for women in 2012 [
2]. Non-small cell lung cancer (NSCLC) accounts for 80–85% of all lung malignancies. In contrast to the steady increase in survival for most cancers, advances have been slow for lung cancer, with a corresponding 5-year relative survival of 18% [
3]. Depending on the stage of cancer, treatment options for people with NSCLC include surgical resection, chemotherapy, radiation therapy, targeted therapy and immunotherapy [
4,
5]. Increasing focus has been placed on the development of immunotherapies, including the directed targeting of specific immune suppressors, such as cytotoxic T-lymphocyte antigen-4 protein (CTLA-4) and programmed cell death-1 protein receptor (PD-1) [
5‐
7]. Another important group of immunotherapeutics have been developed based on interferons (IFNs). IFNs are naturally occurring cellular cytokines that activate immune responses and have been shown to have anti-proliferative, anti-angiogenic and pro-apoptotic effects [
8,
9].
IFN receptor signaling induces the up-regulation of many ISG-s (interferon stimulated genes), including genes with antiviral properties, such as protein kinase R (
PKR), 2,5-oligoadenylate synthetase (
OAS) and myxovirus resistance protein (
MX) family genes [
10‐
14]. In addition to the ISG-s implicated in anti-viral, anti-angiogenic, immunomodulatory and cell cycle inhibitory effects, oligonucleotide microarray studies have identified ISG-s with apoptotic functions, such as XIAP associated factor-1 (
XAF1), caspase-4, caspase-8, death activating protein kinases (
DAPKs) and
IRFs [
15‐
18].
Human BNC2 is an evolutionarily conserved C2H2 zinc finger protein, which has been suggested to be involved in the regulation of mRNA splicing, processing [
19,
20] or transcription [
19‐
21]. BNC2 has been detected in a wide range of tissues: it is abundantly expressed in the ovary, skin, uterus, and kidneys, and its expression has also been detected in the testis, prostate, and lung [
19,
20,
22]. BNC2 expression has been detected in cell lines, including primary human keratinocytes, the keratinocyte cell line HaCaT, and HeLa and HEK293 cells [
19].
Little is known about the expression and function of BNC2 in tumor progression. Genetic variations in the
BNC2 gene have been associated with skin cancer risk [
23‐
25], susceptibility to ovarian cancer [
26‐
28] and prostate cancer development [
29,
30]. The deletion of the
BNC2 gene and the corresponding decreased expression of BNC2 mRNA have been detected in Barrett’s esophagus [
31], hepatocellular carcinoma [
32] and high-grade serous ovarian carcinoma [
33]. In esophageal adenocarcinoma cells, the stable expression of BNC2 caused the growth arrest of tumor cells [
31], suggesting that
BNC2 might also be a tumor suppressor gene. Thus far, there is no evidence of the role of BNC2 in lung cancer.
In this study, the mRNA expression of BNC2 was analyzed in lung squamous cell carcinoma tissue samples and a lung cancer cell line. In addition, the effect of transfected BNC2 on gene expression and cell viability was investigated in the human lung carcinoma cell line A549.
Methods
Tumor samples
Lung squamous cell carcinoma (SCC) and corresponding adjacent non-tumor tissue samples were collected from 8 patients who had undergone curative resection and been histologically characterized by a clinical pathologist in Tartu University Lung Hospital (Tartu, Estonia). The study was approved by the Research Ethics Committee of the University of Tartu, and written informed consent was obtained from all patients. Tissue specimens of appropriate sizes (1–2 cm3) were cut from tumorous and morphologically tumor-free lung tissue. One half of each sample was fixed in formalin and used for pathological examination. The other half of each specimen was snap frozen and stored at −80 °C until use.
