Integrated Analysis Of miRNAs Research Articles

The RNA expression signature of the HepG2 cell line as determined by the integrated analysis of miRNA and mRNA expression profiles.

Understanding miRNAs' regulatory networks and target genes could facilitate the development of therapies for human diseases such as cancer. Although much useful gene expression profiling data for tumor cell lines is available, microarray data for miRNAs and mRNAs in the human HepG2 cell line have only been compared with that of other cell lines separately. The relationship between miRNAs and mRNAs in integrated expression profiles for HepG2 cells is still unknown. To explore the miRNA-mRNA correlations in hepatocellular carcinoma (HCC) cells, we performed miRNA and mRNA expression profiling in HepG2 cells and normal liver HL-7702 cells at the genome scale using next-generation sequencing technology. We identified 193 miRNAs that are differentially expressed in these two cell lines. Of these, 89 miRNAs were down-regulated in HepG2 cells compared with HL-7702 cells, while 104 miRNAs were up-regulated. We also observed 3035 mRNAs that are significantly dys-regulated in HepG2 cells. We then performed an integrated analysis of the expression data for differentially expressed miRNAs and mRNAs and found several miRNA-mRNA pairs that are significantly correlated in HepG2 cells. Further analysis suggested that these differentially expressed genes were enriched in four tumorigenesis-related signaling pathways, namely, ErbB, JAK-STAT, mTOR, and WNT, which until now had not been fully reported. Our results could be helpful in understanding the mechanisms of HCC occurrence and development.

Integrated analysis of miRNA and mRNA during differentiation of human CD34+ cells delineates the regulatory roles of microRNA in hematopoiesis

In the process of human hematopoiesis, precise regulation of the expression of lineage-specific gene products is critical for multiple cell-fate decisions that govern cell differentiation, proliferation, and self-renewal. Given the important role of microRNAs (miRNAs) in development and differentiation, we examined the global expression of miRNA in CD34(+) cells during lineage specific hematopoiesis and found 49 miRNAs to be differentially expressed, with functional roles in cellular growth and proliferation, and apoptosis. miR-18a was upregulated during erythropoiesis and downregulated during megakaryopoiesis. miR-145 was upregulated during granulopoiesis and down regulated during erythropoiesis. Megakaryopoitic differentiation resulted in significant alteration in the expression of many miRNAs that are believed to play critical roles in the regulation of B and T cell differentiation. Target prediction analyses on three different miRNA databases indicated that TargetScan outperformed microCosm and miRDB in identifying potential miRNA targets associated with hematopoietic differentiation process. An integrated analysis of the observed miRNAs and messenger RNAs (mRNAs) resulted in 87 highly correlated miRNA-mRNA pairs that have major functional roles in cellular growth and proliferation, hematopoietic system development, and Wnt/B-catenin and Flt 3 signaling pathways. We believe that this study will enhance our understanding on the regulatory roles of miRNA in hematopoiesis by providing a library of mRNA-miRNA networks.

Genome-Wide Small RNA Sequencing and Gene Expression Analysis Reveals a microRNA Profile of Cancer Susceptibility in ATM-Deficient Human Mammary Epithelial Cells

Deficiencies in the ATM gene are the underlying cause for ataxia telangiectasia, a syndrome characterized by neurological, motor and immunological defects, and a predisposition to cancer. MicroRNAs (miRNAs) are useful tools for cancer profiling and prediction of therapeutic responses to clinical regimens. We investigated the consequences of ATM deficiency on miRNA expression and associated gene expression in normal human mammary epithelial cells (HME-CCs). We identified 81 significantly differentially expressed miRNAs in ATM-deficient HME-CCs using small RNA sequencing. Many of these have been implicated in tumorigenesis and proliferation and include down-regulated tumor suppressor miRNAs, such as hsa-miR-29c and hsa-miR-16, as well as over-expressed pro-oncogenic miRNAs, such as hsa-miR-93 and hsa-miR-221. MicroRNA changes were integrated with genome wide gene expression profiles to investigate possible miRNA targets. Predicted mRNA targets of the miRNAs significantly regulated after ATM depletion included many genes associated with cancer formation and progression, such as SOCS1 and the proto-oncogene MAF. While a number of miRNAs have been reported as altered in cancerous cells, there is little understanding as to how these small RNAs might be driving cancer formation or how they might be used as biomarkers for cancer susceptibility. This study provides preliminary data for defining miRNA profiles that may be used as prognostic or predictive biomarkers for breast cancer. Our integrated analysis of miRNA and mRNA expression allows us to gain a better understanding of the signaling involved in breast cancer predisposition and suggests a mechanism for the breast cancer-prone phenotype seen in ATM-deficient patients.

