Background
Lung cancer is one of the most common causes of cancer-related deaths, and its incidence is increasing [
1‐
3]. Approximately 80% of diagnosed lung cancer cases are non-small-cell lung cancer (NSCLC) [
4,
5]. Despite improvements in diagnosis and treatment, the long-term survival rate has only marginally improved.
Bone morphogenetic protein 2 (BMP-2) was originally identified as an osteoinductive cytokine, and was subsequently reported to have an important role in cell migration, proliferation, and differentiation [
6,
7]. Serum BMP-2 levels from NSCLC samples were higher compared to healthy controls, and positively correlated with poor prognosis, stage, and metastatic burden [
8,
9]. BMP-2 protein expression in human NSCLC is higher than in normal lung tissue, and recombinant BMP-2 promotes cell migration and invasiveness [
10]. Additionally, tumor growth was promoted in nude mice that were injected with A549 cells that were transfected with recombinant BMP-2 [
11]. Finally, BMP-2 is overexpressed in the majority of lung carcinomas and stimulates the growth and progression of lung tumors [
12]. However, the effects of silencing
BMP-2 on lung cancer cell proliferation and migration were not clear.
In this study, we used siRNA to silence BMP-2 to observe the effect on proliferation and migration of the lung cancer cell lines A549 and H460. Moreover, we analyzed the correlation between BMP-2 mRNA expression and the clinicopathological characteristics of 61 patients with NSCLC.
Methods
Clinical sample collection
In this study, 61 patients with NSCLC from the First Affiliated Hospital of Zhengzhou University were enrolled between 2003 and 2008. Patients who had recurrent or primary NSCLC but received chemoradiotherapy before surgery were excluded. Of the 61 patients, 32 were female and 29 were male. Twenty-eight cases had lymph node metastases, whereas 33 cases did not. We obtained paired NSCLC and adjacent non-tumor lung tissues (located more than 5 cm away from the tumors) from 61 patients who underwent primary surgical resection of NSCLC. All patients provided informed consent. Both tumor and non-tumor samples were confirmed as such by pathological examinations. These samples were snap-frozen in liquid nitrogen after resection. The Human Research Ethics Committee of Zhengzhou University approved this study (Table
1).
Table 1
Clinicopathological characteristics and
BMP-2
mRNA expression in NSCLC patients (N = 61)
Sex | | | 0.415 |
Male | 29 | 0.8383 ± 0.13541 |
Female | 32 | 0.8692 ± 0.15649 |
Age (years) | | | 0.312 |
≥60 | 33 | 0.8369 ± 0.12853 |
<60 | 28 | 0.8753 ± 0.16512 |
Differentiation | | | |
Well | 19 | 0.8704 ± 0.15222 | 0.573 |
Moderate-poor | 42 | 0.8473 ± 0.14509 | |
Tumor stage | | | 0.014* |
T1 | 18 | 0.8260 ± 0.13904 |
T2 | 31 | 0.8290 ± 0.14797 |
T3–4 | 12 | 0.9632 ± 0.1066 |
TNM stage | | | 0.341 |
I | 26 | 0.8356 ± 0.13043 |
II | 22 | 0.8458 ± 0.17847 |
III | 13 | 0.9071 ± 0.11028 |
Nodal status | | | 0.026* |
Positive | 28 | 0.8994 ± 0.15196 |
Negative | 33 | 0.8164 ± 0.13218 |
Cell lines and cell culture
The human lung cancer cell lines A549 and H460 were maintained in DMEM supplemented with 10% fetal bovine serum (FBS; Gibco), 100 units/mL penicillin, and 100 μg/mL streptomycin in a humidified incubator of 5% CO2 at 37°C.
