miRNA Profiling Studies Research Articles

Abstract 1847: Molecular profiles of microRNA in endometrial carcinoma using paraffin-embedded (FFPE) specimens.

Abstract MicroRNAs (miRNAs) are small non-coding RNAs that regulate mRNA stability and protein expression at post-transcriptional level. Certain miRNAs have been demonstrated to act either as oncogenes or tumor suppressors. The specific expression profiles of miRNAs have been found in many human cancers, but the role of miRNAs in endometrioid endometrial cancer (EEC) remains poorly understood. Most miRNA profiling studies have used fresh tissue samples. This study was to provide the candidate miRNAs for further confirming the role of miRNAs in carcinogenesis of EEC and to explore whether FFPE material would be suitable for miRNA profiling. We found the differences in miRNA expression profile using human miRNA microarray in EECs and normal endometrium. Of those, the miR-200a*, miR-200b*, miR-141, miR-182, and miR-205 were greatly enriched. The expressions of these 5 miRNAs were validated using quantitative real time reverse transcription-PCR (qRT-PCR). Then, we performed qRT-PCR profiling of miR expression in 30 microdissected formalin-fixed paraffin-embedded (FFPE) specimens (20 EECs, 10 non-tumor specimens) and reconfirmed the results of differential expression between cancer and normal tissue. We next tested whether specific inhibition of overexpressed microRNAs would alter the chemosensitivity. In in vitro cell viability assay, anti-miR200b* slightly enhanced cisplatin cytotoxicity compared with negative control although it showed marginal statistical significance (p=0.07). This information provided the candidate miRNAs for further confirming the role of miRNAs in carcinogenesis of EEC and potentially serving as a diagnostic or therapeutic tool. FFPE specimens of endometrial tissues are suitable as a source for miRNA microarray profiling. Citation Format: Taek Sang Lee, Hye-Won Jeon, Yong-Beom Kim. Molecular profiles of microRNA in endometrial carcinoma using paraffin-embedded (FFPE) specimens. [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 1847. doi:10.1158/1538-7445.AM2013-1847

Abstract 859: miR-638, a novel tumor suppressor for ER-negative breast cancer.

Abstract MicroRNAs (miRNAs) are endogenous non-coding RNAs of 19∼25 nt in length, which regulate many protein-coding genes. miRNA dysregulation is observed in a variety of cancers, including breast cancer. miRNAs have emerged as an important set of biomarkers. miRNA profiling studies have led to the identification of miRNAs that are aberrantly expressed in human breast cancer, with miR-10b, miR-125b and miR-145 being down-regulated and miR-21, and 155 being up-regulated. In our previous miRNA expression profiling studies using microdissected FFPE tissues, we identified 8 clusters of miRNAs, which discrete the normal and breast lesions such as ADH, DCIS and IDC. miR-638 is one of the differentially expressed miRNAs during breast cancer progression. We first sought to determine the expression of miR-638 in different breast cancer cell lines such as MCF-7, Hs578T, T47D and MDA-MB-231 using qRT-PCR, which shows a very low expression compared to normal tissues. This led us to hypothesize that miR-638 may function as a tumor suppressor during breast cancer development. To determine the role of miR-638 in both ER+ and ER- breast cancer cells, we transfected miR-638 mimic oligos and scrambled oligo mocks into MCF-7 (ER+), T47D (ER+), MDA-MB-231 (ER-) and Hs578T (ER-) cells. After 48 hours, we found that the proliferation rate was significantly down-regulated in ER- cells but not in ER+ cells by MTT assay. Our preliminary matrigel analysis shows that miR-638 overexpressing MDA-MB-231 cells had 3∼4 fold invasiveness decrease compared to the control. Further, our TargetScan analysis revealed that BRCA1 is one of the direct targets of miR-638 among the 30 conservative target genes. We found that overexpression of miR-638 resulted in an upregulation of BRCA1 mRNA expressions in ER- cell lines, MDA-MB-231 and Hs578T but not in ER+ cell lines, T47D and MCF-7. Taken together, these findings showed that miR-638 may function as a tumor suppressor in ER- breast cancer, possibly in part by upregulating BRCA1 tumor suppressor gene. Further functional analysis is underway to decipher the exact role of miR-638 in ER- breast cancer, which could become a therapeutic target. Citation Format: Jin Peng, Yebo Fu, Rachel F. Brem, Christine B. Teal, Sidney W. Fu. miR-638, a novel tumor suppressor for ER-negative breast cancer. [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 859. doi:10.1158/1538-7445.AM2013-859

