TaqMan miRNA Array Research Articles

Integrative analysis of mRNA and miRNA array data reveals the suppression of retinoic acid pathway in regulatory T cells of Graves' disease.

It is well known that regulatory T cells (Tregs) are abnormal in Graves' disease (GD) and play crucial roles in the breakdown of immune tolerance and GD development. However, there are controversies about whether the quantity and/or function of Tregs is aberrant in GD. The molecular mechanism of Tregs abnormality and its effects on GD development was still unclear, until now. MiRNAs play important roles in the function and development of the immune system including Tregs. To reveal the Tregs abnormality and its molecular mechanism in GD, we systematically studied the quantity and immunosuppressive function as well as the differential expression profiles of miRNA and mRNA of Tregs in newly diagnosed patients with GD using TaqMan miRNA array and mRNA microarray. Our results showed that the quantity and immunosuppressive function of Tregs in initial patients with GD was significantly decreased. More importantly, the retinoic acid (RA) pathway was markedly suppressed and its agonist, all-trans retinoic acid, could notably improve the quantity and immunosuppressive function of Tregs from patients with GD in vitro. In addition, many other pathways including protein ubiquitination and circadian rhythm were also significantly regulated in Tregs of GD. This integrative study first revealed the expression profiles of mRNA/miRNA in Tregs of initial GD and RA pathway might play important roles in GD development. Our results implied that all-trans RA, which had been used for a long time in the clinical setting, had potential value in the treatment of GD and was worthy of additional study.

Abstract 4008: cDNA and miRNA microarray analysis comparing gastric and metastatic liver gastrointestinal stromal tumors

Abstract Background: Gastrointestinal stromal tumor (GIST) is the most common mesenchymal tumor of the gastrointestinal tract. The majority of GISTs have a gain-of-function mutation of the c-kit or PDGFRA gene in the interstitial cells of Cajal. Although surgery is the most effective treatment for resectable primary GISTs, postoperative recurrence or metastasis has been observed after surgical resection in 40-90% of patients without adjuvant therapy. Metastatic GISTs are found most commonly in the liver, which are difficult to cure with surgical treatment alone. Although tyrosine kinase inhibitor (TKI) imatinib mesylate (Gleevec) is thought to be the most effective agent for treating GISTs, secondary resistance often arises during therapy due to secondary mutations. In this era of TKIs, control of liver metastasis remains to be an important issue in the treatment of GISTs and mechanisms of liver metastasis need to be elucidated. In this study, we compared gene expression and micro RNA (miRNA) expression profiles between gastric GISTs and metastatic liver GISTs to address mechanisms of GIST liver metastasis and to detect novel molecular markers for prediction of the postoperative prognosis of GIST patients. Methods: Frozen tissue specimens of three gastric GISTs and four metastatic liver GISTs were utilized for cDNA microarray analysis. None of the patients had received imatinib therapy before surgery. Formalin-fixed paraffin-embedded (FFPE) tissues of 104 primary GISTs and 13 metastatic GISTs from 107 patients who had undergone surgery were included for immunohistochemistry investigations. GIST cells were isolated from FFPE tissues of five gastric GISTs at low risk, five gastric GISTs at high risk and six metastatic liver GISTs surgically resected, and miRNA expression was analyzed using TaqMan miRNA array. Results: Expression levels were higher for 165 genes and lower for 146 genes in metastatic liver GIST. The upregulation of five oncogenes including versican and downregulation of four tumor suppressor genes, Cadherin8, Protocadherin10, NRCAM and CD9 were confirmed by qRT-PCR. Immunohistochemistry in 117 GISTs revealed that protein levels of versican and CD9 were higher and lower, respectively, in metastatic GIST. High expression of versican and low expression of CD9 in 104 primary gastric GISTs correlated with poor disease-free survival (P= 0.0078 and P= 0.0018). In miRNA array analysis, miR-122 was the most highly expressed in liver metastasis compared with primary gastric GISTs, consistent with our earlier papers that showed the overexpression of miR-122 and concomitant suppression of CAT1 in colorectal liver metastasis (Cancer Sci 104,624-30,2013). Conclusions: In addition to the c-kit gene mutation, genetic or epigenetic changes other than c-kit and alteration of miRNA expression such as miR-122 play important roles in the metastatic process of gastric GIST. Citation Format: Hirotoshi Kikuchi, Ichirota Iino, Shinichiro Miyazaki, Yusuke Ozaki, Yoshihiro Hiramatsu, Manabu Ohta, Kinji Kamiya, Takanori Sakaguchi, Satoshi Baba, Haruhiko Sugimura, Mitsutoshi Setou, Hiroyuki Konno. cDNA and miRNA microarray analysis comparing gastric and metastatic liver gastrointestinal stromal tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4008. doi:10.1158/1538-7445.AM2014-4008