Cell culture
The adenocarcinomic human alveolar basal epithelial cell line A549 and human normal lung epithelial cell line BEAS-2B were purchased from the American Type Culture Collection (Manassas, VA, USA). A549 cells were grown in RPMI-1640 medium (PAA Laboratories, Linz, Austria) supplemented with 10% fetal bovine serum (FBS) (Biochrom AG, Berlin, Germany) and penicillin/streptomycin (PAA Laboratories, Linz, Austria). BEAS-2B cells were grown in DMEM (Lonza, Cologne, Germany) medium supplemented with 3% FBS (Biochrom AG, Berlin, Germany) and penicillin/streptomycin (PAA Laboratories, Linz, Austria). Both cell lines were cultured in a humidified tissue culture incubator with 5% CO2 at 37 °C.
Plasmids and transfections
The expression plasmid containing full-length human BNC2 coding sequence and corresponding empty plasmid pCMV-HA (
https://www.addgene.org/32530/) were kindly provided by Dr. Satrajit Sinha (State University of New York, NY, USA). For transient transfection, 10
6 A549 cells were electroporated with 5 µg plasmid DNA in 250 µl Ingenio electroporation solution (Mirus Bio LLC, Madison, WI, USA) using the Gene Pulser Xcell Electroporation System (Bio-Rad, Stockholm, Sweden) under the following conditions: 280 V, 950 µF and ∞ Ω. After electroporation, cells were plated and harvested every 24 h for 3 days.
Cell viability assay
For the viability assay, 2 × 104 A549 cells per well were seeded in a 24-well plate. The next day, cells were transfected with expression plasmid containing a full-length human BNC2 coding sequence and corresponding empty plasmid pCMV-HA using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to manufacturer’s instructions. Cell proliferation was measured 48 h after transfection using CellTiter-Glo® Luminescent Cell Viability Assay (Promega, Madison, WI, USA), where the Luciferase activity was proportional with the quantity of cellular adenosine triphosphate (ATP).
RNA extraction and RT-qPCR
Total RNA was isolated using the Ambion RNA extraction kit (Ambion Inc., Austin, TX, USA) according to the manufacturer’s instructions. One microgram of total RNA was converted to cDNA using the First Strand cDNA Synthesis kit (Fermentas, Vilnius, Lithuania). Real-time PCR was performed using SYBR Green ROX mix (Fermentas, Vilnius, Lithuania) and ABI 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA, USA). Data were analyzed using SDS 2.2.2 software (Applied Biosystems, Foster City, CA, USA). The primer sequences for RT-qPCR amplifications are shown in Table
1. Gene expression levels were determined by the 2
−ΔΔCT method [
34] after normalization to ESD (Esterase D) [
35]. Relative gene expression was calculated as a fold change compared to the control transfection.
Table 1
List of oligonucleotide primers
Esterase D (ESD) | ATTTGCTCCAATTTGCAACC | TCACAAGGTGGGTAGCATCA |
Basonuclin 2 (BNC2) | TGTGAAACTTCACTACAGGAACG | GAGGCGTCTTCCCTGACATC |
Guanylate binding protein 1, interferon-inducible (GBP1) | CCAGATGACCAGCAGTAGAC | AAGCTAGGGTGGTTGTCCTT |
Myxovirus (influenza virus) resistance 2 (mouse) (MX2) | TGAGTGCTGTGTAAGTGATGG | GGACCGGCTAACAGTCACTA |
2′-5′-oligoadenylate synthetase 2, 69/71 kDa (OAS2) | GGTAGCGCATCTTGATTCCA | GAGTATGTAGGGTGGCAAGC |
Interferon regulatory factor 7 (IRF7) | ATCTTCAAGGCCTGGGCTG | CAGCGGAAGTTGGTTTTCCA |
Interferon induced transmembrane protein 1 (IFITM1) | CTGCAACCTTTGCACTCCA | TGTAGACAGGTGTGTGGGTA |
Aconitase 1, soluble (ACO1) | GCTCACAGGGCAAGAACGAT | TCATGACAGCCTGGAAGGTC |
