Relationship between DLK1 gene promoter region DNA methylation and non-small cell lung cancer biological behavior.

We investigated the possible association between DLKI gene promoter region methylation and the increased invasion capacity of non-small cell lung cancer (NSCLC). Lung cancer cell line H1299, as well as the gene transfection and RNA interference technology were used to build DLK gene overexpression and knockdown cells. An in vitro invasion assay was performed to observe the changes in the invasion ability of lung cancer cells. Western blot analysis was used to verify Notchl and matrix metalloproteinase-9 (MMP-9) expression levels and a sulfurous acid sequencing technique was used to test the DNA methylation level in the promoter region. Our results showed that the invasion ability of cells in the overexpression group was significantly enhanced. This ability was considerably reduced in the knockdown group. The Notchl and MMP-9 expression level increased significantly in the overexpression group, while it was reduced considerably in the knockdown group. We detected significantly lower levels of DNA methylation in the promoter region in the overexpression group. It was concluded that methylation of the DLK1 gene promoter region increased the invasion ability of NSCLC. Furthermore, it is possible that this process is related to the Notch signaling pathway.

Introduction

Lung cancer is a malignant tumor that ranks first in morbidity and mortality worldwide. An increase in the level of pollution, especially air pollution, has exacerbated the problem (1). The effect of early surgical resection of lung cancer and chemoradiotherapy is very limited. However, advances made in targeted therapy and biological therapy have offered new hope for patients suffering from lung cancer. Environmental and genetic factors have been shown to be involved in the formation of malignant lung tumors (2).

Previous studies demonstrated that DLK1 expression in non-small cell lung cancer (NSCLC) tumor cells were significantly higher than the expression in the para-carcinoma and normal tissues (3). Additionally, a positive expression was located in the cytoplasm and closely associated with clinical features, pathological stage and prognosis. In the present study, the molecular mechanism in NSCLC was determined.

Materials and methods

Source of cells

The NSCLC cell line H1299 [(American Type Culture Collection (ATCC), Manassas, VA, USA)] was treated with conventional cell recovery and serial subcultivation until confluence reached 95%. The culture medium was discarded and phosphate-buffered saline (PBS) was added, washed 3 times, followed by the addition of digestive juice (0.05% of pancreatin + 0.02% of EDTA). After 5 min, fresh culture medium was added to suspend cells. Cell suspension was then centrifuged at 800 × g for 5 min, the supernatant was discarded and fresh medium was added. Serial subcultivation was carried out at a dilution ratio of 1:5. The experiment was divided into three groups: i) overexpression; ii) control; and iii) knockdown groups.

Gene transfection and RNA interference technology

Lipofectamine™ 2000 was used as per the manufacturer's instructions. Cells were cultivated in 10% of RPMI-1640 of fetal calf serum, 24 h prior to transfection and transfection was carried out when the confluence reached 90–95%. The culture medium was discarded and the cells were washed with PBS followed by the addition of serum-free medium (DMEM). Opti-MEM (250 µl) was added to two centrifuge tubes. Moderate vectors were introduced in one tube and Lipofectamine™ 2000 in the second one. Tubes were agitated for 5 min at room temperature and the content of the two tubes was transferred to the cell culture and incubated at 37°C with 5% CO2.

Lipofectamine RNAiMAX was used as per the manufacturer's instructions. Transfection was initiated when the confluence reached 30–50%. siRNA was diluted with 1X annealing buffer. Two tubes with l00 µl Opti-MEM in each were prepared and moderate siRNA was added to one tube and RNAiMAX to the other tube. The tubes were incubated for 5 min at room temperature and culture media were added. The media were replaced after 4 h of incubation at 37°C in the presence of 5% CO2.

Transwell test

A Transwell test was conducted according to the BD Biocoat™ Matrigel™ Invasion Chamber (Discovery Labware, Inc., Two Oak Park, Bedford, MA, USA) instructions. Then 0.5 ml of warm RPMI-1640 culture medium were added to the super- and substratum of the Transwell chamber. The membrane was hydrated after the culture medium was incubated for 2 h at 37°C, and the cells were transfected for 12 h. Digestion was conducted using pancreatin followed by washing with PBS. After resuspension in serum-free medium, density was measured and the medium was extracted from the chambers and transferred into empty wells. Moderate cells and serum-free medium (total volume of 500 µl) were added to the superstratum and complete medium containing serum (total volume of 750 µl) was then added to the substratum. The cells were cultivated for 22 h at 37°C with 5% CO2. The two sides of the membrane were washed twice with normal saline and non-transmembrane cells and Matrigel in the superstratum was rinsed. Cells in the substratum were immersed in cold methanol for 20 min at room temperature and fixed with 4% paraformaldehyde and the membrane was washed with normal saline. Cells in the substratum were immersed in 0.2% crystal violet and stained for 20 min at room temperature. The membrane was then washed 3 times with normal saline and was sectioned and placed on a glass slide. The glass slide was sealed with resin, examined under a light microscope (Olympus, Tokyo, Japan) and the number of transmembrane cells was counted.