Challenges for MicroRNA Microarray Data Analysis

Microarray is a high throughput discovery tool that has been broadly used for genomic research. Probe-target hybridization is the central concept of this technology to determine the relative abundance of nucleic acid sequences through fluorescence-based detection. In microarray experiments, variations of expression measurements can be attributed to many different sources that influence the stability and reproducibility of microarray platforms. Normalization is an essential step to reduce non-biological errors and to convert raw image data from multiple arrays (channels) to quality data for further analysis. In general, for the traditional microarray analysis, most established normalization methods are based on two assumptions: (1) the total number of target genes is large enough (>10,000); and (2) the expression level of the majority of genes is kept constant. However, microRNA (miRNA) arrays are usually spotted in low density, due to the fact that the total number of miRNAs is less than 2,000 and the majority of miRNAs are weakly or not expressed. As a result, normalization methods based on the above two assumptions are not applicable to miRNA profiling studies. In this review, we discuss a few representative microarray platforms on the market for miRNA profiling and compare the traditional methods with a few novel strategies specific for miRNA microarrays.

MiR-145 overexpression suppresses the migration and invasion of metastatic melanoma cells

MicroRNAs (miRNAs) are post-transcriptional modulators of gene expression which play important roles in tumorigenesis and cancer metastasis. Since they are often highly deregulated in various types of cancer, miRNAs may be effective treatment targets. miRNA profiling studies of melanoma have led to the identification of several tumor suppressor miRNAs. One of these include miR-145, although functional data proving its specific function are limited. Therefore, in this study, we examined the expression levels of miR-145 in three melanoma cell lines (BLM, FM3P and WM793). Additional gain-of-function experiments revealed that miR-145 exerts an anti-proliferative effect in the primary, non-invasive melanoma cell line, WM793, whereas cell migration and the invasive potential of metastatic melanoma cells was suppressed following transfection with miR-145 mimics. In order to investigate the mechanisms by which miR-145 exerts its invasion suppressor function, we examined the expression level of target genes [fascin homolog 1 (FSCN1), myosin‑Va (MYO5A and SOX9] and that of an indirect target (RAB27A) following the overexpression of miR-145. The results showed that SOX9, MYO5A and RAB27A were not involved in the biological effects caused by miR-145 mimics. Surprisingly, we discovered that miR-145 in melanoma, in contrast to many other tumor types, does not necessarily act via the target, FSCN1, since the downregulation of FSCN1 did not inhibit cell proliferation or migration but, on the contrary, increased cell invasion in two out of the three melanoma cell lines examined. Our in vitro data is in accordance with previously reported in vivo data describing the low expression of FSCN1 in malignant melanomas when compared to dysplastic nevi, suggesting that the expression of FSCN1 decreases as the formation and progression stage of melanoma advances. In conclusion, our data provide evidence that miR-145 is an invasion suppressor in metastatic melanoma cells. Despite the fact that it remains unclear which genes or pathways are regulated by miR-145 in melanoma, miR-145 may serve as a useful therapeutic agent in melanoma when re-expressed in situ.

MicroRNA-193b enhances tumor progression via down regulation of neurofibromin 1.

