Differentially methylated microRNAs in prediagnostic samples of subjects who developed breast cancer in the European Prospective Investigation into Nutrition and Cancer (EPIC-Italy) cohort

Carcinogenesis, Oct 2015

The crosstalk between microRNAs (miRNAs) and other epigenetic factors may lead to novel hypotheses about carcinogenesis identifying new targets for research. Because a single miRNA can regulate multiple downstream target genes, its altered expression may potentially be a sensitive biomarker to detect early malignant transformation and improve diagnosis and prognosis. In the current study, we tested the hypothesis that altered methylation of miRNA encoding genes, associated with deregulated mature miRNA expression, may be related to dietary and lifestyle factors and may contribute to cancer development. In a case–control study nested in a prospective cohort (EPIC-Italy), we analysed DNA methylation levels of miRNA encoding genes (2191 CpG probes related to 517 genes) that are present in the Infinium Human Methylation450 BeadChip array in prediagnostic peripheral white blood cells of subjects who developed colorectal cancer (CRC, n = 159) or breast cancer (BC, n = 166) and matched subjects who remained clinically healthy. In the whole cohort, several differentially methylated miRNA genes were observed in association with age, sex, smoking habits and physical activity. Interestingly, in the case–control study, eight differentially methylated miRNAs were identified in subjects who went on to develop BC (miR-328, miR-675, miR-1307, miR-1286, miR-1275, miR-1910, miR-24-1 and miR-548a-1; all Bonferroni-adjusted P < 0.05). No significant associations were found with CRC. Assuming that altered methylation of miRNAs detectable in blood may be present before diagnosis, it may represent a biomarker for early detection or risk of cancer and may help to understand the cascade of events preceding tumour onset.

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Differentially methylated microRNAs in prediagnostic samples of subjects who developed breast cancer in the European Prospective Investigation into Nutrition and Cancer (EPIC-Italy) cohort