Abstract 3102: miRNA-mRNA regulatory networks by integrating miRNA and mRNA expression profiling of esophageal squamous cell carcinoma.

Abstract Background: miRNA expression profiling of esophageal squamous cell carcinoma(ESCC) has identified some miRNAs related to the clinical diversity of the disease and this potentially provides novel diagnostic and prognostic tools for ESCC therapy. In order to further understand the associations between miRNA expression and their target mRNA in ESCC, we performed integrating miRNA and mRNA expression profiling in ESCC. Material & Methods: Eight esophageal squamous carcinoma of Stage T2N1MO matched with their corresponding normal tissues was included in the study. Microdissection was performed to assure that more than 70% tumor components were selected for RNA extraction. Microarray technology (Agilent Human miRNA microarray and Agilent whole Human gene expression microarray) was used for miRNA expression and genome wide mRNA profiling. Integrated analysis of miRNAs and their target mRNAs by multi-step bioinformatics methods. Seven important differentiate expressed miRNAs (miR-21, miR-196b, miR-1, miR-29c, miR-125b, miR-195, let-7c) and 4 predicted target mRNAs(CDK-6, VEGFA, IGF1, and FLNC) were selected for future validation in an extended cohort of 50 ESCC by qRT-PCR. The relationship of the miRNA and genes were calculated by their differentiate expression values, and according to the interactions of MicroRNA and genes in Sanger MicroRNA database to create the MicroRNA-Gene-Network by the bioinformatic tools. Results:Compared to esophageal normal tissues, there were 67 differentiately expressed miRNAs and 2992 mRNAs in ESCC. The differentially expressed miRNAs and mRNAs were confirmed by real-time PCR. These differentiated miRNAs and their target genes were associated with many biological processes such as signal transduction, angiogenesis, cell proliferation, cell-cell signaling, response to drug. In the MicroRNA-Gene-Network, the significant pathways were Pathways in cancer, Cell cycle, TGF-beta signaling pathway, p53 signaling pathway, MAPK signaling pathway, mTOR signaling pathway and Jak-STAT signaling pathway. Conclusions: It is the first large-scale study to identify miRNA-mRNA regulatory networks in ESCC by combining expression profiles and bioinformatics analysis demonstrating that miRNA expression is highly associated with the proliferation, prognosis and metastasis of ESCC. Our findings provided a valuable source for future studying the differentially expressed miRNAs and their promising target genes, pathways and processes that may be useful for diagnostic or therapeutic applications in ESCC. Citation Format: Qiao Wei, Kuaile Zhao, Weiwei Yu, Guangqi Qin, Zhuanxu Zhang, Xiang Du, Menghong Sun. miRNA-mRNA regulatory networks by integrating miRNA and mRNA expression profiling of esophageal squamous cell carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3102. doi:10.1158/1538-7445.AM2013-3102

A systematic evaluation of miRNA:mRNA interactions involved in the migration and invasion of breast cancer cells