Despite improvements in therapeutic approaches for head and neck squamous cell carcinomas (HNSCC), clinical outcome has remained disappointing, with 5-year overall survival rates hovering around 40–50%, underscoring an urgent need to better understand the biological bases of this disease. We chose to address this challenge by studying the role of micro-RNAs (miRNAs) in HNSCC. MiR-193b was identified as an over-expressed miRNA from global miRNA profiling studies previously conducted in our lab, and confirmed in HNSCC cell lines. In vitro knockdown of miR-193b in FaDu cancer cells substantially reduced cell proliferation, migration and invasion, along with suppressed tumour formation in vivo. By integrating in silico prediction algorithms with in vitro experimental mRNA profilings, plus mRNA expression data of clinical specimens, neurofibromin 1 (NF1) was identified to be a target of miR-193b. Concordantly, miR-193b knockdown decreased NF1 transcript and protein levels significantly. Luciferase reporter assays confirmed the direct interaction of miR-193b with NF1. Moreover, p-ERK, a downstream target of NF1 was also suppressed after miR-193b knockdown. FaDu cells treated with a p-ERK inhibitor (U0126) phenocopied the reduced cell proliferation, migration and invasion observed with miR-193b knockdown. Finally, HNSCC patients whose tumours expressed high levels of miR-193b experienced a lower disease-free survival compared to patients with low miR-193b expression. Our findings identified miR-193b as a potentially novel prognostic marker in HNSCC that drives tumour progression via down-regulating NF1, in turn leading to activation of ERK, resulting in proliferation, migration, invasion, and tumour formation.

A Decade of Global mRNA and miRNA Profiling of HPV-Positive Cell Lines and Clinical Specimens

For more than a decade, global gene expression profiling has been extensively used to elucidate the biology of human papillomaviruses (HPV) and their role in cervical- and head-and-neck cancers. Since 2008, the expression profiling of miRNAs has been reported in multiple HPV studies. Two major strategies have been employed in the gene and miRNA profiling studies: In the first approach, HPV positive tumors were compared to normal tissues or to HPV negative tumors. The second strategy relied on analysis of cell cultures transfected with single HPV oncogenes or with HPV genomes compared to untransfected cells considered as models for the development of premalignant and malignant transformations.In this review, we summarize what we have learned from a decade of global expression profiling studies. We performed comprehensive analysis of the overlap of the lists of differentially expressed genes and microRNAs, in both tissue samples and cell culture based studies. The review focuses mainly on HPV16, however reports from other HPV species are used as references. We discuss the low degree of consensus among different studies and the limitation of differential expression analysis as well as the fragmented miRNA-mRNA target correlation evidence. Furthermore, we propose an approach for future research to include more comprehensive miRNA-mRNA target correlation analysis and to apply systems biology/gene networks methodology.

Comparative microarray analysis of microRNA expression profiles in primary cutaneous malignant melanoma, cutaneous malignant melanoma metastases, and benign melanocytic nevi

Perturbations in microRNA (miRNA) expression profiles have been reported for cutaneous malignant melanoma (CMM) predominantly when examined in cell lines. Despite the rapidly growing number of newly discovered human miRNA sequences, the availability of up-to-date miRNA expression profiles for clinical samples of primary cutaneous malignant melanoma (PCMM), cutaneous malignant melanoma metastases (CMMM), and benign melanocytic nevi (BMN) is limited. Specimens excised from the center of tumors (lesional) from patients with PCMM (n=9), CMMM (n=4), or BMN (n=8) were obtained during surgery. An exploratory microarray analysis was performed by miRNA expression profiling based on Agilent platform screening for 1205 human miRNAs. The results from the microarray analysis were validated by TaqMan quantitative real-time polymerase chain reaction. In addition to several miRNAs previously known to be associated with CMM, 19 unidentified miRNA candidates were found to be dysregulated in CMM patient samples. Among the 19 novel miRNA candidates, the genes hsa-miR-22, hsa-miR-130b, hsa-miR-146b-5p, hsa-miR-223, hsa-miR-301a, hsa-miR-484, hsa-miR-663, hsa-miR-720, hsa-miR-1260, hsa-miR-1274a, hsa-miR-1274b, hsa-miR-3663-3p, hsa-miR-4281, and hsa-miR-4286 were upregulated, and the genes hsa-miR-24-1*, hsa-miR-26a, hsa-miR-4291, hsa-miR-4317, and hsa-miR-4324 were downregulated. The results of this study partially confirm previous CMM miRNA profiling studies identifying miRNAs that are dysregulated in CMM. However, we report several novel miRNA candidates in CMM tumors; these miRNA sequences require further validation and functional analysis to evaluate whether they play a role in the pathogenesis of CMM.

