133 DNA methylation profiles of tumor suppressor genes promoters involved in papillary thyroid carcinoma

133 DNA methylation profiles of tumor suppressor genes promoters involved in papillary thyroid carcinoma

S10 Abstracts 132 POSTER Hypermethylation of microRNA-124 genes is associated with unfavorable prognosis of non-small cell lung cancers 134 POSTER ...

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132 POSTER Hypermethylation of microRNA-124 genes is associated with unfavorable prognosis of non-small cell lung cancers

134 POSTER Correlation of circular RNA abundance with proliferation − exemplified with human normal, benign and malignant tissues

D.S. Kim1 , Y.H. Kim2 , J.Y. Park3 . 1 Kyungpook National University Medical School, Anatomy, Daegu, Korea; 2 Kyungpook National University School of Medicine, Anatomy, Daegu, Korea; 3 Kyungpook National University School of Medicine, Internal Medicine, Daegu, Korea

A. Bachmayr-Heyda1 , A.T. Reiner1 , K. Auer1 , N. Sukhbaatar1 , T. Bachleitner-Hofmann2 , I. Mesteri3 , R. Zeillinger1 , D. Pils1 . 1 Medical University of Vienna, Department of Obstetrics and Gynecology, Vienna, Austria; 2 Medical University of Vienna, Department of Surgery, Vienna, Austria; 3 Medical University of Vienna, Department of Pathology, Vienna, Austria

Background: Many non-small cell lung cancer (NSCLC) patients still develop tumor metastasis and recurrence after pulmonary resection, being the primary causes of failure of lung cancer treatment and death. Recent work has shown that microRNAs (miRs) have central roles during tumor metastasis and many miR genes are potentially subjected to control by DNA methylation in multiple tumor types. Recently, miR-124 family has been demonstrated as the potential regulators of the metastasis process in several types of cancers. Material and Methods: In this study, we studied the frequency and clinical effect of miR-124 methylation in tumor and corresponding non-malignant lung tissue specimens with 157 NSCLCs by using methylation-specific PCR. Results: The methylation frequencies of miR-124−1, miR-124−2, and miR-124−3 were 30.6% (48/157), 49.7% (78/157) and 51.6% (81/157) in NSCLC tissues, respectively. miR-124−2 and miR-124−3 methylation was significantly associated with worse survival in total patients (adjusted HR for miR-124−2 = 1.99, 95% CI = 1.19–3.32, P = 0.009; for miR-124−3 = 2.10, 95% CI = 1.24–3.55, P = 0.006). Conclusion: In summary, our results suggest that DNA methylation of the miR-124 loci may be a tumor-associated frequent event during NSCLC tumorigenesis and may be a powerful marker for prognosis of NSCLC. No conflict of interest. 133 POSTER DNA methylation profiles of tumor suppressor genes promoters involved in papillary thyroid carcinoma A. Botezatu1 , I.V. Iancu1 , D. Manda2 , A. Plesa1 , I. Huica1 , S. Vladoiu2 , G. Anton1 , C. Badiu3 . 1 “Stefan S. Nicolau” Institute of Virology, Molecular Virology, Bucharest, Romania; 2 “CI Parhon” National Institute of Endocrinology, Research Laboratory, Bucharest, Romania; 3 “CI Parhon” National Institute of Endocrinology, Department of Endocrinology, Bucharest, Romania Background: Thyroid carcinoma is the most common endocrine malignancy worldwide with the most common histological type − papillary thyroid cancer (PTC). Epigenetic modifications can result in inappropriate activity or inhibition of various signaling pathways, leading to cancer. According to previous studies, epigenetic modification is reported in numerous types of cancers and aberrant methylation of tumor suppressor genes (TSG) is a hallmark for many types of cancers. We aim to profile tumor samples and evaluate the methylation status for 22 TSG promoters (APC, BRCA1, CDH1, CDH13, CDKN2A, DAPK, ESR1, FHIT, GSTP1, MGMT, MLH1, NEUROG1, PDLIM4, PTEN, RARB, RASSF1, RUNX3, SOCS1, TIMP3, TP73, VHL, WIF1), to correlate methylation level with biological phenotypes. Material and Methods: In order to obtain a comprehensive DNA methylation signature in PTC, we performed a methylation analysis (Human TSG EpiTect Methyl II Signature PCR Array-Qiagen) in PTC samples compared with normal thyroid tissue. Preliminary, we evaluated the promoter methylation for four TSG (CDH1, RARB, DAPK, CDKN2A) in 24 patients samples, consisting of 12 PTC specimens and their adjacent normal tissue as controls, in qMS-PCR using bisulphite treated DNA samples (EpiTect Bisulfite Kit − Qiagen). Results: The methylation percentage was found to be increased in PTC samples than in control (p < 0.001). Higher methylation percentage values were found for TP73 (85.16%) BRCA1 (76.94%), RARB (75.8%), APC (74.67%), CDKN2A (74.6%), PDLIM4 (67.74%). A significantly patients number was observed presenting methylated promoters for RARB (41.7%; 5/12) and CDH1 (58.3%; 7/12), while 33.3% (4/12) display DAPK promoter hypermethylation and 16.7% (2/12) of patients presented methylation for CDKN2A promoter. The methylation level was correlated with tumor grade. Conclusions: These results illustrate the involvement of epigenetic alterations in thyroid oncogenesis. We consider that the TSG promoters methylation profile may be a starting point for diagnosis, prognosis of PTC. This study was supported by PCCA 135/2012. No conflict of interest.