In this study we performed a systematic evaluation of functional miRNA-mRNA interactions associated with the invasiveness of breast cancer cells using a combination of integrated miRNA and mRNA expression profiling, bioinformatics prediction, and functional assays. Analysis of the miRNA expression identified 11 miRNAs that were differentially expressed, including 7 down-regulated (miR-200c, miR-205, miR-203, miR-141, miR-34a, miR-183, and miR-375) and 4 up-regulated miRNAs (miR-146a, miR-138, miR-125b1 and miR-100), in invasive cell lines when compared to normal and less invasive cell lines. Transfection of miR-200c, miR-205, and miR-375 mimics into MDA-MB-231 cells led to the inhibition of in vitro cell migration and invasion. The integrated analysis of miRNA and mRNA expression identified 35 known and novel target genes of miR-200c, miR-205, and mir-375, including CFL2, LAMC1, TIMP2, ZEB1, CDH11, PRKCA, PTPRJ, PTPRM, LDHB, and SEC23A. Surprisingly, the majority of these genes (27 genes) were target genes of miR-200c, suggesting that miR-200c plays a pivotal role in regulating the invasiveness of breast cancer cells. We characterized one of the target genes of miR-200c, CFL2, and demonstrated that CFL2 is overexpressed in aggressive breast cancer cell lines and can be significantly down-regulated by exogenous miR-200c. Tissue microarray analysis further revealed that CFL2 expression in primary breast cancer tissue correlated with tumor grade. The results obtained from this study may improve our understanding of the role of these candidate miRNAs and their target genes in relation to breast cancer invasiveness and ultimately lead to the identification of novel biomarkers associated with prognosis.

Integrated analysis of miRNA and mRNA reveals that acrolein modulates GPI anchor biosynthesis in human primary endothelial cells

Acrolein (ACR) is a highly reactive α, β-unsaturated carbonyl compound that is related to several diabetic vascular complications. ACR, a ubiquitous aldehyde pollutant, is a product of incomplete combustion reactions and a major component of cigarette smoke. ACR is also endogenously generated during lipid peroxidation. Glycosylphosphatidylinositol (GPI)-anchored proteins is a membrane bound protein, which have a variety of cellular functions. It is reported that some of them showed the reduced level under diabetic conditions. In this study, we explored whether ACR may affect GPI anchor related gene expression in human umbilical vein endothelial cells (HUVECs) by modulation of miRNA expression that play a significant role in the post-transcriptional regulation of genes. We performed global gene expression analysis, acquired altered expression levels of 27 miRNAs and 21 mRNAs that are relevant to GPI anchored proteins, and observed their anti-correlations. Gene Ontology (GO) terms that are closely associated with GPI anchor biosynthesis were highest when analysis was based on biological processes characterized by enriched GO categories. Our results revealed that ACR may control miRNAs to regulate genes involving in GPI anchor biosynthesis in human endothelial cells.

Modulation of the Osteosarcoma Expression Phenotype by MicroRNAs

BackgroundOsteosarcomas are the most common primary malignant tumors of bone and show multiple and complex genomic aberrations. miRNAs are non-coding RNAs capable of regulating gene expression at the post transcriptional level, and miRNAs and their target genes may represent novel therapeutic targets or biomarkers for osteosarcoma. In order to investigate the involvement of miRNAs in osteosarcoma development, global microarray analyses of a panel of 19 human osteosarcoma cell lines was performed.Principal findingsWe identified 177 miRNAs that were differentially expressed in osteosarcoma cell lines relative to normal bone. Among these, miR-126/miR-126*, miR-142-3p, miR-150, miR-223, miR-486-5p and members of the miR-1/miR-133a, miR-144/miR-451, miR-195/miR-497 and miR-206/miR-133b clusters were found to be downregulated in osteosarcoma cell lines. All miRNAs in the paralogous clusters miR-17-92, miR-106b-25 and miR-106a-92 were overexpressed. Furthermore, the upregulated miRNAs included miR-9/miR-9*, miR-21*, miR-31/miR-31*, miR-196a/miR-196b, miR-374a and members of the miR-29 and miR-130/301 families. The most interesting inversely correlated miRNA/mRNA pairs in osteosarcoma cell lines included miR-9/TGFBR2 and miR-29/p85α regulatory subunit of PI3K. PTEN mRNA correlated inversely with miR-92a and members of the miR-17 and miR-130/301 families. Expression profiles of selected miRNAs were confirmed in clinical samples. A set of miRNAs, miR-1, miR-18a, miR-18b, miR-19b, miR-31, miR-126, miR-142-3p, miR-133b, miR-144, miR-195, miR-223, miR-451 and miR-497 was identified with an intermediate expression level in osteosarcoma clinical samples compared to osteoblasts and bone, which may reflect the differentiation level of osteosarcoma relative to the undifferentiated osteoblast and fully differentiated normal bone. Significance: This study provides an integrated analysis of miRNA and mRNA in osteosarcoma, and gives new insight into the complex genetic mechanisms of osteosarcoma development and progression.

