Genome-wide microRNA expression profiling identifies miR-9-3 and miR-193a as targets for DNA methylation in non-small cell lung cancers
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE31579 | GEO | 2012/03/13
SECONDARY ACCESSION(S): PRJNA145611
REPOSITORIES: GEO
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Project description:Purpose: The major aim of this study was to investigate the role of DNA methylation (referred to as methylation) on microRNA (miRNA) silencing in non-small cell lung cancers (NSCLC). Experimental Design: We performed microarray expression analyses of 856 miRNAs in NSCLC A549 cells before and after treatment with the DNA methyltransferase inhibitor 5-aza-2´-deoxycytidine (Aza-dC) and with a combination of Aza-dC and the histone deacetylase inhibitor trichostatin A. MiRNA methylation was determined in 11 NSCLC cell lines and in primary tumors and corresponding non-malignant lung tissue samples of 101 stage I-III NSCLC patients. Results: By comparing microarray data of untreated and drug treated A549 cells, we identified 33 miRNAs whose expression was upregulated after drug treatment and which are associated with a CpG island. Thirty (91%) of these miRNAs were found to be methylated in at least 1 of 11 NSCLC cell lines analysed. Moreover, miR-9-3 and miR-193a were found to be tumor-specifically methylated in NSCLC patients. We observed a shorter disease-free survival of miR-9-3 methylated lung squamous cell carcinoma (LSCC) patients compared to miR-9-3 unmethylated LSCC patients by multivariate analysis (HR = 3.8, 95% CI = 1.3 to 11.2, p = 0.017) and a shorter overall survival of miR-9-3 methylated LSCC patients compared to miR-9-3 unmethylated LSCC patients by univariate analysis (p = 0.013). Conclusions: Overall, our results suggest that methylation is an important mechanism for inactivation of certain miRNAs in NSCLCs and that miR-9-3 methylation may serve as a prognostic parameter in LSCC patients. MiRNA expression (LC Sciences, mirBASE12) was analyzed before and after treatment of A549 cells with 5-aza-2´-deoxycytidine (Aza-dC) and a combination of Aza-dC and trichostatin A (TSA). Experiments were performed in duplicates.
2012-03-13 | E-GEOD-31579 | biostudies-arrayexpress
Project description:Epigenetic changes largely contribute to the regulation of gene expression in cancer cells. DNA methylation is part of the epigenetic gene regulation complex which is relevant for the pathogenesis of cancer. We performed a genome-wide search for methylated CpG islands in tumors and corresponding non-malignant lung tissue samples of 101 stage I-III non-small cell lung cancer (NSCLC) patients by combining methylated DNA immunoprecipitation and microarray analysis using NimbleGenM-BM-4s 385K Human CpG Island plus Promoter arrays. By testing for differences in methylation between tumors and corresponding non-malignant lung tissues, we identified 298 tumor-specifically methylated genes. From many of these genes epigenetic regulation was unknown so far. Gene Ontology analysis revealed an over-representation of genes involved in regulation of gene expression and cell adhesion. Expression of 182 of 298 genes was found to be upregulated after 5-aza-2M-BM-4-deoxycytidine (Aza-dC) and/or trichostatin A (TSA) treatment of 3 NSCLC cell lines by Affymetrix microarray analysis. In addition, methylation of selected genes in primary NSCLCs and corresponding non-malignant lung tissue samples were analyzed by methylation-sensitive high resolution melting analysis (MS-HRM). Our results obtained by MS-HRM analysis confirmed our data obtained by MeDIP-chip analysis. Moreover, by comparing methylation results from MeDIP-chip analysis with clinico-pathological parameters of the patients we observed methylation of HOXA2 as potential parameter for shorter disease-free survival of NSCLC patients. In conclusion, using a genome-wide approach we identified a large number of tumor-specifically methylated genes in NSCLC patients. Our results stress the importance of DNA methylation for the pathogenesis of NSCLCs. Overall, samples of 3 untreated, with Aza-dC treated and with Aza-dC/TSA treated NSCLC cell lines were hybridized to Affymetrix HG-U133_plus_2.0 microarrays (18 in total).