Effects of remote ischemic preconditioning and myocardial ischemia on microRNA-1 expression in the rat heart in vivo.

Remote ischemic preconditioning (RIPC) is an easily applicable method for protecting the heart against a subsequent ischemia and reperfusion (I/R) injury. However, the exact molecular mechanisms underlying RIPC are unknown. We examined the involvement of microRNAs (miRNAs) and in particular the expression of miRNA-1 (miR-1) in RIPC and myocardial ischemia. Remote ischemic preconditioning was conducted by four cycles of 5-min bilateral hind-limb ischemia in male Wistar rats. Cardiac ischemia was induced by ligation of the left anterior descending coronary artery for 35 min followed by 2 or 6 h of reperfusion. MicroRNA expression was analyzed by Taqman miRNA arrays and quantitative polymerase chain reaction assays. Luciferase assays were performed to validate the miR-1 target gene brain-derived neurotrophic factor (BDNF). Brain-derived neurotrophic factor mRNA and protein levels were analyzed by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. Remote ischemic preconditioning led to a differential expression of miRNAs. The most abundant cardiac miRNA, miR-1, was downregulated by RIPC without following ischemia as well as after I/R and RIPC followed by I/R after 2 h of reperfusion. After 6 h of reperfusion, RIPC led to an upregulation of miR-1, whereas ischemia had no effect on miR-1 expression. Luciferase assays confirmed the interaction of miR-1 with BDNF, a protein that has been shown to exert cardioprotective effects. Brain-derived neurotrophic factor protein levels in rat hearts measured by enzyme-linked immunosorbent assay were not significantly altered after 2 or 6 h of reperfusion in all intervention groups. Remote ischemic preconditioning leads to changes in the expression levels of the most abundant cardiac miRNA, miR-1. MicroRNA 1 levels did not correlate with protein levels of BDNF, a known miR-1 target, in vivo. Further studies are needed to explore the biological significance of changes in miR-1 expression levels and the potential interaction with BDNF in RIPC-induced cardioprotection.

Downregulated microRNA-148b in circulating PBMCs in chronic myeloid leukemia patients with undetectable minimal residual disease: a possible biomarker to discontinue imatinib safely.

BackgroundA subset of patients with chronic myeloid leukemia (CML) can sustain a complete molecular response after discontinuing imatinib mesylate (IM). We focused on microRNAs (miRNAs), with the aim of finding a molecular biomarker to discriminate which patients can safely and successfully discontinue IM use.MethodsTo identify miRNAs that showed altered expression in patients who had discontinued IM (STOP-IM group), we first screened miRNA expression of peripheral blood mononuclear cells by using a TaqMan miRNA array on samples from five unselected patients from the STOP-IM group, seven CML patients receiving IM (IM group), and five healthy volunteers. We then performed miRNA quantification in 49 CML patients with deep molecular response. Mann–Whitney U and chi-square tests were used to determine statistical significance for comparisons between the control (healthy volunteers) and test groups (STOP-IM and IM groups). Multiple groups were compared by one-way analysis of variance.ResultsDownregulation of miR-148b was noted in patients in the STOP-IM group and in a subset of the IM group. We then subdivided the IM patients into two groups: one with downregulated miR-148b expression (IM-1; less than the cut-off value) and the other without downregulated miR-148b expression (IM-2; greater than the cut-off value). The number of patients who had a sustained stable molecular response was significantly lower in IM-2 group. This group also had a significantly lower percentage of natural killer cells.ConclusionDownregulated miR-148 may contribute to immune surveillance in STOP-IM patients and may therefore have potential as additive information in managing CML patients undergoing treatment with IM.