Abstract 5738: Expression profiling of microRNA and mRNA on hepatic inflammation in high-fat induced murine model

Abstract The inflammation has been identified as a risk factor for cancer and even as a means to prognose/diagnoses cancer at the inception of the inflammatory process. Therefore, it is necessary to discover novel molecules that might play a critical role in the regulation of inflammation, which can be the target of anti-inflammation. Recent reports indicate that microRNAs (miRNAs) have a significant role in the mechanism of inflammation in chronic inflammatory diseases. The goal of this study was to identify and characterize of miRNA in the regulation of inflammatory responses of high-fat induced inflammation mouse model. In this study, we established a mouse model of high-fat-diet-induced inflammation to gain understanding on the biological functions deregulated by aberrant gene expression based on miRNA and mRNA expression profiling analysis. And then we looked for direct miRNA targets by performing pair-wise correlation coefficient analysis on expression levels of mRNAs and by comparing these results with predicted miRNA targets from TargetScan5.1. We used quantitative PCR technology to evaluate the expression of miRNAs and mRNA on liver tissue. Inflammatory foci were observed over 6 months after high-fat induced inflammation mouse. In cDNA microarray analysis, total 2287 genes were differentially expressed between high-fat induced inflammatory group and normal group. Among those genes, the genes related-metabolic pathway were up-regulated at 3 months while those related inflammatory response were up-regulated at late stage of over 6 months in high-fat induced inflammation mouse model. Furthermore, we identified 15 new miRNAs in high-fat induced inflammation model by bioinformatics analysis and further confirmed their expression by stem-loop RT-PCR. GO analysis indicated that many of the identified miRNA targets may function in high-fat induced inflammation model. For three miRNAs (miR-34a, miR-21 and miR-1224), correlated genes were concordant with predicted targets. A gene set enrichment analysis on these genes demonstrated significant enrichment in biological processes related to lipid and glucose metabolism and inflammation. This study represents, to the best of our knowledge, the first integrated analysis of miRNA and mRNA expression in high-fat induced inflammation model. We identified a distinct profile of miRNAs of which expression differed between inflammation and normal, making these miRNAs candidate markers. Supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (No. 2011-0014620). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5738. doi:1538-7445.AM2012-5738

An integrated analysis of miRNA and gene copy numbers in xenografts of Ewing's sarcoma

BackgroundXenografts have been shown to provide a suitable source of tumor tissue for molecular analysis in the absence of primary tumor material. We utilized ES xenograft series for integrated microarray analyses to identify novel biomarkers.MethodMicroarray technology (array comparative genomic hybridization (aCGH) and micro RNA arrays) was used to screen and identify copy number changes and differentially expressed miRNAs of 34 and 14 passages, respectively. Incubated cells used for xenografting (Passage 0) were considered to represent the primary tumor. Four important differentially expressed miRNAs (miR-31, miR-31*, miR-145, miR-106) were selected for further validation by real time polymerase chain reaction (RT-PCR). Integrated analysis of aCGH and miRNA data was performed on 14 xenograft passages by bioinformatic methods.ResultsThe most frequent losses and gains of DNA copy number were detected at 9p21.3, 16q and at 8, 15, 17q21.32-qter, 1q21.1-qter, respectively. The presence of these alterations was consistent in all tumor passages. aCGH profiles of xenograft passages of each series resembled their corresponding primary tumors (passage 0). MiR-21, miR-31, miR-31*, miR-106b, miR-145, miR-150*, miR-371-5p, miR-557 and miR-598 showed recurrently altered expression. These miRNAS were predicted to regulate many ES-associated genes, such as genes of the IGF1 pathway, EWSR1, FLI1 and their fusion gene (EWS-FLI1). Twenty differentially expressed miRNAs were pinpointed in regions carrying altered copy numbers.ConclusionIn the present study, ES xenografts were successfully applied for integrated microarray analyses. Our findings showed expression changes of miRNAs that were predicted to regulate many ES associated genes, such as IGF1 pathway genes, FLI1, EWSR1, and the EWS-FLI1 fusion genes.