2012-11-26 | E-GEOD-32496 | biostudies-arrayexpress
Project description:Epigenetic changes largely contribute to the regulation of gene expression in cancer cells. DNA methylation is part of the epigenetic gene regulation complex which is relevant for the pathogenesis of cancer. We performed a genome-wide search for methylated CpG islands in tumors and corresponding non-malignant lung tissue samples of 101 stage I-III non-small cell lung cancer (NSCLC) patients by combining methylated DNA immunoprecipitation and microarray analysis using NimbleGen´s 385K Human CpG Island plus Promoter arrays. By testing for differences in methylation between tumors and corresponding non-malignant lung tissues, we identified 298 tumor-specifically methylated genes. From many of these genes epigenetic regulation was unknown so far. Gene Ontology analysis revealed an over-representation of genes involved in regulation of gene expression and cell adhesion. Expression of 182 of 298 genes was found to be upregulated after 5-aza-2´-deoxycytidine (Aza-dC) and/or trichostatin A (TSA) treatment of 3 NSCLC cell lines by Affymetrix microarray analysis. In addition, methylation of selected genes in primary NSCLCs and corresponding non-malignant lung tissue samples were analyzed by methylation-sensitive high resolution melting analysis (MS-HRM). Our results obtained by MS-HRM analysis confirmed our data obtained by MeDIP-chip analysis. Moreover, by comparing methylation results from MeDIP-chip analysis with clinico-pathological parameters of the patients we observed methylation of HOXA2 as potential parameter for shorter disease-free survival of NSCLC patients. In conclusion, using a genome-wide approach we identified a large number of tumor-specifically methylated genes in NSCLC patients. Our results stress the importance of DNA methylation for the pathogenesis of NSCLCs.
2012-11-26 | GSE32496 | GEO
Project description:Objective: The purpose of this study was to investigate the miRNAs basis of tumorigenesis in LSCC, and analyze the physiological processes of target genes predicted by the screened miRNAs that may be useful for the effective therapeutic strategies. Methods: A total number of 6 patients who underwent surgery for primary laryngeal squamous cell carcinoma were recruited for miRNA array analysis. LSCC tissues compared with corresponding adjacent non-neoplastic tissues were analysed by the Affymetrix GeneChip miRNA Array 3.0 to screen effective miRNAs, then TargetScan,PITA and microRNAorg were used for target gene prediction in GeneSpring 12.5 software.Moreover, pathway-based methods were adopted to analyse physiological processes of the target genes. The screened miRNAs and the significant target genes were also validated by qRT-PCR in another 36 patients diagnosed for LSCC. Results: Analysed by the miRNAs arrays, there were seven miRNAs (hsa-miR-224, hsa-miR-183, hsa-miR-23a, hsa-miR-675, hsa-miR-27a, hsa-miR-503 and hsa-miR-4800-3p) significantly related to tumorigenesis ,and all the screened miRNAs in laryngeal squamous cell carcinoma tissues were significantly up-regulated(P<0.05). The expressions of these miRNAs were also validated by qRT-PCR. Then analysed by GeneSpring 12.5 software, there were 72 putative target genes corresponding to the seven significant miRNAs. Moreover,analysed by String database,the result indicated that most target genes could be composed of gene networks; Analysed by GO database, we observed that these target genes were involved in processes such as metabolic process, biological regulation,membrane,protein binding,ion binding and so on (P <0.05); Analysed by KEGG pathways database, MAPK signaling pathway, adherens junction and pathways in cancer played especially important role in tumorigenesis of LSCC (P<0.05). As the two important genes in MAPK signaling pathway which plays pivotal roles in tumorigenesis, FGF2 and MAP2K4 were validated by qRT-PCR, and they were significantly down-regulated(P<0.05). Conclusions: The research revealed seven miRNAs expression signature(hsa-miR-224, hsa-miR-183, hsa-miR-23a, hsa-miR-675, hsa-miR-27a, hsa-miR-503 and hsa-miR-4800-3p) of tumorigenesis in laryngeal squamous cell carcinoma,and analysed the physiological processes of the predicted target genes regulated by screened miRNAs in LSCC. The result will contribute to the understanding of the molecular basis of LSCC and help to improve the treatment. A total number of 6 patients who underwent surgery for primary laryngeal squamous cell carcinoma were recruited for miRNA array analysis. LSCC tissues compared with corresponding adjacent non-neoplastic tissues were analysed by the Affymetrix GeneChip miRNA Array 3.0 to screen effective miRNAs, then TargetScan,PITA and microRNAorg were used for target gene prediction in GeneSpring 12.5 software.Moreover, pathway-based methods were adopted to analyse physiological processes of the target genes. The screened miRNAs and the significant target genes were also validated by qRT-PCR in another 36 patients diagnosed for LSCC.
