NGX6 gene mediated by promoter methylation as a potential molecular marker in colorectal cancer

Human colon carcinoma cell lines HT-29 and SW480 were from American Type Culture Collection (ATCC, Rockville, MD) cell bank. Cos7 cells were provided by the Cancer Research Institute, Xiangya School of Medicine, Central South University (Human, P.R. China). All cells were cultured in RPMI1640 medium containing 10% heat-inactivated fetal bovine serum (FBS) and incubated at 37°C in a humidified incubator containing 5% CO₂.

Fresh colorectal cancer tissues and adjacent normal colorectal tissues were obtained from patients treated by primary surgery for colorectal cancer at Third Xiangya hospital surgery department (Hunan, People's Republic of China). All patients gave informed consent for the study to retain and analyze their tissue for research purposes. The samples were snap-frozen immediately following resection and stored in liquid nitrogen until processing. The 19 male and 21 female were aged from 18 to 81 years (mean 54.7 ± 15.1 years). For the colorectal portion, we obtained approval from the Ethic Committee of Central South University.

The NGX6 promoter region in the 5' flanking region was predicted using the PromoterInspector and FirstEF programs. The CpG island was found using CpGplot of the European Molecular Biology Open Software Suite. To obtain the 5' flanking region of the NGX6 gene, PCR amplification on human genomic DNA (forward primer 5'-CGAGCCCAGAGGGTTTACTT-3', reverse primer 5'-GCCTCAATCTTCCCTGCTTC-3') was preformed in a 50-μl reaction mixture. After an initial denaturation step at 94°C for 10 min, the PCR reactions were carried out for 30 cycles at 94°C for 30 sec, 58°C for 1 min, 72°C for 2 min, with a final extension of 10 min at 72°C. The PCR product was purified using a Gel Purification Kit and cloned into the T/A cloning vector pGEM T-Easy (Promega). Positive clones of pT/A -357/+769 were isolated and sequenced.

All NGX6 promoter fragments were cloned into pGL3 enhancer vector (Promega). Construct naming is based on the positions of the promoter fragments. Five deletion constructs of the NGX6 promoter region were created. These progressive deletion constructs (pGL3 -357/+769, pGL3 -357/+276, pGL3 +276/+769, pGL3 -357/-159, pGL3 -159/+276), originating from the construct pT/A -357/+769, were amplified by PCR using the primers listed in Table 1. All the primers included 9-bp non complementary extensions capable of generating KpnI or HindIII restriction sites. The promoter fragments were then subcloned into the KpnI/HindIII sites of pGL3 enhancer vector and sequenced.

Transfections were performed with Lipofectamine 2000 (Invitrogen, CarIsbad, CA); 5 × 10⁵ cells were seeded in each well of 12-well tissue culture plate 24 h prior to transfection. The cells were transfected with 1 μg of various NGX6 promoter constructs, pGL3-control plasmid, or pGL3-enhancer plasmid, and 0.5 μg β-galactosidase vector for normalizing transfection efficiency per well according to manufacturer's instructions. Firefly luciferase activity was measured in cell lysates 48 h after transfection by using Luciferase Assay Kit (Promega). β-Galactosidase activity was measured in cell lysates by the β-galactosidase Enzyme Assay System (Promega). Experiments were repeated at least three times with three replicates per sample for each experiment. Results are normalized against β-galactosidase activity.

Genomic DNA from cells and tissues was prepared using a DNA Extraction Kit (TaKaRa) according to the manufacturer's instructions. Five hundred nanograms of genomic DNA was modified and purified using an EZ DNA Methylation-Gold Kit (ZYMO REAEARCH), following the manufacturer's protocol. Modified DNA was used immediately or stored at -80°C for up to six months.