Integrated analysis of miRNA and mRNA profiles enables target acquisition in human cancers

Evaluation of: Buffa FM, Camps C, Winchester L et al. miRNA-associated progression pathways and potential therapeutic targets identified by integrated mRNA and miRNA expression profiling in breast cancer. Cancer Res. 71(17), 5635–5645 (2011).miRNAs play a role in post-transcriptional gene regulation by translational repression and/or mRNA degradation in a very tissue-specific manner. In order to understand the function of a miRNA, it is best to identify the genes that it regulates. Putative mRNA targets of miRNAs identified from seed sequence matches are available using computational algorithms in various web-based databases. However, these tend to have high false-positive rates and, owing to a whole-genome approach, cannot identify tissue/tumor specificity of regulation. The evaluated article presents a large amount of data analyzing global RNA expression in breast cancer and examines whether miRNAs are prognostic due to their effects on mRNA targets. This valuable and important resource of combined miRNA and mRNA expression in breast cancer and its subtypes has been summarized. Many studies have now investigated the integrated analysis of miRNA:mRNA profiles in human malignancies, the goal as always being to identify novel biomarkers and therapeutic targets for each tumor.

Inflammation-Mediated Regulation of MicroRNA Expression in Transplanted Pancreatic Islets

Nonspecific inflammation in the transplant microenvironment results in β-cell dysfunction and death influencing negatively graft outcome. MicroRNA (miRNA) expression and gene target regulation in transplanted islets are not yet well characterized. We evaluated the impact of inflammation on miRNA expression in transplanted rat islets. Islets exposed in vitro to proinflammatory cytokines and explanted syngeneic islet grafts were evaluated by miRNA arrays. A subset of 26 islet miRNAs was affected by inflammation both in vivo and in vitro. Induction of miRNAs was dependent on NF-κB, a pathway linked with cytokine-mediated islet cell death. RT-PCR confirmed expression of 8 miRNAs. The association between these miRNAs and mRNA target-predicting algorithms in genome-wide RNA studies of β-cell inflammation identified 238 potential miRNA gene targets. Several genes were ontologically associated with regulation of insulin signaling and secretion, diabetes, and islet physiology. One of the most activated miRNAs was miR-21. Overexpression of miR-21 in insulin-secreting MIN6 cells downregulated endogenous expression of the tumor suppressor Pdcd4 and of Pclo, a Ca2+ sensor protein involved in insulin secretion. Bioinformatics identified both as potential targets. The integrated analysis of miRNA and mRNA expression profiles revealed potential targets that may identify molecular targets for therapeutic interventions.

Integrated Analysis of miRNA and mRNA Expression in Childhood Medulloblastoma Compared with Neural Stem Cells

Medulloblastoma (MB) is the most common malignant brain tumor in children and a leading cause of cancer-related mortality and morbidity. Several molecular sub-types of MB have been identified, suggesting they may arise from distinct cells of origin. Data from animal models indicate that some MB sub-types arise from multipotent cerebellar neural stem cells (NSCs). Hence, microRNA (miRNA) expression profiles of primary MB samples were compared to CD133+ NSCs, aiming to identify deregulated miRNAs involved in MB pathogenesis. Expression profiling of 662 miRNAs in primary MB specimens, MB cell lines, and human CD133+ NSCs and CD133− neural progenitor cells was performed by qRT-PCR. Clustering analysis identified two distinct sub-types of MB primary specimens, reminiscent of sub-types obtained from their mRNA profiles. 21 significantly up-regulated and 12 significantly down-regulated miRNAs were identified in MB primary specimens relative to CD133+ NSCs (p<0.01). The majority of up-regulated miRNAs mapped to chromosomal regions 14q32 and 17q. Integration of the predicted targets of deregulated miRNAs with mRNA expression data from the same specimens revealed enrichment of pathways regulating neuronal migration, nervous system development and cell proliferation. Transient over-expression of a down-regulated miRNA, miR-935, resulted in significant down-regulation of three of the seven predicted miR-935 target genes at the mRNA level in a MB cell line, confirming the validity of this approach. This study represents the first integrated analysis of MB miRNA and mRNA expression profiles and is the first to compare MB miRNA expression profiles to those of CD133+ NSCs. We identified several differentially expressed miRNAs that potentially target networks of genes and signaling pathways that may be involved in the transformation of normal NSCs to brain tumor stem cells. Based on this integrative approach, our data provide an important platform for future investigations aimed at characterizing the role of specific miRNAs in MB pathogenesis.