MiR-422a as a potential cellular microRNA biomarker for postmenopausal osteoporosis.

BackgroundMicroRNAs (miRNAs) are a class of short non-coding RNA molecules that regulate gene expression by targeting mRNAs. Recently, miRNAs have been shown to play important roles in the etiology of various diseases. However, little is known about their roles in the development of osteoporosis. Circulating monocytes are osteoclast precursors that also produce various factors important for osteoclastogenesis. Previously, we have identified a potential biomarker miR-133a in circulating monocytes for postmenopausal osteoporosis. In this study, we aimed to further identify significant miRNA biomarkers in human circulating monocytes underlying postmenopausal osteoporosis.Methodology/Principal FindingsWe used ABI TaqMan miRNA array followed by qRT-PCR validation in human circulating monocytes from 10 high BMD and 10 low BMD postmenopausal Caucasian women to identify miRNA biomarkers. MiR-422a was up-regulated with marginal significance (P = 0.065) in the low compared with the high BMD group in the array analysis. However, a significant up-regulation of miR-422a was identified in the low BMD group by qRT-PCR analysis (P = 0.029). We also performed bioinformatic target gene analysis and found several potential target genes of miR-422a which are involved in osteoclastogenesis. Further qRT-PCR analyses of the target genes in the same study subjects showed that the expression of five of these genes (CBL, CD226, IGF1, PAG1, and TOB2) correlated negatively with miR-422a expression.Conclusions/SignificanceOur study suggests that miR-422a in human circulating monocytes (osteoclast precursors) is a potential miRNA biomarker underlying postmenopausal osteoporosis.

Circulating microRNA profiles altered in mice after 28 d exposure to perfluorooctanoic acid

Perfluorooctanoic acid (PFOA) is a stable man-made compound with many industrial and commercial uses. Recently, however, concern has been raised that it may induce various toxicological effects such as hepatotoxicity, immunotoxicity, and developmental toxicity. Because levels of circulating microRNAs (miRNAs) can be altered in several clinical diseases, they may serve as potential novel biomarkers. Here, we explored differences in the profiles of circulating miRNAs in mice after PFOA exposure. Using TaqMan miRNA arrays, we determined that the levels of 24 circulating miRNAs were altered in mice dosed with PFOA at 1.25mg/kg/d and 73 were altered in mice dosed with 5mg/kg/d. Eight miRNAs were further validated using TaqMan Real-Time PCR assays. Results were consistent with those obtained from the TaqMan miRNA arrays, except for miR-199a-3p. The most remarkable of the circulating miRNAs (miR-26b-5p and miR-199a-3p) were also up-regulated in the serum of occupational workers in our previous epidemiological study. We also found similar patterns in mice exposed to PFOS. These results demonstrated that circulating miRNA profiles were altered after exposure to high concentrations of PFOA and miR-28-5p, miR-32-5p, miR-122-5p, miR-192-5p, and miR-26b-5p in serum may be linked to effects of PFOA, especially in occupationally exposed people.