The methylation-specific PCR primers were designed according to the promoter-active DNA sequence using Methyl Primer Express v1.0. Modified DNA was amplified by two different primer pairs specific to the unmethylated (u) and methylated (M) NGX6 promoter sequences, respectively. Methylation-specific primers (forward) 5'-AGGGATTAATCGAGTCGGTC-3', (reverse) 5'-ATAACCTCCGATATCCTCGC-3'. Unmethylation-specific primers (forward) 5'-GGTTTTATTGATAGGGATTA-3', (reverse) 5'-TATCCTCACAAACCCAAA-3'. PCR amplification was performed for a total of 38 cycles with an annealing temperature of 65°C and 55°C, respectively. Non-methylated and methylated human DKO DNA (ZYMO RESEARCH) was used as negative and positive control, respectively. Methylation-specific PCR products were analyzed by a 2% agarose gel and stained with ethidium bromide.

According to the result of CpGplot program, a 385-bp sequence which covers the whole sequence of MSP product was amplified. The primers were forward 5'-TGTGAGGATAGGGTTTTTTTGAGAT-3' and reverse 5'-CACCCCCRAAAAATAACCTC-3'. The PCR amplification was performed for a total of 38 cycles with annealing temperature of 60°C. PCR products were gel-purified and cloned into the T/A cloning vector pGEMT-Easy (Promega). Ten subclones were isolated and identified by double digestion and sequencing.

Human colon carcinoma cells HT-29 was grown for 5 days in the presence of various concentrations of 5-Aza-dC (0, 0.625, 1.25, 2.5, 5, and 10 μM). Fresh drug was add every 24 h. RNA and genomic DNA were separately isolated.

RNA was isolated from harvested cells with Trizol (Invitrogen) reagent and then treated with DNase (Roche) to eliminate possible contaminated DNA. Reverse transcription of the RNA was performed according to the instructions of Promega. 2 μl cDNA was used for each PCR using NGX6-special primers. The forward primer was 5'-AGAACCGCCATCCCTT-3', and the reverse primer was 5'-CACCTCGTGAGTCAAGCA-3'. The primers for GAPDH were as follows: forward, 5'-AGGTCGGAGTCAACGGATTTG-3'; reverse, 5'-GTGATGGCATGGACTGTGGT-3'. The GAPDH primers were added to the PCR at the end of the tenth cycle as control experiments. Ten microliters of each reaction was then run on 2% agarose gel and stained with ethidium bromide.

Then quantification of relative Quantification of relative transcript levels for NGX6 was performed using the BIO-RAD IQ™ 5 Muticolor Real Time PCR Detection System. cDNA samples were amplified with SYBR^® Premix Ex Taq™. The thermal cycling profile consisted of initial denaturation at 95°C for 30 s and 40 cycles at 95°C for 5 s, 54°C for 15 s, and 72°C for 30 s. Each sample was processed in triplicate. The expression level of NGX6 was normalized to GAPDH using IQ5 software.

Statistical analyses were carried out using SPSS 16.0. Comparisons of categorical variables were made using χ² test or Fisher's exact test as appropriate. Differences were considered statistically significant when P-values were below 0.05.

To clone the NGX6 promoter region, a database search against the human genomic DNA database using NGX6 (Genbank accession AF188239) as query http://​www.​ncbi.​nlm.​nih.​gov/​BLAST was performed to reveal the 5' upstream sequence of the NGX6 gene. Several bioinformatics tools were used to identify the potential promoter region of the NGX6 gene. A 248-bp region spanning positions -157 to +91 was identified as the potential promoter region of the NGX6 gene using PromoterInspector [11], whereas two regions located from -257 to +313 and -163 to +407 were identified as the NGX6 promoter using FirstEF program [12]. The 5' upstream sequence of the NGX6 gene was submitted for analysis to the CpGplot program [13]. Typical CpG islands were defined as ≥ 200 bp of sequence that had a C+G content of ≥ 50% and a value of >0.6 for the ratio (CpG observed)/(CpG expected) [14]. A CpG island that spanned positions -107 to +299 was detected using the program (Figure 1). Finally, a genomic DNA fragment that spanning positions -357 to +769 (the genomic sequence number 35819222 of the NGX6 gene is defined as +1) was amplified by PCR. The fragment was then cloned into the T/A cloning vector pGEM-T-Easy (Promega).