Main materials
DMEM (coring, USA); Cell Counting Kit-8 (Dojindo Laboratories, Japan); hematoxylin (sigma, USA); quantitative real time PCR assay kit(SYBR Premix Ex Taq)(TaKaRa, Japan); Trizol (Invitrogen, USA); AMV reverse transcriptase (Promega, USA); the first antibody of BMP-2, β-actin (Santa Cruz Corp, USA); the goat anti-rabbit horseradish peroxidase-labeled secondary antibody (Bio-Rad, USA); chemiluminescence substrate kit (Amersham, USA); NSCLC-derived cell lines A549 and H460 were obtained from (ATCC, USA).
RNA oligo-ribonucleotides and cell transfection
siBMP-2 and negative control (NC) sequences were as follows: siBMP-2: sense: 5′-AATAGCAGTTTCCATCACCGA-3′; anti-sense: 3′-TTATCGTCAAAGGTAGTGGCT-5′; negative control: sense: 5′-ATACTATTCCGAGCGACATAC-3′; anti-sense: 3′-TA TGATAAGGCTCGCTGTATG-5′. Sequences were chemically synthesized by Shanghai GenePharma Co., Ltd. A549 and H460 cells were seeded into six-well plates (2 × 105 cells/well). Transfection was performed by electroporation. Three groups were generated for the ensuing experiments: non-transfected group (blank control), siRNA negative control-transfected group (NC), and siBMP-2 transfected group (siRNA BMP-2). Cells were harvested for experiments 24–48 h post-transfection.
Cell counting kit-8 assays
We used the Cell Counting Kit-8 (Dojindo Laboratories, Japan) according to the manufacturer’s instructions to determine cell viability. Briefly, cells were seeded at a density of 2 × 103 cells/well in 96-well plates (in three replicate wells) and treated daily for 4 consecutive days with 10 μl/well of Cell Counting Kit-8 solution. Optical density was measured at 450 nm to estimate the number of viable cells.
Transwell migration assays
We assayed the migration ability of cells using 6.5 mm diameter transwell chambers with 8 μm membranes (Corning, USA). Twenty-four hours post-transfection, A549 and H460 cells were seeded in the upper chambers, and the bottom wells were coated with 1 mg/ml matrigel for migration assays. Media containing 10% FBS were added to the bottom chambers. After 24 h at 37°C in a 5% CO2 humidified atmosphere, cells in the upper chamber were carefully scraped off using a cotton swab, and cells that had migrated to the basal side of the membrane were fixed in methanol, stained with hematoxylin, and counted. Each test was performed in triplicate.
RNA extraction and quantitative real-time RT-PCR
We isolated total RNA from tissue samples and transfected cells using Trizol, and cDNAs were generated using AMV reverse transcriptase. BMP-2 primers were designed using Oligo 7.0 software according to the BMP-2 mRNA sequence (NM_001200). Sequences were as follows: BMP-2 forward 5′-ATAGCAGTTTCCATCACCGAA-3′, reverse 5′-ACTTCCACCACGAAT CCAT-3′; β-actin forward 5′-AAAGACCTGTACGCCAACACA-3′, reverse 5′-CGATCCACACGGAGTACTTGC-3′. Primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. Real-time RT-PCR was performed in triplicate on the ABI 7500 Fast Real-time PCR system. Cycling parameters were 35 denaturation cycles of 95°C for 15 s, annealing at 55°C for 30 s, and elongation at 72°C for 30 s. Gene expression was quantified using the comparative CT method, normalizing CT values to the housekeeping gene β-actin. After amplification, melting curve analyses were performed to ensure the specificity of the products.
Western blotting
Total protein was extracted from transfected cells, and protein concentrations were measured using Bradford assays. Protein lysates (25 μg) were subjected to SDS-PAGE. Electrophoresed proteins were transferred to nitrocellulose membranes (Whatman, USA). After blocking in 5% non-fat milk, membranes were washed at room temperature and incubated with the following primary antibodies: BMP-2 (1:1000; Santa Cruz Biotechnology, USA) and β-actin (1:1000; Santa Cruz Biotechnology). Following extensive washing, membranes were incubated for 1 h with the goat anti-rabbit horseradish peroxidase-labeled secondary antibody (1:3000; Bio-Rad, USA). An enhanced chemiluminescence substrate kit (Amersham, USA) was used to detect signals with autoradiography film (Amersham).