Identification of MicroRNAs in Human Follicular Fluid: Characterization of MicroRNAs That Govern Steroidogenesis in Vitro and Are Associated With Polycystic Ovary Syndrome in Vivo

Human follicular fluid is a combination of proteins, metabolites, and ionic compounds that is indicative of the general state of follicular metabolism and is associated with maturation and quality of oocytes. Deviations in these components are often associated with reproductive diseases. There has been no report of microRNAs (miRNAs) in human follicular fluids. We hypothesized that human follicular fluid may contain miRNAs. We sought to identify cell-free miRNAs in human follicular fluid and to investigate the function of these miRNAs in vitro and any roles they play in polycystic ovary syndrome (PCOS). Genome-wide deep sequencing and TaqMan miRNA arrays were used to identify miRNAs, and the roles of the highly expressed miRNAs in steroidogenesis were investigated in KGN cells. Quantification of candidate miRNAs in follicular fluids of PCOS and controls was performed using TaqMan miRNA assays. We identified miRNAs in microvesicles and the supernatant of human follicular fluid. Bioinformatics analysis showed that the most highly expressed miRNAs targeted genes associated with reproductive, endocrine, and metabolic processes. We found that miR-132, miR-320, miR-520c-3p, miR-24, and miR-222 regulate estradiol concentrations and that miR-24, miR-193b, and miR-483-5p regulate progesterone concentrations. Finally, we showed that miR-132 and miR-320 are expressed at significantly lower levels in the follicular fluid of polycystic ovary patients than in healthy controls (P = .005 and P = .0098, respectively). These results demonstrate that there are numerous miRNAs in human follicular fluids, some of which play important roles in steroidogenesis and PCOS. This study substantially revises our understanding of the content of human follicular fluid and lays the foundation for the future investigation of the role of miRNAs in PCOS.

Abstract 404: Effect of miR-122 and its target gene cationic amino acid transporter 1 on colorectal liver metastasis.

Abstract Background: Control of liver metastasis has been an important issue in the treatment of colorectal cancers. Although microRNA (miRNA) has been shown to be involved in the development of many cancers including colorectal cancers, only few studies have investigated the roles of miRNAs in the process of colorectal liver metastasis. In this study, we compared miRNA expression between primary colorectal cancers and their corresponding liver metastasis to identify miRNAs involved in colorectal liver metastasis and to detect novel molecular markers for prediction of the postoperative prognosis of colorectal cancer patients. Methods: Cancer cells were isolated from formalin-fixed paraffin-embedded (FFPE) tissues of primary colorectal cancers and their corresponding metastatic liver tumors in 6 patients using laser capture microdissection methods. miRNA expression was analyzed using TaqMan miRNA array. Protein levels of cationic amino acid transporter 1 (CAT1), a negative target gene of miR-122, in 132 primary colorectal cancers and 22 colorectal liver metastases were analyzed by immunohistochemical staining. Relationship between clinicopathological factors and CAT1 expression levels in 132 primary colorectal cancers were evaluated. Results: miR-122 was the most highly expressed in liver metastasis compared with primary tumor. Immunohistochemical analysis revealed that the expression levels of CAT1 were lower in liver metastases than primary tumors (P <0.001). Expression levels of CAT1 in 132 primary tumors were negatively correlated with the existence of synchronous liver metastasis (P=0.0333) and tumor stage (P <0.0001). In the analysis of 121 colon cancer patients without synchronous liver metastasis, patients with CAT1-low colon cancer had significantly shorter liver metastasis-free survival (P=0.0258) but not overall survival or disease-free survival. Univariate analysis, for clarifying the risk factors of metachronous colorectal liver metastasis after primary resection, revealed that the 5-year liver metastasis-free survival rate was negatively correlated with the existence of lymph node metastasis and positively correlated with expression levels of CAT1 protein in the primary tumors. Conclusions: Overexpression of miR-122 and concomitant suppression of CAT1 in the primary tumor appear to play important roles in the development of colorectal liver metastasis. CAT1 expression in the primary colorectal cancer has a potential to be a novel biomarker with which to predict the risk of postoperative liver metastasis of the patients with colorectal cancer. Citation Format: Ichirota Iino, Hirotoshi Kikuchi, Shinichiro Miyazaki, Yoshihiro Hiramatsu, Manabu Ohta, Kinji Kamiya, Takanori Sakaguchi, Mitsutoshi Setou, Hiroyuki Konno. Effect of miR-122 and its target gene cationic amino acid transporter 1 on colorectal liver metastasis. [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 404. doi:10.1158/1538-7445.AM2013-404 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Abstract 3052: Ovarian carcinoma derived exosomes: miRNA signatures and role in cancer progression .

