MMP-9 overexpression is associated with intragenic hypermethylation of MMP9 gene in melanoma

Computational identification of an intragenic methylation hotspot in the MMP9 gene

Four CpG islands were identified within the MMP9 locus using the bioinformatic tool CpG Islands Tracks available in UCSC Genome browser (https://genome-euro.ucsc.edu). These regions were named CpG-1, CpG-2, CpG-3, CpG-4 according to their relative position in the MMP9 sequence (Figure 1A, Table S1). In addition, the methylation analysis of MMP9 gene was performed comparing 6 normal cell lines with 7 cancer cell lines using the bioinformatic ENCODE DNA Methylation RRBS (Figure 1B). As reported in the Figure 1B, the cancer cell lines showed a hypermethylated region (red and yellow bars) inside the annotated CpG-2 island compared to the normal cell lines (green bars). This region, located at chromosomal position CH20:46012338-46012584 (GRCh38 Primary Assembly), was reported in this study as a CpG-2 hotspot region.

Figure 1. MMP9 methylation pattern. (A) Computational detection of CpG islands within the MMP9 locus. (B) Methylation status of MMP9 gene, performed by ENCODE RRBS tool, in 6 normal cells compared to 7 cancer cells.

MMP-9 correlated levels of intragenic hypermethylation and mRNA expression in melanoma: computational analysis

The transcript levels of MMP9 in the samples analyzed in GSE31879 melanoma dataset were about 1.5 fold higher in melanoma samples compared to melanocyte controls (p<0.01) (Figure S1A). Afterwards, we proceeded to stratify melanoma samples into low (below 30th percentile), medium (between 30th and 70th percentile) and high (above 70th percentile) groups according to the levels of MMP-9 mRNA expression. Statistical analysis revealed that in the high and medium groups, MMP-9 levels were significantly overexpressed as compared to both the low group and melanocytes (p<0.01) (Figure S1B). Furthermore, Pearson correlation analysis was performed between MMP-9 expression levels and methylation intensity of MMP9-specific probes in selected samples included in GSE31879 dataset. The statistical analysis reveals a moderate positive correlation (p<0.05) between MMP-9 levels and methylation status of probes belonging to CpG-2 group (Figure 2, Table S2). When the melanoma samples were stratified in high, moderate and low group according the levels of MMP-9 expression, the CpG-2 region was hypermethylated in the MMP-9 high-expression melanoma samples (box 4) compared to other groups, including melanocyte controls (box 1-3) (Figure 3A). The cumulative statistical analysis of methylation levels of MMP9 showed a significant difference among the four groups in CpG-2 region (p<0.01); while, in GpG-1 island statistical significance difference was observed only for high vs medium (p<0.01) and high vs low (p<0.05) (Figure 3B).

Figure 2. Correlation between MMP-9 expression and methylation status of MMP9 gene. Pearson correlation analysis between methylation levels of each probeset and MMP-9 expression performed in all samples included in GSE31879 dataset.

Figure 3. Differential analysis of MMP-9 expression and methylation status of MMP9 gene according to MMP-9 expression levels. (A) Methylation analysis of MMP9 locus in the MMP-9 expression groups of melanoma samples, and melanocyte controls. (B) Cumulative statistical analysis of methylation levels of MMP9 in stratified expression groups.

Positive correlation between MMP-9 expression and hyper-methylation of CpG-2 hotspot in melanoma cell lines

Protein and mRNA levels of MMP-9 were tested in A375, A2058, M14 and MEWO melanoma cell lines by ELISA test and RT-qPCR, respectively. Real-time analysis revealed that MMP-9 gene expression was 100-fold higher in A375 compared to other cell lines (Figure 4A). Similar results were obtained by ELISA. Soluble MMP-9 levels were 2024.6 pg/mL for A375 and 13.2 pg/mL for A2580, whereas M14 and MEWO cell lines showed MMP-9 protein levels undetectable by the ELISA kit used in this study (Figure 4B).

Figure 4. Correlation of MMP-9 CpG-2 hotspot with MMP-9 expression in melanoma cell lines. (A) RT-qPCR analysis of MMP-9 transcription levels. (B) ELISA determination of released MMP-9 in cell medium supernatant. (C) Methylation analysis of MMP9 CpG-2 hotspot in melanoma cell lines by MSRE assay. One-way ANOVA test was performed to analyze statistical significance.