Abstract 4937: Integrated miRNA and mRNA expression profiling of breast cancer cell lines identifies the signatures of miRNA-mRNA pairs in aggressive breast cancer cells

Abstract MicroRNAs (miRNAs) are a class of small, non-coding RNAs that regulate gene expression by translational repression or mRNA degradation. Although the functions of specific miRNAs in breast cancer development and progression have been extensively investigated, few studies have focused on the systematic identification of miRNAs and their target genes related to breast cancer aggressiveness. In this study, we sought to discover the signatures of miRNA-mRNA target pairs in aggressive breast cancer cell lines through a combination of integrated miRNA and mRNA expression profiling, bioinformatics prediction and functional assays. MiRNA and mRNA expression profiles of 12 breast cancer cell lines including 8 less aggressive lines (MCF10A, MCF12A, BT474, MCF7, MDA-MB-468, SK-BR3, T-47D and ZR-75-1) and 4 aggressive lines (BT549, HS578T, MDA-MB-231 and SUM159) were generated using Affymertrix miRNA Array and U133 Plus 2.0 Array, respectively. The miRNA expression array analysis has shown that 44 out of 847 miRNAs were differentially expressed (p&amp;lt;0.01) between less aggressive and aggressive cell lines. Among the 44 differentially expressed miRNAs, 30 miRNAs (i.e. mir-141, mir-200c, mir-203, mir-205 and mir-34a) were down-regulated in aggressive lines as compared to less aggressive lines, while 14 miRNAs (i.e. mir-100, mir-125b, mir-138 and mir-146a) were up-regulated. The integrated analysis of miRNA and mRNA expression was performed in Partek Genomic Suites and the predicted miRNA-mRNA target pairs by TargetScan5.1 that demonstrated negative correlations were identified. Twelve candidate targets for mir-146a, mir-200c, mir-203, mir-205 and mir-375, including IRAK1, TRAF6, FOXF1, SHOX2, CDH11, ZEB1, FYN, ETS1, CSF1, ACSL4, SNAIL2 and BMI1, were selected for functional analysis. Quantitative RT-PCR analysis confirmed the differential expression patterns of the 5 miRNAs and their 12 target genes in the above-mentioned 12 cell lines. We have further established stable clones overexpressing individual mir-200c, mir-203, mir-205 and mir-375 in MDA-MB-231 cell line. The change in the aggressiveness of miRNA-overexpressing cells was evaluated by migration, invasion and soft agar colony formation assays. qRT-PCR and western blot validation for the potential miRNA-mRNA target pairs and 3′UTR reporter assays in miRNA-overexpressing clones are currently underway. In summary, we have identified a group of negatively correlated miRNA-mRNA target pairs that are differentially expressed in aggressive breast cancer cells. The results obtained from this study may advance our understanding of the role of these candidate miRNAs and their target genes in breast cancer aggressiveness and lead to identification of novel biomarkers associated with prognosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4937. doi:10.1158/1538-7445.AM2011-4937

Abstract LB-348: Integrated analysis of microRNA and mRNA expression in microsatellite-stable colon cancer using next-generation sequencing and cDNA microarrays