Abstract Ovarian cancer (OC) is the leading cause of death from gynecological cancer in Western countries. The disease is asymptomatic in the early stages, and is usually diagnosed at an advance stage, leading to an extremely poor prognosis. Primary solid tumor, solid metastases, and effusions to the peritoneal and pleural cavities characterize the tumor as it progresses. MicroRNAs (miRNAs, miRs) are small, non-coding RNAs. They regulate gene expression by binding to the 3′UTR of a target mRNA, and decrease protein levels by inhibition of protein translation. In the recent years, miRNAs were found to play an important role in cancer, both as oncogenes and as tumor suppressor genes, depending on the target mRNA and the type of cancer. In addition to cells, miRNAs are present in body fluids in exosomes, 30-90 nm vesicles secreted by cells. Exosomes contain proteins, mRNA and miRNA. Exosomal RNA content can be shuttled from one cell to another, affecting the recipient cell. It has been implicated that an "exosomal messenger system" exhibits paracrine bioactivities that facilitate tumor communication within the local tumor microenvironment. We compared the expression of miRNAs present in effusion fluid-derived exosomes from 50 pre-chemo and 50 post-chemo patients of which 37 were chemo-resistant and 31chemo-sensitive, respectively. The patient cohort was in addition divided into 28 long and 72 short survivors, respectively. TaqMan miRNA arrays were used to identify miRNA profiles, and the qPCR miScript system for validation of the obtained results. Web-based algorithms were used for further bioinformatics analysis. We identified differentially expressed miRNAs in OC effusion-derived exosomes compared to exosomal miRNAs from sera, malignant mesothelioma effusions and reactive effusions. Fifty-seven miRNAs were differentially expressed in specimens from patients with long vs. short survival and 62 differentially expressed miRNAs in specimens obtained before vs. after chemotherapy treatment. We tested the effects of effusion derived exosomes on cell behavior in-vitro and on tumor spread in-vivo. Our results show that exosomes affect the interaction between cancer cells and their microenvironment. Pre-injection of exosomes to female SCID mice, followed by injection of cancer cells, caused shorter life span and larger tumor burden, compared to mice not pre-treated with exosomes. OC effusion derived exosomes might have autocrine and/or paracrine effects on cells, thus enhancing the oncogenic potential of cancer cells, or sensitizing the body to malignant dissemination. This is the first study to evaluate the differential expression of miRNAs in exosomes derived from OC ascites fluid. We believe that the differential expression and the distinct signatures of miRNAs possess diagnostic, prognostic and therapeutic potential. Citation Format: Olga Vaksman (Goreshnik), Ben Davidson, Claes G. Trope’, Reuven Reich. Ovarian carcinoma derived exosomes: miRNA signatures and role in cancer progression . [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 3052. doi:10.1158/1538-7445.AM2013-3052

Effect of miR-122 and its target gene cationic amino acid transporter 1 on colorectal liver metastasis.