Methylation status of MMP9 at CpG-2 hotspot sequence, as identified by computational approach, was analyzed using the methylation-specific restriction enzyme (MSRE) assay. This was performed as a one-step protocol using the methylation-sensitive HpaII restriction enzyme, that is able to digest the unmethylated DNA but not methylated at 5′-CCGG-3′sites. Digested DNA and non-digested DNA (control reference), of each melanoma cell line samples were subjected to q-PCR to amplify the putative CpG-2 hotspot region, containing 6 HpaII consensus sites.

As expected, higher amplification levels of CpG-2 hotspot sequence were observed in A375 compared to other cell lines. A2058 and M14 showed lower relative methylation levels (< 50%) compared to those observed in A375. While, no amplification signal was observed in the MEWO cell line (Figure 4C).

Computational identification of CpG islands and methylation status of MMP9 gene

The CpG islands of MMP9 gene were identified by computational approaches available on UCSC Genome browser (https://genome.ucsc.edu) (Table S1 and Figure 1A). This tool shows the genomic region satisfying the following conditions: CG content greater than or equal to 50%, length greater than 200 bp and ratio is greater than 0.6 of observed number of CG dinucleotides to the expected number on the basis of the number of Gs and Cs in the segment [27].

Moreover, computational analysis of CpG methylation of MMP9 locus were explored using ENCODE DNA Methylation by Reduced Representation Bisulfite Sequencing (RRBS) Tracks developed by Encyclopedia of DNA Elements, available on UCSC Genome browser (Figure 1B). This track reports the percentage of DNA fragments that exhibit methylation at specific CpG dinucleotides. DNA methylation status was assayed using RRBS. Briefly, Genomic DNA, extracted from several cell lines, was digested with the methyl-insensitive restriction enzyme MspI. After purification, the small genomic DNA fragments were used to construct an Illumina sequencing library. This genomic library was treated with sodium bisulfite and amplified by PCR to convert every unmethylated cytosine in a thymidine while methylated cytosines were protected from bisulfite conversion. The sequenced fragments were aligned to the reference genome sequence to calculate the percentage of sequence that showed methylated CpG dinucleotides [28].

Melanoma dataset analysis for MMP-9 mRNA expression and methylation

Publicly available Gene Expression Omnibus (GEO) datasets of melanoma were analyzed to evaluate the association between MMP-9 mRNA expression and methylation status of MMP9 locus. Only datasets with both gene expression and DNA methylation profiling by genome tiling array were considered for the analysis. According to these criteria only the dataset GSE31879 was suitable for the present study. Expression data were obtained using Affymetrix Human Genome U133 Plus 2.0 while methylation profiling data were evaluated by Human DNA Methylation 3x720K CpG Island Plus RefSeq Promoter Array platform (Roche NimbleGen, Inc. - Germany).

In this dataset only 10 of 11 melanoma specimens and 3 of 5 melanocyte cells samples exhibited both mRNA expression levels and DNA methylation status. Melanoma samples were stratified into 3 groups: low (below 30th percentile), medium (between 30th and 70th percentile) and high (above 70th percentile) according to MMP-9 mRNA expression (Figure S1). Methylation-sensitivity probes specific for MMP9 gene, were grouped into Promoter, Intron-1, CpG-1, CpG-2, CpG-3 and CpG-4 regions according to alignment position of each probe with the promoter, the first intron and the previously named CpG islands of MMP9 gene (Table S2, Figure 2 and 3).

Cell lines and culture conditions

Melanoma cell lines A375, A2058 and MEWO were obtained from the ATCC (Rockville, MD, USA). M14 cell line was available at the Department of Biomedical and Biotechnological Sciences of the University of Catania.

Cells were maintained in a humidified 5% CO₂ incubator at 37°C with RPMI-1640 for A375, A2058 and M14 while EMEM was used for MEWO. Both media were supplemented with 2 mmol/L L-glutamine, 100 IU penicillin and 100 μg/ml streptomycin and 10% fetal bovine serum (FBS). All media and supplements were provided from Lonza (Walkersville, USA).