MicroRNA profiling: approaches and considerations

MicroRNAs (miRNAs) are small RNAs that post-transcriptionally regulate the expression of thousands of genes in a broad range of organisms in both normal physiological contexts and in disease contexts. miRNA expression profiling is gaining popularity because miRNAs, as key regulators in gene expression networks, can influence many biological processes and also show promise as biomarkers for disease. Technological advances have spawned a multitude of platforms for miRNA profiling, and an understanding of the strengths and pitfalls of different approaches can aid in their effective use. Here, we review the major considerations for carrying out and interpreting results of miRNA-profiling studies.

Abstract 5037: Identifying differentially expressed microRNAs in retinoblastoma

Abstract Retinoblastoma, the most common pediatric cancer of the eye, is a devastating and sometimes fatal pediatric cancer. Within the United States, the majority of retinoblastoma patients are diagnosed before their 2nd birthday, and many lose their sight due to this disease. Approximately 75% of the retinoblastoma patients in the United States have unilateral disease, and treatment for many unilateral patients with advanced disease involves enucleation (removal of the eye). Therefore, new more effective targeted therapies for retinoblastoma could save a child's quality of life by preserving their sight. Outside of the United States, advanced retinoblastoma is an even greater clinical challenge in low-income countries, where the mortality rate among children diagnosed with advanced retinoblastoma is as high as 70%. Currently, the chemotherapeutic treatments for retinoblastoma involve the use of broad-based drugs. However, therapies targeted directly to aberrant signaling pathways may provide more effective therapy for this disease. MicroRNAs, short noncoding RNA transcripts that can regulate the expression of target genes, are known to affect the progression of many different tumor types. MicroRNAs (miRNAs) are also ideal therapeutic targets because they are amenable to therapeutic manipulation. To identify miRNAs associated with retinoblastoma progression, we have conducted miRNA profiling studies on 12 late-stage human retinoblastoma tumors. Our analysis has led to the identification of 19 differentially expressed miRNAs (p<0.05) in retinoblastoma as compared to normal human retina. From those 19 miRNAs, 15 miRNAs are newly associated as being differentially expressed in retinoblastoma. These miRNAs could serve as potential therapeutic targets for retinoblastoma due to their known involvement in viability, proliferation, cell cycle regulation, and apoptosis in other tumor types. This is an essential step in the discovery of miRNAs associated with retinoblastoma progression, and in the identification of potential therapeutic targets for this disease. 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 5037. doi:1538-7445.AM2012-5037

MicroRNA‐16 regulates ENaC expression in alveolar epithelial cells

ObjectiveMicroRNAs (miRNAs) have emerged as a novel class of gene regulators which play a critical role in complex diseases like acute lung injury (ALI). Our miRNA profiling studies in animal models of ALI revealed that miR‐146a, mir‐16 and miR‐155 were dysregulated. Mir16 targets Serotonin transporter (SERT) which is involved in serotonin uptake. Elevated levels of serotonin in ALI are known to regulate epithelial sodium channel (ENac) expression. The aim of this study is to investigate the functional effects of mir16 on SERT and ENaC in human alveolar epithelial cells (A549) in vitro.MethodsA549 cells were commercially obtained and cultured according to the manufacturer instructions. Cells were transfected with plasmid containing miR16 expressing or control vector by Lipofectamine. q PCR and immunoblot analysis were performed to assess the effects of miR‐16 on SERT, ENaC and other downstream signalling molecules.ResultsA549 cells transfected with miR16 decreased the expression levels of SERT when compared to controls. Dose dependant regulation of SERT was observed in untransfected A549 cells treated with serotonin. Increased expression of ENaC β was observed in miR16 transfected cells compared to controls.ConclusionmiR16 over expression altered SERT and ENaC expression in human alveolar epithelial cells.Research supported by:RO1HL105932(NIH) and 09SDG2260957(AHA)