Control of liver metastasis is an important issue in the treatment of colorectal cancer (CRC). MicroRNAs have been shown to be involved in the development of many cancers, but little is known about their role in the process of colorectal liver metastasis. We compared miRNA expression between primary colorectal tumors and liver metastasis to identify those involved in the process of metastasis. Cancer cells were isolated from formalin-fixed paraffin-embedded primary CRC samples and their corresponding metastatic liver tumors in six patients using laser capture microdissection, and miRNA expression was analyzed using TaqMan miRNA arrays. The most abundant miRNA in liver metastasis compared with primary tumors was miR-122. Immunohistochemical analysis revealed that the expression levels of cationic amino acid transporter 1 (CAT1), a negative target gene of miR-122, were lower in liver metastases than primary tumors (P < 0.001). Expression levels of CAT1 in 132 primary tumors were negatively correlated with the existence of synchronous liver metastasis (P = 0.0333) and tumor stage (P < 0.0001). In an analysis of 121 colon cancer patients without synchronous liver metastasis, patients with CAT1-low colon cancer had significantly shorter liver metastasis-free survival (P = 0.0258) but not overall survival or disease-free survival. Overexpression of miR-122 and concomitant suppression of CAT1 in the primary tumor appears to play important roles in the development of colorectal liver metastasis. Expression of CAT1 in the primary CRC has the potential to be a novel biomarker to predict the risk of postoperative liver metastasis of CRC patients.

MiRNA Biomarkers in Cyst Fluid Augment the Diagnosis and Management of Pancreatic Cysts

The diagnosis of pancreatic cystic lesions has increased dramatically. Most are benign, whereas some, such as intraductal papillary mucinous neoplasms (IPMN), represent precursors of pancreatic adenocarcinoma. Therapeutic stratification of IPMNs is challenging without precise information on dysplasia grade and presence of invasion. We assessed the diagnostic benefit of using miRNAs as biomarkers in pancreatic cyst fluid, focusing on IPMNs because of their frequency and malignant potential. RNA was extracted from 55 microdissected formalin-fixed, paraffin-embedded (FFPE) IPMN specimens, and 65 cyst fluid specimens aspirated following surgical resection. Expression of 750 miRNAs was evaluated with TaqMan miRNA Arrays using 22 FFPE and 15 cyst fluid specimens. Differential expression of selected miRNA candidates was validated in 33 FFPE and 50 cyst fluid specimens using TaqMan miRNA Assays. We identified 26 and 37 candidate miRNAs that distinguish low-grade from high-grade IPMNs using FFPE and cyst fluid specimens, respectively. A subset of 18 miRNAs, selected from FFPE and cyst fluid data, separated high-grade IPMNs from low-grade IPMNs, serous cystadenomas (SCA) and uncommon cysts, such as solid pseudopapillary neoplasms (SPN) and cystic pancreatic neuroendocrine tumors (PanNET). A logistic regression model using nine miRNAs allowed prediction of cyst pathology implying resection (high-grade IPMNs, PanNETs, and SPNs) versus conservative management (low-grade IPMNs, SCAs), with a sensitivity of 89%, a specificity of 100%, and area under the curve of 1. We found candidate miRNAs that helped identify patients with high-grade IPMN and exclude nonmucinous cysts. These classifiers will require validation in a prospective setting to ultimately confirm their clinical usefulness.

Abnormal MicroRNA Expression in Ts65Dn Hippocampus and Whole Blood: Contributions to Down Syndrome Phenotypes

Down syndrome (DS; trisomy 21) is one of the most common genetic causes of intellectual disability, which is attributed to triplication of genes located on chromosome 21. Elevated levels of several microRNAs (miRNAs) located on chromosome 21 have been reported in human DS heart and brain tissues. The Ts65Dn mouse model is the most investigated DS model with a triplicated segment of mouse chromosome 16 harboring genes orthologous to those on human chromosome 21. Using ABI TaqMan miRNA arrays, we found a set of miRNAs that were significantly up- or downregulated in the Ts65Dn hippocampus compared to euploid controls. Furthermore, miR-155 and miR-802 showed significant overexpression in the Ts65Dn hippocampus, thereby confirming results of previous studies. Interestingly, miR-155 and miR-802 were also overexpressed in the Ts65Dn whole blood but not in lung tissue. We also found overexpression of the miR-155 precursors, pri- and pre-miR-155 derived from the miR-155 host gene, known as B cell integration cluster, suggesting enhanced biogenesis of miR-155. Bioinformatic analysis revealed that neurodevelopment, differentiation of neuroglia, apoptosis, cell cycle, and signaling pathways including ERK/MAPK, protein kinase C, phosphatidylinositol 3-kinase, m-TOR and calcium signaling are likely targets of these miRNAs. We selected some of these potential gene targets and found downregulation of mRNA encoding Ship1, Mecp2 and Ezh2 in Ts65Dn hippocampus. Interestingly, the miR-155 target gene Ship1 (inositol phosphatase) was also downregulated in Ts65Dn whole blood but not in lung tissue. Our findings provide insights into miRNA-mediated gene regulation in Ts65Dn mice and their potential contribution to impaired hippocampal synaptic plasticity and neurogenesis, as well as hemopoietic abnormalities observed in DS.