In order to identify the proximal promoter region of NGX6 gene, four constructs of progressive deletion were generated and cloned to the upstream of the luciferase reporter gene in the pGL3-enhancer vector (Promega). Transient transfection experiments were carried out using promoter deletion constructs spanning positions -357 to +769. Plasmid pGL3-357/+276, pGL3+276/+769, pGL3 -357/-159 and pGL3-159/+276 were transfected with Lipofectamine 2000 (Invitrogen) into Cos7 and HT-29 cells, respectively. The luciferase activity driven by NGX6 promoter constructs was measured 48 h after transfection. Expression levels were corrected for variable transfection efficiencies by cotransfection with a plasmid directing the β-galactosidase expression.

As shown in Figure 2, in Cos7 and HT-29 cells, the reporters driven by the shorter 433-bp fragment (pGL3-157/+276) which should contain the core promoter, showed as high luciferase expression as the SV40 promoter of pGL3-control. Luciferase expression driven by the construct pGL3 +276/+769 and pGL3 -357/-159 exhibited as extremely low as pGL3-enhancer. Thus, the sequence spanning positions -159 to +276 was identified as a promoter. MethPrimer Program analysis showed that the NGX6 promoter region had high G/C content and characteristic of a CpG island [15]. In addition, no canonical TATA boxes, but two CAAT boxes were found in this promoter region by the MatInspector program (data not shown).

Epigenetic modification is a frequent mechanism of inactivation of tumor suppressor genes in colorectal cancer [16]. To clarify the mechanism of NGX6 gene inactivation in colorectal cancer cells and tissues, methylation-specific PCR was used to examine the methylation status of the NGX6 promoter. Both HT-29 and SW480 cell lines showed methylation of the NGX6 promoter (Figure 3A).

To identify whether aberrant methylation of the NGX6 promoter in colon cancer cell lines reflects an epigenetic event occurring in primary colorectal cancer tissues, we further examined the NGX6 promoter mathylation status in the 40 pairs of colorectal cancers and adjacent normal colorectal mucosa using methylation-specific PCR (some are shown in Figure 3B). The methylated PCR product was detected in 18/40 (45%) cancer samples and 7/40 (17.5%) adjacent normal samples (Table 2).

To clarify whether the NGX6 methylation status of the colorectal samples was correlated with clinicopathological features of colorectal cancer patients, univariate analyses were carried out to correlate the methylation status of the NGX6 promoter with various clinicopathological parameters. As shown in Table 3, only methylation of the NGX6 promoter was found to correlate with age in colorectal cancer (p < 0.05).

To determine a more detailed map of the methylation in the NGX6 promoter, we performed bisulfite sequencing around the promoter region of the NGX6 gene in colon cancer cell lines and some of the colorectal cancer. Specific primers without CpG sites were used to amplify the region spanning position -233 to +150. The sequence including 34 CpG sites was showed in Figure 4. Bisulfite sequencing of 10 individual clones of PCR products from primary colon cancer biopsies (T38) and cell lines (HT-29 and sw480) revealed densely methylated CpGs within the promoter region compared to normal colon tissue (N38).

In order to clarify the functional association between NGX6 promoter methylation and its aberrant expression in CRC, a DNA demethylating agent, 5-Aza-dC was used to treat HT-29 cells. After 5 days treatment, we isolated mRNA and genomic DNA from the cells, and then detected NGX6 mRNA re-expression in these cells using RT-PCR assay (Figure 5A) and Real-time PCR (Figure 5B). NGX6 expression increased with increasing dosage of 5-Aza-dC. NGX6 expression was higher when induced by 2.5 μM 5-Aza-dC. To confirm that reactivation of NGX6 mRNA expression in HT-29 cell line was caused by demethylation of the NGX6 promoter, methylation-specific PCR was used to detect methylation status changes in the NGX6 promoter in HT-29 cell lines after 5-Aza-dC treatment. The results showed that 2.5 μM 5-Aza-dC could reverse the methylation of NGX6 promoter in HT-29 cells (Figure 5C).