2014-10-30 | E-GEOD-62819 | biostudies-arrayexpress
Project description:Background: Laryngeal squamous cell carcinoma (LSCC) is the most common type in head and neck squamous cell carcinoma (HNSCC), and the development of LSCC are multistep processes accompanied by changes of molecular biology. Objective: The purpose of this study was to investigate the miRNAs basis of tumorigenesis in LSCC, and analyze the microRNAs dysregulation related with the six targeted genes (CDK1,CDK2, CDK4, MCM2, MCM3, MCM4) which were related to tumorigenesis from the screened miRNAs. Methods: A total number of 5 patients who underwent surgery for primary laryngeal squamous cell carcinoma were recruited for miRNA array analysis. LSCC tissues compared with corresponding adjacent non-neoplastic tissues were analysed by the Affymetrix GeneChip miRNA Array 3.0 to screen effective miRNAs, then mirfocus 3.0 database (http://mirfocus.org/index.php) was adopted to select putative regulated miRNAs of the six targeted genes. Moreover, the selected putative regulated miRNAs were also validated by qRT-PCR in another 36 patients diagnosed for LSCC. Results: Analysed by the miRNAs arrays, there were 127 miRNAs significantly related to tumorigenesis ,and 78 showed a higher expression in tumor than in non-tumor tissue while 49 presented the contrasting pattern(P<0.01).Then analyzed by mirfocus 3.0 database, there were 2 putative regulated miRNAs, hsa-miR-24-3p and hsa-miR-183-5p, corresponding to three of the six targeted genes. Among the 2 putative miRNAs, hsa-miR-24-3p regulated MCM4,CDK1 and CDK4,while hsa-miR-183-5p regulated MCM4 only. Another miRNA we should focus on is hsa-miR-30a-5p, a tumor-suppressive factor, which was down-expressed obviously analysed by the miRNAs arrays. The expression of the 3 putative regulated miRNAs were also validated by qRT-PCR in another 36 patients (P<0.05). Conclusions: The research revealed 127 miRNAs expression signature of tumorigenesis in laryngeal squamous cell carcinoma,and analyzed 3 putative regulated miRNAs, hsa-miR-24-3p, hsa-miR-183-5p and hsa-miR-30a-5p, as potential diagnostic and therapeutic markers in LSCC. The result will contribute to the understanding of the molecular basis of LSCC and help to improve the treatment.
2014-10-30 | GSE62819 | GEO
Project description:Purpose: Generate genome-wide methylation profiles of non-small cell lung carcinomas (NSCLC) and their matching lung tissues for detection of hypermethylated and hypomethylated regions present in the tumors. Methods: MethylCapture followed by next-generation sequencing (Illumina GAIIx) of 7 nsclc tumor samples and paired lung tissues in replicated, plus one cell line, 2 fully artificially methylated and 2 fully artificially unmethylated controls. Normalization of methylation reads based on CpG coupling factor–method. Relative methylation scores (rms) in 500bp non-overlapping windows. 90th percentile of rpm (reads per million) values for all 500bp genome-wide windows, with rpm <1.33 were considered. Distributions of 10bp bins rms values within each 500bp genomic region were compared using both one-sided Student’s t-test and one-sided Wilcoxon rank-sum test. Testing was done separately for hypo- and hypermethylation and p-value threshold of 10-18. Results: MethylCap-seq data revealed strong positive correlation between replicate experiments and between paired tumor/lung samples. 14472 differentially methylated regions (DMR) with non-overlapping 500 bp windows were found. 57 DMRs were present in all NSCLC tumors. 287 were unique for squamous-cell carcinomas and 26 unique for adenocarcinomas. While hypomethylated DMRs did not correlate to any particular functional category of genes, the hypermethylated DMRs were strongly associated with genes encoding transcriptional regulators. Furthermore, subtelomeric regions and satellite repeats were hypomethylated in the NSCLC samples. Conclusion: We provide a resource containing genome-wide DNA methylation maps of NSCLC and their paired lung tissues, and comprehensive lists of known and novel DMRs and associated genes in NSCLC. The DMRs can be in further studies to develop sensitive biological markers for NSCLC, which may enable non-invasive diagnosis and early detection of the disease, and potentially allow histological classification. MethylCap-seq of 7 nsclc tumor samples and paired lung tissues, plus 2 fully methylated and 2 fully unmethylated controls.