Western blot analysis

The method of conventional cell lysates (RIPA, containing 1% of protease inhibitor PMSF and 1% of protease inhibitor cocktail) was used to extract total protein. BCA™ Protein Assay kit was utilized to carry out protein quantification. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), semi-dry protein transfer method and Ponceau-S stain reagent was used. After electric transfer, the PVDF membrane was placed in PBST blocking buffer containing 5% skim milk powder and then sealed for 1 h at room temperature. The primary antibody was diluted with blocking buffer and added to the membrane followed by overnight incubation at 4°C. After washing the PVDF membrane with PBST buffer for 10 min (3 times), secondary goat anti-rabbit (HRP) IgG antibody (dilution, 1:2,000; catalog no. ab6721) was added and the membrane was incubated for 1 h at room temperature. The PVDF membrane was then washed (PBST buffer for 10 min, 3 times). Super ECL Plus allergic luminous fluid was used to develop the image.

Sulfurous acid sequencing technique

Sulfite transversion was applied to genomic DNA according to the protocol of the EZ DNA Methylation-Gold kit. The working solution of the CT conversion reagent was prepared (one CT conversion reagent, 900-µl deionized water, 300-µl M-dilution buffer and 50-µl M-dissolving buffer). The reagent was kept in the dark and dissolved for 10 min at room temperature. Genomic DNA (500 ng) was dissolved in 20-µl deionized water and 130-µl CT conversion reagent was added to the deionized water. The deionized water was kept in the dark and incubated for 10 min at 98°C and incubated for 2.5 h at 64°C, followed by 20-h incubation at 4°C in the dark. M-binding buffer (600 µl) was added to the upper section, mixed and then centrifuged at 8,000 × g for 30 sec at room temperature. The supernatant was discarded and 100-µl M-wash buffer was added and mixed to wash DNA, followed by centrifugation at 8,000 × g for 30 sec at room temperature. Again the supernatant was discarded and 200-µl M-desulphonation buffer was added to wash DNA, which was centrifuged at 8,000 × g for 30 sec at room temperature, after which the supernatant was discarded. DNA was washed once more and 10-µl M-elution buffer was added to elute the DNA. It was centrifuged for 1 min at 8,000 × g at room temperature. Finally, the eluent was collected and DNA was kept at −20°C.

Statistical analysis

SPSS 19.0 statistical software (Chicago, IL, USA) was used for statistical analysis. Quantitative data were expressed as mean ± standard deviation. A comparison of groups was analyzed by single factor ANOVA and qualitative data were expressed as the number of cases or percentage (%). A comparison of the groups was made using the χ2 test. Statistical significance was set at P<0.05.

Results

Comparison of cell invasion ability

The invasion ability of cells in the overexpression group was significantly enhanced, while the invasion ability of cells in the knockdown group decreased significantly. The differences were statistically significant (P<0.05) (Figs. 1 and 2).

Figure 1.

Cell invasion ability experiment (magnification, ×40). (A) Overexpression, (B) control and (C) gene knockdown groups.

Figure 2.

Cell invasion ability.

Expression level comparison between Notch1 and matrix metalloproteinase-9 (MMP-9) protein

The expression level of Notch1 and MMP-9 protein in the overexpression group increased significantly. Notch1 and MMP-9 expression level in the knockdown group was markedly reduced. Differences had statistical significance (P<0.05) (Figs. 3 and 4).

Figure 3.

Detection of protein expression level by western blot analysis.

Figure 4.

Relative quantification of protein expression.

Comparison of DNA methylation level in the promoter region

The content of CG in DLKI gene promoter region was high forming CpG island composed of 89 pairs of CG dinucleotide. We designed two pairs of methylation-specific polymerase chain reaction (MSP) primers based on the promoter region and used PCR amplification to reflect DNA methylation of CpG island (Fig. 5). DNA methylation level in promoter region in the overexpression group was reduced significantly and the difference had statistical significance (P<0.05) (Fig. 6).

Figure 5.

CpG island MSP electrophoretogram (dlk1 CpG1 and CpG2 are two pairs of primer located in different locations within the island). Methylation level in the overexpression group was lower while the methylation level in the control and knockdown groups was higher. MSP, methylation-specific polymerase chain reaction.

Figure 6.

