(beneath 30th percentile), medium (in between 30th and 70th percentile) and high
(under 30th percentile), medium (amongst 30th and 70th percentile) and high (above 70th percentile) groups in line with the levels of MMP-9 mRNA expression. Statistical evaluation revealed that in the high and medium groups, MMP-9 levels had been drastically overexpressed as in comparison with both the low group and melanocytes (p0.01) (Figure S1B). Additionally, Pearson correlation analysis was performed in between MMP-9 expression levels and methylation intensity of MMP9-RESULTSComputational identification of an methylation hotspot inside the MMP9 gene intragenicFour CpG islands had been identified inside the MMP9 locus employing the bioinformatic tool CpG Islands TracksFigure 1. MMP9 methylation pattern. (A) Computational detection of CpG islands inside the MMP9 locus. (B) Methylationstatus of MMP9 gene, performed by ENCODE RRBS tool, in 6 typical cells when compared with 7 cancer cells.impactaging.com934 AGING, May well 2016, Vol. 8 No.specific probes in selected samples incorporated in GSE31879 SCF, Human dataset. The statistical analysis reveals a moderate positive correlation (p0.05) between MMP-9 levels and methylation status of probes belonging to CpG-2 group (Figure 2, Table S2). When the melanoma samples have been CD19, Human (HEK293, Fc) stratified in higher, moderate and low group according the levels of MMP-9 expression, the CpG-2 region was hypermethylated in the MMP-9 highexpression melanoma samples (box 4) when compared with other groups, which includes melanocyte controls (box 1-3) (Figure 3A). The cumulative statistical analysis of methylation levels of MMP9 showed a substantial distinction among the four groups in CpG-2 area (p0.01); although, in GpG-1 island statistical significance distinction was observed only for high vs medium (p0.01) and high vs low (p0.05) (Figure 3B). Positive correlation in between MMP-9 expression and hyper-methylation of CpG-2 hotspot in melanoma cell lines Protein and mRNA levels of MMP-9 had been tested in A375, A2058, M14 and MEWO melanoma cell lines by ELISA test and RT-qPCR, respectively. Real-time evaluation revealed that MMP-9 gene expression was 100-fold larger in A375 in comparison with other cell lines (Figure 4A). Related final 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 employed within this study (Figure 4B).Methylation status of MMP9 at CpG-2 hotspot sequence, as identified by computational method, was analyzed making use of the methylation-specific restriction enzyme (MSRE) assay. This was performed as a onestep protocol applying the methylation-sensitive HpaII restriction enzyme, that is in a position to digest the unmethylated DNA but not methylated at 5′-CCGG3’sites. Digested DNA and non-digested DNA (handle reference), of each melanoma cell line samples had been subjected to q-PCR to amplify the putative CpG-2 hotspot region, containing six HpaII consensus websites. As anticipated, larger amplification levels of CpG-2 hotspot sequence were observed in A375 in comparison to other cell lines. A2058 and M14 showed reduced relative methylation levels ( 50 ) compared to these observed in A375. While, no amplification signal was observed within the MEWO cell line (Figure 4C).Figure two. Correlation among MMP9 expression and methylation status of MMP9 gene. Pearson correlation analysisbetween methylation levels of every single probeset and MMP9 expression performed in all samples included in GSE31879 dataset.impactaging.com935.