Stability analysis of liver cancer-related microRNAs

MicroRNAs (miRNAs) are non-coding, single-stranded RNAs of approximately 22 nt and constitute a novel class of gene regulators that are found in both plants and animals. Several studies have demonstrated that serum miRNAs could serve as potential biomarkers for the detection of various cancers and other diseases. A few documents regarding the stability of liver cancer-related miRNAs in serum are available. A systemic analysis of the stability of miRNA in serum is quite necessary. The purpose of this study was to evaluate the stability of miRNAs from three different sources, cultured liver cancer Huh-7 cell line, clinical liver cancer, and serum under different experimental conditions, including different temperature, time duration, pH values, RNase A digestion, DNase I digestion, and various freeze-thaw cycles. The qRT-PCR analysis demonstrated that liver cancer-related miRNAs were detectable under each of test conditions, indicating that miRNAs were extremely stable and resistant to destruction and degradation under harsh environmental conditions. However, ribosomal RNA was fragile and easily degraded by demonstrating sharp decrease of relative expression under the non-physiological test conditions. We also established a robust procedure for serum RNA extraction, which is greatly important not only for the miRNA profiling studies but also for the disease prognosis based on abnormal miRNA expression.

MicroRNA expression in maturing murine megakaryocytes

AbstractMicroRNAs are small noncoding RNAs that regulate cellular development by interfering with mRNA stability and translation. We examined global microRNA expression during the differentiation of murine hematopoietic progenitors into megakaryocytes. Of 435 miRNAs analyzed, 13 were up-regulated and 81 were down-regulated. Many of these changes are consistent with miRNA profiling studies of human megakaryocytes and platelets, although new patterns also emerged. Among 7 conserved miRNAs that were up-regulated most strongly in murine megakaryocytes, 6 were also induced in the related erythroid lineage. MiR-146a was strongly up-regulated during mouse and human megakaryopoiesis but not erythropoiesis. However, overexpression of miR-146a in mouse bone marrow hematopoietic progenitor populations produced no detectable alterations in megakaryocyte development or platelet production in vivo or in colony assays. Our findings extend the repertoire of differentially regulated miRNAs during murine megakaryopoiesis and provide a useful new dataset for hematopoiesis research. In addition, we show that enforced hematopoietic expression of miR-146a has minimal effects on megakaryopoiesis. These results are compatible with prior studies indicating that miR-146a inhibits megakaryocyte production indirectly by suppressing inflammatory cytokine production from innate immune cells, but cast doubt on a different study, which suggests that this miRNA inhibits megakaryopoiesis cell-autonomously.

DNA Methylation and miRNA Expression Profiling in Childhood B-Cell Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemia (ALL) is the most common cancer in children in the modern world. Recent efforts in characterizing the genetic contribution to this disease through uncovering gene mutations, deletions and structural variation by genome-scale methods have only accounted for a modest proportion of children with ALL. This suggests that either further genetic contributions to ALL have yet to be characterized or other factors, such as epigenetic aberrations are involved. A number of DNA methylation and miRNA profiling studies have investigated the role of both in childhood ALL. Here, we review these profiling efforts, summarize their major findings and speculate as to what the future may hold.

Normalization strategies for microRNA profiling experiments: a ‘normal’ way to a hidden layer of complexity?

MicroRNA (miRNA) profiling is a first important step in elucidating miRNA functions. Real time quantitative PCR (RT-qPCR) and microarray hybridization approaches as well as ultra high throughput sequencing of miRNAs (small RNA-seq) are popular and widely used profiling methods. All of these profiling approaches face significant introduction of bias. Normalization, often an underestimated aspect of data processing, can minimize systematic technical or experimental variation and thus has significant impact on the detection of differentially expressed miRNAs. At present, there is no consensus normalization method for any of the three miRNA profiling approach. Several normalization techniques are currently in use, of which some are similar to mRNA profiling normalization methods, while others are specifically modified or developed for miRNA data. The characteristic nature of miRNA molecules, their composition and the resulting data distribution of profiling experiments challenges the selection of adequate normalization techniques. Based on miRNA profiling studies and comparative studies on normalization methods and their performances, this review provides a critical overview of commonly used and newly developed normalization methods for miRNA RT-qPCR, miRNA hybridization microarray, and small RNA-seq datasets. Emphasis is laid on the complexity, the importance and the potential for further optimization of normalization techniques for miRNA profiling datasets.