Carcinogenesis Differentially methylated microRNAs in prediagnostic samples of subjects who developed breast cancer in the European Prospective Investigation into Nutrition and Cancer (EPIC-Italy) cohort Francesca Cordero Giulio Ferrero 1 Silvia Polidoro 0 Giovanni Fiorito 5 6 Gianluca Campanella 4 Carlotta Sacerdot 3 eAmalia Mattiell 8 o Giovanna Masala 7 Claudia Agnol i Graziella Frasc a Salvatore Panic 8 o Domenico Palli 7 Vittorio Krogh 2 Rosario Tumino Paolo Vineis 0 Alessio Naccarat 0 i 0 lMy,olecular and Genetic Epidemiology Unit an 1 MRC-PHE Centre for Environment and Health, Imperial College London , St Mary's Campus Norfolk Place W2 1PG London , UK 2 yE,pidemiology and Prevention Unit Fondazione IRCCS Istituto Nazionale dei Tumori , Via Venezian 1, 20133 Milan , Italy 3 UKn,it of Cancer Epidemiology, AO Citta' della Salute e della Scienza-University of Torino and Center for Cancer Prevention (CPO) , Via Santena 7, 10126 Turin , Italy 4 Daleyp,artment of Epidemiology and Biostatistics, Imperial College London , St Mary's Campus Norfolk Place W2 1PG London, U 5 Department of Clinical and Biological Sciences, University of Torino , Via Santena 7, 10126 Turin, It 6 dGenomic Variation in Human Populations and Complex Diseases Unit , Human Genetics Foundation, Via Nizza 52, 10126 Turin , Italy 7 Molecular and Nutritional Epidemiology Unit, Cancer Research and Prevention Institute - ISPO , Via delle Oblate 4, 50141 Florence, Ital 8 ,Department of Clinical Medicine and Surgery, Federico II University , Via S. Pansini 5, 80131 Naples , Italy The crosstalk between microRNAs (miRNAs) and other epigenetic factors may lead to novel hypotheses about carcinogenesis identifying new targets for research. Because a single miRNA can regulate multiple downstream target genes, its altered expression may potentially be a sensitive biomarker to detect early malignant transformation and improve diagnosis and prognosis. In the current study, we tested the hypothesis that altered methylation of miRNA encoding genes, associated with deregulated mature miRNA expression, may be related to dietary and lifestyle factors and may contribute to cancer development. In a case-control study nested in a prospective cohort (EPIC-Italy), we analysed DNA methylation levels of miRNA encoding genes (2191 CpG probes related to 517 genes) that are present in the Infinium Human Methylation450 BeadChip array in prediagnostic peripheral white blood cells of subjects who developed colorectal cancer (C R=C 1,5n9) or breast cancer (BC, n = 166) and matched subjects who remained clinically healthy. In the whole cohort, several differentially methylated miRNA genes were observed in association with age, sex, smoking habits and physical activity. Interestingly, in the case-control study, eight differentially methylated miRNAs were identified in subjects who went on to develop BC (miR-328, miR-675, miR-1307, miR1286, miR-1275, miR-1910, miR-24-1 and miR-548a-1; all Bonferroni-adjusted P < 0.05). No significant associations were found with CRC. Assuming that altered methylation of miRNAs detectable in blood may be present before diagnosis, it may represent a biomarker for early detection or risk of cancer and may help to understand the cascade of events preceding tumour onset. b a a t Discriminatory ability of identified DMmiRNAs The RPMM method identified seven clusters based on the methylation profiles of the 36 probes associated to the DMmiRNAs We verified the methylation levels of the eight previously identified DMmiRNAs in 734 primary BC and 98 normal breast tissues collected by the TCGA consortium. Four probes (cg15317267, cg18002519, cg12974668 and cg05797594) out of the 36 associated to the eight DMmiRNAs were excluded since no data were available. A comparison of methylation levels between tumour and normal tissues showed, in agreement with our findings, a significant hypomethylation for four miRNAs: miR-675, miR548a1, miR-1910 and miR-1275 (P < 0.001 for all miRNAs, except miR-1275 P  =  0.041) while for miR-1307 hypomethylation was non-significant. In contrast, miR-24-1, miR-328 and miR-1286 resulted hypermethylated in neoplastic tissue compared to control (Supplementary Figure 4, available at Carcinogenesis Online). Target genes and gene enrichment analysis We investigated targets of the identified DMmiRNAs by using the mirWalk database (21), considering only the validated target annotations. Three DMmiRNAs (miR-328, miR-1910 and miR548a) had Dicer1 as common target, while two DMmiRNAs (miR328 and miR-675) had the KRAS gene. No other overlap emerged (see Supplementary Table 4, available at Carcinogenesis Online for the complete list of targets). We performed a functional enrichment analysis on the identified validated targets using the Enrichr algorithm (22). GO biological process terms associated with a significant P (P < 0.001) are reported iFnigure 2B. Notably, significant terms were mainly related to apoptosis (GO:0006917, induction of apoptosis, adjusted P  <  0.001) and growth pathways (GO:0030308, negative regulation of cell growth, adjusted P < 0.001). Discussion Aberrant DNA methylation of miRNA encoding genes has received attention as an emerging mechanism for miRNA deregulation in cancer (23–25). If the DNA methylation patterns significantly correlate with repression/upregulation of relevant miRNAs, the biological effects should be greater than the effect on a single protein-coding gene since a single miRNA can regulate multiple downstream target genes. These amplified effects indicate that miRNAs may be potentially more sensitive biomarkers to detect early malignant transformation and improve diagnosis and prognosis. In the present study, we investigated the methylation levels of miRNA encoding genes in blood samples of subjects from two prospective case–control studies nested in the EPIC-Italy cohort. The main finding is that we have identified eight DMmiRNAs in prediagnostic samples of subjects who developed BC during follow-up, in comparison with healthy subjects matched for main potential confounders. Interestingly, all significant DMmiRNAs resulted less methylated in subjects who developed BC and they, independently of other well-known investigated risk factors, contributed to increase sensitivity and specificity of logistic regression models. In contrast, no DMmiRNAs emerged among subjects who developed CRC. Among the DMmiRNAs, we have found the intergenic miR-548a1, represented only by one TSS probe and coding for miR-548-3p. This miRNA is part of the miR-548 family, whose encoding genes are located across several human chromosomes (26). To the best of our knowledge, there are no published studies reporting an association between this miRNA and BC. More evidence is available for other miRNAs. miR-675 is located in the first exon of the long non-coding RNA H19 gene that is imprinted and maternally expressed. Long non-coding RNAs and internal miRNAs may have versatile roles in multiple biological processes, including tumorigenesis, as potential non-coding RNA regulatory molecules (27). In particular, H19 has been observed several times in association with different cancers, among which BC, but the underlying mechanism of action remains unclear (27). miR675 is known to target the tumour suppressor retinoblastoma 25. Lehmann, U. et  al. ( 2008 ) Epigenetic inactivation of microRNA gene 39 . Heijmans , B. T . et al. ( 2012 ) Commentary: The seven plagues of epigehsa-mir-9-1 in human breast cancer . J. 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Francesca Cordero, Giulio Ferrero, Silvia Polidoro, Giovanni Fiorito, Gianluca Campanella, Carlotta Sacerdote, Amalia Mattiello, Giovanna Masala, Claudia Agnoli, Graziella Frasca, Salvatore Panico, Domenico Palli, Vittorio Krogh, Rosario Tumino, Paolo Vineis, Alessio Naccarati. Differentially methylated microRNAs in prediagnostic samples of subjects who developed breast cancer in the European Prospective Investigation into Nutrition and Cancer (EPIC-Italy) cohort, Carcinogenesis, 2015, 1144-1153, DOI: 10.1093/carcin/bgv102