Abstract Alterations in the expression of microRNAs (miRNAs) contribute to the development and progression of colon cancer, which arises from the accumulation of genetic and epigenetic alterations in the colon tissue. With the advent of next-generation sequencing (NGS), new depths can be reached, allowing for comprehensive profiling of known and novel miRNAs. To profile the miRNAs and their associated target genes in colon cancer, we examined differential expression of miRNAs and predicted target genes between tumor and normal tissue of microsatellite stable (MSS) and CpG island methylator phenotype (CIMP)-negative colon cancer. Ten fresh-frozen colon tumors and 10 adjacent normal mucosa were profiled for miRNAs using the SOLiD NGS platform and mRNA expression analysis was conducted using Affymetrix arrays. To identify differentially expressed miRNAs in tumor versus normal tissue, analysis of variance was conducted that included Bonferroni correction. Integrated expression analysis of miRNAs and their target mRNAs was conducted using Pearson's correlation and the Benjamini and Hochberg's false discovery rate. Both analyses were done using Partek Genomics Suite software. Nineteen miRNAs were significantly differentially expressed in tumor versus normal tissue (fold change ≥ ±2 and p&amp;lt;0.05). Six of these 19 miRNAs have previously been associated with colon cancer (miR-30a, miR-31, miR-135b, miR-182, miR-183 and miR-202). Integrated analysis of miRNA and predicted target mRNA expression yielded 97 significantly correlated miRNA::mRNA pairs for 14 miRNAs with 92 genes (absolute r = 0.71–0.88, q&amp;lt;0.05). Within these miRNA::mRNA pairs, expression levels of the 6 colon cancer-associated miRNAs were correlated with 33 target genes and 8 miRNAs newly identified to be differentially expressed in colon cancer were correlated with 57 target genes. The top functional Ingenuity Pathway Analysis gene ontology categories (p&amp;lt;0.05) for the 92 genes were gastrointestinal disease (18 genes), genetic disorder (21genes), inflammatory disease (15 genes) and cancer (21 genes). The correlated target genes included several genes implicated in colon cancer such as CDH3, NKD1, RGS2, TCFL5 and ZEB2. In summary, deep miRNA sequencing among MSS/CIMP-negative colon tumors confirmed differential expression of 6 colon cancer-associated miRNAs and identified 13 miRNAs not previously reported in colon cancer. Differential expression of most of the discovered miRNAs was correlated with the expression of genes potentially relevant for colon cancer. If confirmed, these miRNAs may serve as new biomarkers for colon cancer and may further our understanding of the epigenetic changes and pathways involved in the development of colon cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-348. doi:10.1158/1538-7445.AM2011-LB-348

Abstract 1149: Clinically relevant interactions between micro-RNAs and genes in malignant mesothelioma characterized by an integrated analysis

Abstract Objective: We hypothesize that certain microRNA (miRNA)-mRNA interactions are associated with malignant transformation and progression of MM. To date, no integrated analysis has been performed to identify mechanistic interactions between miRNA-mRNA that may drive the malignant phenotype. Method: Total RNA was extracted from 24 specimens of biopsy proven human MM and 6 specimens of normal pleura. Paired global transcriptional profiles of miRNA and genes (mRNA) expression were generated using Illumina microarrays. To optimize detection of causal associations, Spearman correlations between miRNA and mRNA expression were calculated among MM samples. Correlation p-values were converted to false discovery rates (FDR q-values) to account for multiple comparisons; associations with q&amp;lt;=0.05 were considered significant. To better determine the genes of relevance for a given process, sets of genes whose expression levels negatively correlated with specific miRNAs were identified as likely direct targets of that miRNA. Gene Set Enrichment Analysis (GSEA) was used to find target gene sets that were significantly repressed or overexpressed in MM vs normal in the opposite direction to expression of the miRNA itself. Independent data sets were interrogated with GSEA to refine target gene sets whose behavior was consistent across the data and identify which were associated with patient survival. Associations of mRNA expression levels and disease-specific survival were evaluated by univariate Cox proportional hazard regression. Result: We found multiple miRNA (total = 16) showing negative correlation in expression to multiple genes, and having the opposite expression pattern in MM vs normal compared to their putative targets. We arrived at this total by assessing the association between gene expression and outcome (disease-specific survival), and retaining only miRNA whose targets were significant (q&amp;lt;0.05). An example: expression of miR-192 (highly expressed in lung cancer) negatively correlated with expression of 156 mRNA (FDR&amp;lt;0.05). This miRNA was expressed 4-fold more in MM (FDR&amp;lt;0.01), while its gene targets showed an opposite pattern, being repressed in MM (FDR&amp;lt;0.001 in independent data). Functional annotation of target genes showed that they were enriched for targets of the Polycomb complex gene Ezh2, and for genes that are downregulated by KRAS in lung cancer. High expression of the genes negatively-correlated with miR-192 associated with better overall survival in an independent dataset, indicating that high expression of this miRNA affects poorer patient outcomes. Conclusion: Integrated analysis of miRNA and mRNA expression profiles in MM revealed novel miRNA associated with MM and identified putative interactions that may underlie the malignant phenotype. These miRNA-mRNA are candidates for functional validation. miR-192 is a putative oncogenic miRNA in MM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1149. doi:10.1158/1538-7445.AM2011-1149