All cell lines were plated in triplicate into 100 mm cell-culture dishes (Thermo Fisher Scientific Inc., USA) and grown under normal culture conditions to 80% confluency. Supernatants of each cell culture were harvested and centrifugated to remove cellular debris. Adherent cells were washed once and scraped in 1X DPBS (Lonza, Walkersville, USA). Cellular pellets were collected via centrifugation and stored at −80°C.

RT-qPCR analysis

Total cellular RNAs were extracted from cultured cell lines with The PureLink® RNA Mini Kit (Thermo Fisher Scientific Inc., USA) according to the manufacturer's instructions. The concentration and purity of the RNAs were ascertained on a NanoDrop spectrophotometer (Thermo Scientific). Reverse transcription was carried out using M-MLV reverse transcriptase (Invitrogen) and random primers (Invitrogen). SYBR green-based real time PCR was conducted with the Applied Biosystem 7500 Real-Time PCR System using SYBR Green PCR Master Mix (Applied Biosystem, USA). The amplification of MMP9 and the Phosphoglycerate Kinase 1 (PGK1) cDNAs, were performed in triplicate using primers and amplification conditions reported in Table S3. The ddCt relative quantification method was performed to quantify the expression of MMP-9 using PGK-1 signal value as control reference.

ELISA

MMP-9 protein levels were detected in supernatants of each melanoma cell culture using MMP-9 Human ELISA Kit (Invitrogen) according to the datasheet. Briefly, the plate was coated in duplicate with 100 μL of supernatants, standards and controls. The plate was left at RT for 2 hours. After washing four times with wash buffer, 100 μL of streptoavidin-HRP conjugated were added to plate and incubated for 30 min at RT. Then, the plate was washed four times and 100 μL of stabilized chromogen were added to each well. After 30 min incubation, the substrate reaction was stopped with stop solution. Finally, optical density (OD) was measured by Tecan ELISA plate reader (Tecan, Switzerland). The averages of duplicate readings of standards and controls were used to generate the standard curve by linear regression analysis. MMP-9 concentrations were calculated fitting the average of duplicate ODs of each sample with standard curve.

Methylation-specific restriction enzyme assay (MSRE)

Methylation-specific restriction enzyme (MSRE) assay is based on use of endonucleases that are not able to cleave methylated-cytosine residues contained in specific consensus sites, leaving methylated DNA intact. Amplification levels of target sequence depend on its content of methylated CpGs, in particular low amplification signal is observed in hypomethylated DNA while higher signal is detected in hypermethylated sequence.

Genomic DNA was extracted from melanoma cell lines using Illustra triplePrep Kit (GE Healthcare, USA) according to manufacturer's instructions. DNA of each sample was digested with restriction enzymes HpaII (New England Biolabs) as explained below.

Each restriction mixture, equally distributed in 0.2 ml reaction tubes, was composed of 50 units of HpaII (5 μL), 5 μL of corresponding 10X REact 8 (New England Biolabs), 1 μg of DNA sample and filled to 50 μL with DNAse free water. Similarly, for each sample the same restriction mixtures without HpaII enzyme were prepared and used as control reference. The methylation insensitive MspI restriction enzyme was used to test the efficiency of MSRE assay.

The restriction reactions and matching controls were incubated at 37°C for 8 hours. Subsequently, 2 μL of Proteinase K (NEB) (20 mg/mL) were added to each tube and placed at 40°C for 30 min, followed by denaturation at 95°C for 10 min. 5 μL of all samples (digested DNA and controls) were subjected to real SYBR green-based real time PCR. The CpG-2 hotspot region, as identified by bioinformatic approaches, was amplified using primers and amplification conditions as shown in table S3. All analyses were performed in triplicate.

Statistics analysis

No additional normalization procedures were applied to data obtained from GSE31879 dataset included in this study. For each MMP-9 expression group, above described, we performed the average of cumulative methylation probe values of the promoter region, of the intron 1 element, and the annotated CpG islands, previously identified in MMP9 locus. Student's t-Test, one-way ANOVA test and Pearson correlation analysis were performed by R software (www.r-project.org).