MicroRNA-221 Regulates Chondrogenic Differentiation through Promoting Proteosomal Degradation of Slug by Targeting Mdm2

MicroRNAs (miRNAs) are small RNAs that fulfill diverse functions by negatively regulating gene expression. Here, we investigated the involvement of miRNAs in the chondrogenic differentiation of chick limb mesenchymal cells and found that the expression of miR-221 increased upon chondrogenic inhibition. Blockade of miR-221 via peanut agglutinin-based antisense oligonucleotides reversed the chondro-inhibitory actions of a JNK inhibitor on the proliferation and migration of chondrogenic progenitors as well as the formation of precartilage condensations. We determined that mdm2 is a relevant target of miR-221 during chondrogenesis. miR-221 was necessary and sufficient to down-regulate Mdm2 expression, and this down-modulation of Mdm2 by miR-221 prevented the degradation of (and consequently up-regulated) the Slug protein, which negatively regulates the proliferation of chondroprogenitors. These results indicate that miR-221 contributes to the regulation of cell proliferation by negatively regulating Mdm2 and thereby inhibiting Slug degradation during the chondrogenesis of chick limb mesenchymal cells.

Targeting miRNAs for Drug Discovery: A New Paradigm

The discovery of miRNAs and the establishment of it's clinical links with multiple diseases has led to a paradigm shift in the drug development pipeline of major pharmaceutical companies and has given birth to several biotechnology enterprises revolving around these magic molecules. The miRNA profiling studies over the last few years have indicated implicit involvement of miRNAs in the pathobiology of cancer, diabetes, infectious diseases as well as cardiovascular, neurological and immune system disorders. This information is currently being translated into tools for diagnosis, prognosis and predicting response to treatment. In addition, active and vigorous investigations are ongoing in several laboratories across academia and industry to decipher the precise molecular functions and mechanism of action for key miRNAs with therapeutic potential. Knowledge gained from these efforts will surely pave the way for designing effective R&D strategies and will revolutionize the way we diagnose and treat various diseases. The present review attempts to track the evolutionary progression of microRNA research from it's early infancy years to its maturity into a dynamic field of drug discovery.

The role of microRNAs in ovarian cancer initiation and progression

Epithelial ovarian cancer (EOC) has the highest mortality rate of all gynaecological cancers. One of the greatest impediments to improving outcome is an incomplete understanding of the molecular underpinnings of EOC pathogenesis and progression. Recent studies suggest that microRNAs (miRNAs) are involved in ovarian tumorigenesis and cancer development. Several miRNA profiling studies have identified some consensus aberrantly expressed miRNAs in EOC tissues, and these EOC-related miRNAs may play critical roles in the pathogenesis and progression of EOC. Moreover, some of the miRNAs may have diagnostic or prognostic significance. In this review, recent progress to reveal the role of miRNAs in EOC will be addressed, and a model for miRNA functions in ovarian cancer initiation and progression will be proposed.

Circulating microRNAs as Novel Minimally Invasive Biomarkers for Breast Cancer

The development of clinically validated biomarkers for cancer has remained an insurmountable task despite other advances in the field of cancer molecular biology. Mi(cro)RNAs have many characteristics of an ideal biomarker most notably their inherent stability and resilience. Recent blood-based miRNA profiling studies, reporting their presence in serum and plasma, have generated the concept that circulating miRNAs hold much potential as novel noninvasive biomarkers for cancer and other disease processes. The objective of this study was to investigate the potential of circulating microRNAs as novel breast cancer biomarkers. Using a novel approach to extract miRNAs from the circulation followed by real-time quantitative polymerase chain reaction analysis, levels of a panel of 7 candidate miRNAs were quantified in tissue and blood specimens of 148 patients with breast cancer and 44 age-matched and disease free control individuals. We report that cancer-specific miRNAs were detected and significantly altered in the circulation of breast cancer patients, and that increased systemic miR-195 levels in breast cancer patients were reflected in breast tumors. Furthermore, we identified that circulating levels of miR-195 and let-7a decreased in cancer patients postoperatively, to levels comparable with control subjects, following curative tumor resection. Finally, we found that specific circulating miRNAs correlated with certain clinicopathological variables, namely nodal status and estrogen receptor status. These findings suggest that systemic miRNAs have potential use as novel breast cancer biomarkers and may prove useful in clinical management during the perioperative period.