Identification of Genes Regulated by IL-1β Using Integrative microRNA and mRNA Genomic Analysis in Human Articular Chondrocytes

Objective. The physiological and pathogenetic role of microRNAs (miRNAs) in the maintenance of joint homeostasis and in the development of arthritis is recently being elucidated. In this study, we attempted to identify differentially expressed miRNAs in human osteoarthritis (OA) chondrocytes in response to interleukin (IL)-1β. In addition, simultaneous profiling of miRNA and mRNA expression was performed to get an integrated analysis of miRNA and mRNA expression. Methods. Monolayer cultured chondrocytes obtained from knee cartilages of OA patients were stimulated with IL-1β for 4 hours and RNA was isolated. One microgram of total RNA was polyadenylated and converted to cDNA and miRNA microarray was performed. Seven hundred thirty five oligos were used, corresponding to 470 well-annotated human miRNA sequences and 265 potential miRNAs that were identified recently. mRNA microarray was performed simultaneously using the RNA samples that were used for miRNA array. Both sequence and expression information was used to identify regulatory relationship between miRNA and mRNA pairs. Results. Expression profiling of miRNA extracted from IL-1β treated chondrocytes identified 25 miRNA which showed differential expression. We also identified 7190 mRNAs differentially regulated by IL-1β treatment. Among the 25 miRNAs differentially regulated, 13 miRNAs had targets searched by MiRANDA scheme. By combining target search and miRNA-mRNA pairing, we could identify 1043 miRNA-mRNA target pairs. MiR-200a was found to be expressed in human OA and normal cartilages, with downregulation in OA lesion cartilages. Conclusion. It is suggested that miRNA may play a role in the regulation of cartilage degradation in OA.

Integrated miRNA and mRNA expression profiling of the inflammatory breast cancer subtype

Background:MicroRNAs (miRNAs) are key regulators of gene expression. In this study, we explored whether altered miRNA expression has a prominent role in defining the inflammatory breast cancer (IBC) phenotype.Methods:We used quantitative PCR technology to evaluate the expression of 384 miRNAs in 20 IBC and 50 non-IBC samples. To gain understanding on the biological functions deregulated by aberrant miRNA expression, we looked for direct miRNA targets by performing pair-wise correlation coefficient analysis on expression levels of 10 962 messenger RNAs (mRNAs) and by comparing these results with predicted miRNA targets from TargetScan5.1.Results:We identified 13 miRNAs for which expression levels were able to correctly predict the nature of the sample analysed (IBC vs non-IBC). For these miRNAs, we detected a total of 17 295 correlated miRNA–mRNA pairs, of which 7012 and 10 283 pairs showed negative and positive correlations, respectively. For four miRNAs (miR-29a, miR-30b, miR-342-3p and miR-520a-5p), correlated genes were concordant with predicted targets. A gene set enrichment analysis on these genes demonstrated significant enrichment in biological processes related to cell proliferation and signal transduction.Conclusions:This study represents, to the best of our knowledge, the first integrated analysis of miRNA and mRNA expression in IBC. We identified a set of 13 miRNAs of which expression differed between IBC and non-IBC, making these miRNAs candidate markers for the IBC subtype.