Statistical analyses
Statistical analyses were performed using SPSS 17.0 software (SPSS Inc., USA). Data are expressed as the mean ± standard deviation (SD). Student’s t-tests were used to compare the mean between two samples. Logistic analyses were used in the correlation of lymph node metastasis with BMP-2 mRNA expression. Follow-up data were analyzed using the Kaplan–Meier method and log-rank tests. P-values less than 0.05 were considered statistically significant.
Discussion
Bone morphogenetic proteins (BMPs) are members of the TGF-β superfamily and are aberrantly expressed in many types of carcinoma cells, including prostate, lung, breast, gastric, and ovarian [
13‐
16]. BMP-2 is known to stimulate proliferation, differentiation, and migration during embryonic development [
6,
17‐
21]. BMP-2 abrogated the fibrogenic function of TGF-β in pancreatic stellate cells via the Smad1 signaling pathway [
22]. Moreover, high concentrations of BMP-2 strongly enhanced gastric cancer cell motility and invasiveness [
23]. BMP-2 upregulation caused epithelial dysfunction and hyperpermeability [
24], and enhanced the neovascularization of developing lung tumors. BMP-2 is aberrantly expressed in approximately 98% of lung carcinomas [
25]. BMP-2 is highly overexpressed in human NCSLC compared with normal lung tissue and benign lung tumors, and high BMP-2 levels enhanced tumor cell migration and invasion, thereby promoting tumor growth [
10,
11,
26,
27]. Thus, these data indicate that BMP-2 has important biological activity in lung carcinomas and a potential marker of lung carcinomas. Up to now, several lung carcinomas potential markers had reported, such as Tiam1, MAT3, DNA methylome [
28‐
30].
In this study, we observed that the mRNA expression of BMP-2 in tumor tissue was significantly higher than in matched adjacent normal tissues (P < 0.01). Furthermore, we found that BMP-2 expression was related to lymph node metastasis, tumor stage, and survival time. These results suggest that BMP-2 may play a role in tumor metastasis.
High levels of
BMP-2 promote tumorigenesis. However, downregulation of
BMP-2 reduced tumor growth. For example, inhibition of
BMP-2 activity using either recombinant Noggin or a BMP-2 antibody caused a reduction in lung tumor growth [
10]. Blocking BMP signaling with the inhibitor DMH1 reduced lung cell proliferation, promoted cell death, and decreased cell migration and invasion in NSCLC cells [
31].
BMP-2 knockdown by adenovirus inhibited growth and invasion of human lung adenocarcinoma cells by blocking PI3K/AKT signaling [
32]. In this study, we suppressed
BMP-2 activity by siRNA. These data show that suppressing BMP-2 expression significantly inhibited lung tumor cell proliferation and migration (Figures
3 and
4). This outcome is in accordance with previous studies and further confirms the biological function of BMP-2 in lung cancer.
Previous studies of BMP-2 have focused on the expression of BMP-2 in tumor tissues and its function in tumor cell proliferation, invasion, and migration. However, for the first time, we investigated and assessed the relationship between
BMP-2 expression and clinicopathological characteristics. Our analyses found significant correlations between
BMP-2 expression and lymph node metastasis, TNM stage, tumor stage, and survival time (Figure
1). Transwell migration assays also showed that the number of si
BMP-2-transfected cells that migrated decreased. This result suggests that
BMP-2 expression is closely related to lung tumor metastasis.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made.
The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
GJZ, HYC and GQZ: conceived of the study, and participated in its design and coordination and helped to draft the manuscript. HYC, HLL, YB, FRZ, RRC, SSC and YYW: collected the samples. HYC, HLL, HQW, XNC and PL: carried out part of experiments and wrote the manuscript. HYC, YYW, GJZ and GQZ performed the statistical analysis. All authors read and approved the final manuscript.