Systemic MiRNA Levels as Novel Breast Cancer Biomarkers.

Abstract Background: The development of clinically validated biomarkers for breast cancer has remained an insurmountable task despite other advances in the field of molecular biology. Mi(cro)RNAs have many characteristics of an ideal biomarker most notably their inherent stability, resilience, and ease of detection. Recent blood based miRNA profiling studies, reporting their presence in serum and plasma, have generated the concept that circulating miRNAs hold much potential as novel non-invasive biomarkers for cancer and other disease processes. The objective of this study was to investigate the potential of circulating microRNAs as novel breast cancer biomarkers.Materials and Methods: Using a real-time quantitative PCR approach, expression levels of a panel of 7 oncogenic miRNAs were quantified in tissue and blood specimens of 148 patients with breast cancer and 24 age-matched disease free control individuals. Advanced QBase Plus software and SPSS were used for biostatistical analysis of the data, and correlation with clinicopathological variables.Results: Cancer specific miRNAs were detected and significantly altered in the circulation of breast cancer patients preoperatively, most notably miR-195 (12 fold increase) and let-7a (5-fold increase), (p < 0.001 and p<0.001 respectively). Increased systemic miR-195 levels in breast cancer patients were also reflected in breast tumors. Systemic let-7a levels varied inversely with disease stage, being highest in carcinoma in-situ patients and getting progressively lower through to stage IV invasive disease. Furthermore we identified that circulating levels of miR-195 and let-7a decreased in cancer patients post-operatively following curative tumor resection (p<0.001 and p<0.001 respectively). Finally we found that specific circulating miRNAs correlated with certain clinicopathological variables, namely nodal status and estrogen receptor statusDiscussion/Conclusion: Our results demonstrates the novel finding that miRNAs are detectable in the circulation of breast cancer patients, that expression profiles differ between patients and controls, and that the circulating miRNA expression profile correlates with various biopathologic parameters of breast cancer. Also, systemic miRNA levels revert to normal levels following tumour resection. In this, lies the expectation that systemic miRNAs will be clinically useful as a novel minimally invasive tool to aid in the early diagnosis and perioperative management of breast cancer. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 32.

MicroRNA Expression in Melanocytic Nevi: The Usefulness of Formalin-Fixed, Paraffin-Embedded Material for miRNA Microarray Profiling

MicroRNAs (miRNAs) are small, noncoding RNA molecules that regulate cellular differentiation, proliferation, and apoptosis. MiRNAs are expressed in a developmentally regulated and tissue-specific manner. Aberrant expression may contribute to pathological processes such as cancer, and miRNA may therefore serve as biomarkers that may be useful in a clinical environment for diagnosis of various diseases. Most miRNA profiling studies have used fresh tissue samples. However, in some types of cancer, including malignant melanoma, fresh material is difficult to obtain from primary tumors, and most surgical specimens are formalin fixed and paraffin embedded (FFPE). To explore whether FFPE material would be suitable for miRNA profiling in melanocytic lesions, we compared miRNA expression patterns in FFPE versus fresh frozen samples, obtained from 15 human melanocytic nevi. Out of microarray data, we identified 84 miRNAs that were expressed in both types of samples and represented an miRNA profile of melanocytic nevi. Our results showed a high correlation in miRNA expression (Spearman r-value of 0.80) between paired FFPE and fresh frozen material. The data were further validated by quantitative RT-PCR. In conclusion, FFPE specimens of melanocytic lesions are suitable as a source for miRNA microarray profiling.