Expression and DNA methylation alterations of seven cancer-associated 3p genes and their predicted regulator miRNAs (miR-129-2, miR-9-1) in breast and ovarian cancers

• Published in: Gene 2016-01, Vol.576 (1), p.483-491
• Main Authors: Pronina, Irina V., Loginov, Vitaly I., Burdennyy, Alexey M., Fridman, Marina V., Kazubskaya, Tatiana P., Dmitriev, Alexey A., Braga, Eleonora A.
• Format: Article
• Language: English
• Subjects: Breast Neoplasms - genetics; Cancer-associated genes; Chromosomes, Human, Pair 3; Deregulation of gene expression; DNA Methylation; Epigenetic mechanisms; Female; Humans; Methylation of promoter CpG islands; MicroRNAs - genetics; MiRNA target genes; MiRNA–mRNA interaction pairs; Neoplasms - genetics; Ovarian Neoplasms - genetics
• ISSN: 0378-1119; 1879-0038
• DOI: 10.1016/j.gene.2015.10.059

The methylation of promoter CpG islands and interactions between microRNAs (miRNAs) and messenger RNAs (mRNAs) of target genes are considered two crucial epigenetic mechanisms for inducing gene and pathway deregulation in tumors. Here, the expression levels of seven cancer-associated 3p genes (RASSF1(isoform A), RARB(isoform 2), SEMA3B, RHOA, GPX1, NKIRAS1, and CHL1) and their predicted regulator miRNAs (miR-129-2, miR-9-1) were analyzed in breast (BC, 40 samples) and ovarian (OC, 14 samples) cancers using RT-PCR and qPCR. We first revealed a negative correlation between the level of the miR-129-2 precursor and RASSF1(A) and GPX1 mRNA levels in BC (Spearman's correlation coefficient (rs) was −0.26 in both cases). Similar results were observed for the miR-129-2 precursor and the RASSF1(A), GPX1, RARB(2), and CHL1 genes in OC (rs was in the range−0.48 to −0.54). Using methylation-specific PCR, a significant correlation was shown between promoter hypermethylation and the down-regulation of the RASSF1(A), GPX1, RARB(2), SEMA3B, MIR-129-2, and MIR-9-1 genes in BC (rs=0.41 to 0.75) and of the RASSF1(A) gene in OC (rs=0.67). We first demonstrated a high hypermethylation frequency of MIR-129-2 and SEMA3B (up to 45 to 48%) in both BC (69 samples) and OC (41 samples). Moreover, we observed a positive correlation between the hypermethylation of MIR-129-2 and the up-regulation of the RASSF1(A) and GPX1 genes in BC (rs=0.38 and 0.42, respectively). QPCR analysis of the expression of RASSF1(A) and mature miR-129-2 in additional BC sample set (24 samples) revealed a negative correlation between them (rs=−0.41) that strengthened the results obtained during the analysis of miR-129-2 precursor level. In summary, the obtained data indicate the involvement of methylation in the down-regulation of the studied coding and miRNA genes and suggest the involvement of miR-129-2 in the deregulation of RASSF1(A) via a direct interaction or/and mediators in common pathways (according to KEGG, Gene Ontology (FDR