Background: Circular RNAs are a recently (re-)discovered abundant RNA species with presumed function as miRNA sponges, thus part of the competing endogenous RNA network. Some support proposes that they origin from specific back-splicing events guided by specific sequences around splice sites. However, neither their formation mechanism nor their cellular function is completely understood. Material and Methods: We analysed the expression of circular and linear RNAs and proliferation in 31 matched normal colon mucosa and tumour tissues. Additionally, immunohistochemical staining of the proliferation marker Ki-76 was performed with the corresponding formalin-fixed paraffinembedded tissues. The correlation of global circular RNA abundance (the circRNA index) and proliferation was validated in additional human healthy and diseased (benign and malignant) tissues. Results: We predicted >1,800 circular RNAs and proved the existence of five randomly chosen examples using RT-qPCR. Interestingly, the ratio of circular to linear RNA isoforms was always lower in tumour compared to normal colon samples and even lower in colorectal cancer cell lines. Furthermore, this ratio correlated negatively with the proliferation index. The correlation of the circRNA index and proliferation was validated in a non-cancerous proliferative disease, idiopathic pulmonary fibrosis, ovarian cancer cells compared to cultured normal ovarian epithelial cells, and 13 normal human tissues. Conclusion: We are the first to report a global reduction of circular RNA abundance in colorectal cancer cell lines and cancer compared to normal tissues and discovered a negative correlation of global circular RNA abundance and proliferation. This negative correlation seems to be a general principle in human tissues as validated with three different settings. Finally, we present a simple model how circular RNAs could accumulate in non-proliferating cells. No conflict of interest. 135 POSTER Nucleosome occupancy and epigenetic modifications in the alternative splicing site of ZNF518B gene in colorectal cancer A.L. Riffo-Campos1 , J. Castillo1 , S. Siscar-Lewin1 , A. Vallet-Sanchez1 , G. Lopez-Rodas2 , L. Franco2 . 1 Institute of Health Research INCLIVA, Biochemistry, Valencia, Spain; 2 Institute of Health Research INCLIVA and University of Valencia, Biochemistry, Valencia, Spain Background: The colorectal cancer (CRC) is the third most common cancer in the world and the incidence rate is rapidly increasing in areas historically considered as with low risk as in Europe. The most common genes mutated in CRC patients are APC, TP53 and KRAS. The KRAS mutations in codon 12 and 13 resulting in constitutive activation of downstream signaling, independent of external cell signals. Therefore, CRC patients showing this mutation do not respond to treatment with anti-EGFR antibodies (as cetuximab or panitumumab). In consequence KRAS is considered one of the most important targets for anticancer therapy. In a study of RNAseq in our laboratory, a list of genes, with significantly differential expression in presence of KRAS G13D mutation, were identified. Among the list, a zing fingers protein, the gene ZNF518B, is significantly altered in this analysis. The study of the mechanisms controlling gene expression is critical in the case of human diseases, such as cancer. Chromatin structure provides essential functions in eukaryotic transcriptional regulation. Histone post translational modifications and nucleosome remodeling are coordinate events involved in the control of gene expression. ZNF518B gene has been characterized in the present work, analyzing the level of expression in some CRC cell lines, studying the nucleosomal positioning and epigenetic changes in the promoter and in two alternative splicing sites in the gene. Material and Methods: The expression of ZNF518B and their isoforms were study through RT-qPCR in 9 colorectal cancer cell lines. The DLD1, DMUT1 HAE6 (KRAS G13D mutated) and SW48 (control) cell lines was selected for the study of nucleosome occupancy (through MNase digestion) and histone modifications (through Nuc-ChIP) since these lines show a higher changes in the ratio between their isoforms. Results: The results show major differences in ZNF518B gene expression between CRC cell lines depending on the presence or not of the