Differentiation antagonizing non-protein coding RNA (DANCR) | ACTATGTAGCGGGTTTCGGG | TTCCGCAGACGTAAGAGACG |
Leucine rich repeat containing 20 (LRRC20) | CTGCTTGGAGAGTTTGCCCT | GCTTAGGGGCTCACTCACTG |
5′-nucleotidase domain containing 2 (NT5DC2) | CATCTTCCGCACCTTCCACA | TGAAGTCCACGCGGTAGTTG |
Thioredoxin domain containing 12 (endoplasmic reticulum) (TXNDC12) | GCTTGAGCTTCCCTGTTTGC | TGGCTACACCTAGGGCTTGA |
2′-5′-oligoadenylate synthetase 1, 40/46 kDa (OAS1) | CGGACCCTACAGGAAACTTG | GAGGTCTCACCAGCAGAATC |
2′-5′-oligoadenylate synthetase 3, 100 kDa (OAS3) | AGAGTTCTGAGCAGGGCCTA | TGGAAAGAGCCACCTAACTGC |
2′-5′-oligoadenylate synthetase-like (OASL) | ATTCCAAGGCCAAGTCCTG | TCTTCGAGAGGATGAGAGTGT |
XIAP associated factor 1 (XAF1) | GGTTTGCCCAAGGACTACAA | GGGTGTAGGATTCTCCAGGT |
Array analysis
The Illumina
® TotalPrep™ RNA Amplification Kit (Ambion Inc., Austin, TX, USA) was used to generate biotinylated amplified RNA for hybridization with Illumina HumanHT-12 v4 Expression BeadChip (Illumina Inc., San Diego, CA, USA) and the Illumina BeadChip platform (Illumina Inc., San Diego, CA, USA). Experiments were performed according to the manufacturer’s instructions. Raw expression data were collected and background subtracted by Illumina GenomeStudio Gene Expression Module v1.8.0 (Illumina, Inc., San Diego, CA, USA). Data were transformed by variance-stabilizing transformation and quantile normalization using the Lumi package (v 2.14.0) [
36] from Bioconductor (
https://www.bioconductor.org/). Differentially expressed genes were identified using the Limma package (v 3.18.1) [
37]. A
p value of 0.05 was used as threshold for differential expression after multiple testing correction by the Benjamini-Hochberg method [
38].
Gene enrichment analysis
Pathway and gene ontology (GO) enrichment analyses were performed with ingenuity pathway analysis (IPA) Ingenuity Systems (
http://www.ingenuity.com) (Qiagen, Redwood City, CA, USA) and g:Profiler (
http://biit.cs.ut.ee/gprofiler/) [
39] using the default settings and the g:SCS method for statistical analysis. The g:SCS method computes multiple testing corrections for
p values from GO and pathway enrichment analysis using a threshold of 0.05. All reported pathways and biological processes are listed according to their GO enrichment score provided by the two software packages as −log (
p values) and with a false discovery rate (FDR) <0.05%.
Statistical analysis
Statistical significance between the different groups and conditions was assessed with Student’s t-test, the Wilcoxon matched pair test was used to analyze the relative mRNA expression in tumor and matched adjacent non-tumor tissues. Results were considered significant at p < 0.05 (*) and highly significant at p < 0.01 (**). Statistical analysis was performed using GraphPad Prism5 (GraphPad Software, San Diego, CA, USA).
Discussion
Lung cancer is a leading cause of cancer-related death worldwide [
48]. Although improvements in molecular diagnostics and targeted therapies have been achieved in recent decades, the average 5-year survival rate for lung cancer is still below 20% [
3]. New therapeutic targets are eagerly needed for this disease. In the current study, we demonstrate that human BNC2 is down-regulated in the adenocarcinomic alveolar epithelial cell line A549 and in SCC tissue compared to non-cancerous cells and tissue, respectively. The transfection of BNC2 to A549 cells led to the up-regulation of numerous ISGs, of which a subset (
XAF1, IRF7, OAS family) is known to inhibit cancer growth and promote the apoptosis of cancer cells.