Methylation quantification in DLK1 gene.

Discussion

DLK1 gene is located on the long arm of chromosome 14 at a position corresponding to band 14q32. The total length of mRNA is 1,532 bp, encoding 383 amino acids. DLK1 is a highly conserved protein that contains six structural domains of epidermal growth factors (EGFs) (4). A high expression of DLK1 has been detected in embryo, whereas the expression level decreased in adults (5). The abnormal expression of DLK1 has been detected in liver cancer, brain glioma, myelodysplasia syndrome and prostate cancer (3–7). Through the immunohistochemistry tests and PCR amplification on lung tumor cells, especially NSCLC, we showed a high level of DLK1 expression which was closely related to the clinical features, therapeutic effect and prognosis. A high DLKI expression increased the invasion ability of the tumor and was related to the biological behavior of NSCLC.

The DLL1 proteins in DLK1 and Notch/Delta signal pathways are highly homologous, and they only lack the structural domain of the Delta/Serrate/Lag (DSL). The results obtained from an in vitro study revealed that the DLK1 expression level was negatively correlated with Notch signal activity and was positively correlated with the differentiation degree of fat cells (6). These findings provided evidence for DLK1 and Notch signal transduction. It was shown that MMP-9 promoted the tumor invasion ability through Notch signaling (7). Changes in adhesive forces among tumor cells or between tumor cells and extracellular matrix promoted the degradation of extracellular matrix around the tumor and laid the groundwork for the invasion of cancer towards adjacent tissues. There is a significant increase in the level of proteolytic enzymes which can be used as a sign of the presence of the tumor cells (7).

Members of the MMP family often participate in the degradation process of a variety of extracellular matrix and play an important role in the invasion and transfer process of tumor (8). MMP family proteins can also participate in other biological fuctions other than cell invasion. They achieve this by influencing other proteins such as proteins involved in growth proliferation, cell differentiation, angiogenesis and immune response (9). Our results showed that the expression level of Notch-1 and MMP-9 proteins in the overexpression group increased significantly while the expression level of these proteins in the knockdown group was reduced.

Compared with the control cells, the genomic DNA in tumor cells demonstrated a much lower level of DNA methylation. A low level of methylation usually results in chromatin instability and malfunctions at the transcriptional level (10). Extremely high levels of methylation in the specific sites have also been shown in some tumor cells (11). Abnormal DNA methylation can contribute to tumor formation in many ways: i) abnormal methylation of the cancer suppressor gene promoter region that may result in the inactivation of cancer suppressor genes (12). Over 50% of p53 genes have modifications in cytosine residues and the abnormal methylation of cytosine residues leads to dysfunction of p53; ii) extremely low methylation or lack of methylation within c-oncogene promoter regions results in the overexpression of these proteins which may lead to tumor formation (13). For instance, abnormal methylation of MLH1 (gene associated with non-polyposis colorectal cancer) increases the instability of the genome and promotes cancer; iii) abnormal methylation in the gene imprinting region leads to the deletion of the imprinting gene (14); and iv) overexpression of DNA methyltransferase-1 (DNMT1) leads to the high methylation of CpG island in tumor-related genes (15). Thus, the detection of DNA methylation has a diagnostic value. Sputum samples obtained from patients diagnosed with squamous lung cancer, showed that they underwent DNA methylation in CNKN2A and MGMT gene promoter regions.

Previous findings showed that the abnormal DNA methylation in CNKN2A and MGMT promoters in smoking population increased the risk of squamous lung cancer by 15–25% (16). DNA methylation can provide some guidance for clinical treatment, for example in NSCLC treatment, only the patients whose IGFBP3 gene promoter indicates a non-methylation state can react to chemotherapeutics (17). DNA methylation is a potential target for cancer therapy. Currently, the main drug use for DNA methylation therapy is DNA methylation inhibitor. DNA methylation inhibitor is divided into two categories, nucleoside derivatives and non-nucleoside derivatives (18). Azacitidine and decitabine are the most known nucleoside derivatives DNA methylation inhibitors while hydrarazine and procainamide are among non-nucleoside derivatives.

As a type of detection index, DNA methylation has the following advantages: i) DNA structure is stable and do not degrade easily in vitro; ii) the detecting technology has a high sensitivity. MSP is a type of DNA methylation detection technology widely applied in clinical screenings (19); and iii) DNA methylation analysis is a positive detection method, namely, the observation results of abnormal methylation is regarded as a judgment standard and the analysis results are not disturbed by the existence of normal cells (20).

We concluded that methylation of DLK1 gene promoter increased the invasion ability of NSCLC. It is possible that this process is somehow related to the Notch signaling pathway.