2012-10-05 | E-GEOD-41343 | biostudies-arrayexpress
Project description:Brain metastasis (BM) can affect up to 25% of non-small cell lung cancer (NSCLC) patients during their lifetime. Efforts to characterize patients that will develop BM have been fairly disappointing. Small non-coding microRNAs (miRNAs) regulate the expression of target mRNAs by repressing their translation or regulating their sequence-specific degradation. miRNAs play a role in regulating a variety of targets and, consequently, multiple pathways, which makes them a powerful tool to be exploited for early detection of disease, risk assessment, and prognosis. In this study, we investigated miRNAs that may serve as biomarkers to differentiate between NSCLC patients with and without BM. miRNA microarray profiling was performed on samples from clinically matched NSCLC from patients with BM (BM+) and without BM (BM-). miR-328 and miR-330-3p were able to correctly classify BM+ vs. BM- patients. Gene expression analysis comparing NSCLC parental and stably transfected miR-328 cells identified several significantly differentially-expressed genes, whose expression may be directly or indirectly regulated by miR-328. NSCLC Cell Line A549 analysis: Two-condition experiment, A549 GFP Control vs. A549 miR-328 cells. Biological replicates: 2 control replicates, 2 miR-328 replicates. miRNA microarray analysis was performed on RNA extracted from formalin-fixed paraffin-embedded (FFPE) NSCLC tumor specimens from 7 BM+ patients and 5 BM- patients.
2011-02-16 | E-GEOD-23019 | biostudies-arrayexpress
Project description:We have recently found that the human methyltransferase ZCCHC4 is responsible for introducing the single m6A modification in 28S rRNA. The project is aimed at further elucidating the functional consequences of such methylation. To this end, protein mass spectrometry is utilized to identify proteins that bind preferentially to the methylated or unmethylated form of the m6A containing hairpin sequence in 28S rRNA. Specifically, this is done by incubating a HeLa cell extract with an methylated or unmethylated RNA oligonucleotide containing a biotin affinity tag, which is used to pull-down the RNA with associated proteins.
2019-11-22 | PXD013354 | Pride
Project description:In this study, we investigated the dynamics during differentiation of the in vivo binding sites of ZBTB2, a putative reader for unmethylated DNA. We performed DNA pull-downs followed by mass spectrometry, using a genomic sequence containing either unmethylated or methylated CpGs, to study the influence of DNA methylation on ZBTB2 binding. Additionally, we performed interaction proteomics to identify ZBTB2 interaction partners. We found that ZBTB2 recruits a zinc finger module of three proteins to unmethylated DNA.
2018-01-29 | PXD007099 | Pride
Project description:Lack of a standard method for stratifying advanced-stage NSCLC patients receiving platinum combination therapy often results in a number of patients that do not derive benefit yet are still exposed to treatment toxicity. We hypothesized that miRNAs in pre-treatment serum and/or plasma could be used to differentiate non-small cell lung cancer (NSCLC) patients who would have disease progression to first-line carboplatin and gemcitabine chemotherapy at first response assessment. miRNA profiling of mature and precursor miRNAs was performed on total RNA isolated from the pre-treatment serum and plasma of 24 NSCLC patients. Single validated candidates or combinations thereof were selected based on specificity and sensitivity to segregate patients with disease progression at first radiologic response (PD) vs. those without progressed disease (nonPD). Two precursor miRNA were significantly over-expressed in serum (but not plasma) of PD patients: pre-miR-518b and pre-miR-598. Serum miRNAs may serve as a screening tool in predicting chemoresistance to platinum-based combination chemotherapy. miRNA microarray was performed on RNA extracted from matched human serum or plasma obtained from NSCLC patients
2012-12-01 | E-GEOD-25019 | biostudies-arrayexpress