BNC2 was discovered as a gene with a similar domain structure as basonuclin 1, with a serine-rich region, nuclear localization signal (NLS) and three pairs of distinct C
2H
2 zinc fingers [
20]. BNC2 is evolutionarily conserved in vertebrates: there is a remarkable conservation of the amino acid sequence of BNC2 across species as distant as the zebrafish, chicken, and mammals. The level of similarity of amino acids between human and mouse BNC2 is 97% [
20,
21].
Early studies suggested that BNC2 might act as a transcription regulator [
19,
20]. Later, it was proposed that BNC2 has a function in RNA processing [
21] and may regulate the expression of genes essential for the development of craniofacial bones [
49]. Multiple studies have demonstrated the down-regulation of BNC2 in numerous cancers [
31‐
33]. Akagi and colleagues detected the decreased expression of BNC2 mRNA in esophageal adenocarcinoma cells and showed that the stable expression of BNC2 caused the growth arrest of tumor cells, which suggests that BNC2 is a tumor suppressor [
31,
32]. Our results show that BNC2 was significantly down-regulated in the lung adenocarcinoma cell line A549 compared to the human normal bronchial epithelial cell line BEAS-2B, as well as in lung tumor tissue compared to non-tumor tissue. In addition, we also show that the over-expression of BNC2 inhibits the proliferation of A549 cells. Thus, our data are in line with previous studies that report the down-regulation of the
BNC2 gene in cancers of epithelial origin and indicate that BNC2 has a tumor-suppressive function.
Microarray technologies have been intensively used in cancer research [
50‐
53] and are useful to profile gene expression patterns to facilitate diagnosis, predict the response to therapy, find new biomarkers and examine the development of drug resistance in cancer [
54‐
56].
Microarray data from A549 cells transfected with BNC2 show the relationship of BNC2 with the modulation of immune system. Increased BNC2 expression in Th22 cells compared to other T cell subsets [
57] and the suppression of NF-κB basal activity in HEK293 cells [
58] have been reported previously. We determined the relationship of BNC2 with immune regulation with two different pathway analysis programs: IPA and G:profiler, which both revealed that the increased expression of BNC2 primarily affects genes associated with the interferon signaling pathway. Several ISGs with increased expression in BNC2-transfected cells have been associated with the restriction of tumor growth and development. For example,
XAF1 has been shown to inhibit proliferation and to induce the apoptosis of cancer cells as it negatively regulates the caspase-inhibiting activity of XIAP [
42,
47]. Along with
XAF1, we discovered the up-regulation of a subset of genes with the capacity to inhibit cell proliferation and to stimulate cancer cells to undergo apoptosis (
IRFs, IFIT1-
3, ISG12a, IFITM and the OAS family members) [
59‐
61].
The use of interferons (IFNs) could be a potential strategy in the treatment of lung cancer [
8]. Type I IFNs (the IFN-α family and IFN-β) have been used with some success for the treatment of different cancers, including hematological malignancies and solid tumors [
62‐
65]. Type II IFN, IFN-γ, also has antitumor effects in various types of cancers [
66,
67]. In addition to in vitro studies, several pre-clinical and clinical in vivo studies demonstrate the efficacy of type I IFNs alone or in combination with other treatments in cancer therapy [
68‐
74].
Thus, our results suggest that BNC2 has the capacity to increase the expression of IFN-regulated genes and thereby act as a tumor suppressor gene in lung epithelial cells.
Authors’ contributions
EU and AR conceived the study, EU performed the experiments and prepared the manuscript. ER analyzed the array results. All the authors participated in the design of study, data analysis, interpretation of results, writing the manuscript and approved the final version of the manuscript. All authors read and approved the final manuscript.