MicroRNAs are associated with human embryo implantation defects

Repeated implantation failure (RIF) is a major problem encountered in IVF. We have previously reported that RIF-IVF patients have a different endometrial gene expression profile during the window of implantation. Considering microRNA (miRNA) function in post-transcriptional regulation of gene expression, the aim of the study was to evaluate the involvement of miRNA in defects of endometrial receptivity. We used TaqMan miRNA array cards to identify the miRNAs differentially expressed in the secretory endometrium of RIF-IVF patients when compared with fertile women, and bioinformatics tools to identify their predicted targets and the molecular networks they may affect. Comparing miRNA expression profiles, we identified 13 miRNAs, differentially expressed in RIF endometrial samples, that putatively regulate the expression of 3800 genes. We found that 10 miRNAs were overexpressed (including miR 145, 23b and 99a) and 3 were underexpressed. Using our previous gene expression analysis, we paralleled miRNA-mRNA expression profiling. By this means, we identified novel and previously characterized miRNA-regulated molecular pathways such as adherens junctions, cell adhesion molecules, Wnt-signaling, p53 signaling and cell cycle pathways. Consistent with the miRNA-predicted targets, mRNA levels of N-cadherin, H2AFX, netrin-4 and secreted frizzled-related protein-4, belonging to the cell adhesion molecules, Wnt signaling and cell cycle pathways were lower in RIF-IVF patients. To our knowledge, this is the first study to evaluate the differential expression of miRNAs in the secretory endometrium of RIF-IVF patients. We suggest that the RIF-associated miRNAs could be exploited as new candidates for diagnosis and treatment of embryo implantation failures.

Increases In Mirna-145 and Mirna-146a Expression In Patients with IPSS Lower-Risk Myelodysplastic Syndromes and Del(5q) Treated with Lenalidomide.

AbstractAbstract 3631 Introduction:The erythroid differentiation defect observed in 5q– syndrome has been attributed to the RPS14 gene located within the CDR of the long arm of chromosome 5. We have recently demonstrated that RPS14 expression increases during lenalidomide treatment. However, haploinsufficiency of RPS14, which encodes ribosomal protein S14, does not explain clonal dominance. The expression of miRNAs, miR-145 (5q33.1) and miR-146a (5q33.3), in CD34+ bone marrow (BM) cells of individuals with MDS with deletion of the long arm of chromosome 5 (del(5q)) is lower compared to normal controls (Starczynowski et al, Nature Medicine, 2010). miRNAs are small noncoding RNAs that post-transcriptionally repress specific messenger RNA targets through interaction with the 3′ untranslated region (UTR). Loss of noncoding transcripts encoding miRNAs within the CDR may result in haploinsufficiency by loss of inhibition of their targets. Concurrent loss of both miR-145 and miR-146a resulted in activation of innate immune signalling through elevated expression of their respective targets, TIRAP and TRAF6. Furthermore, knockdown of miR-145 and miR-146a or overexpression of TRAF6 in mouse HSPC (Hematopoietic stem and progenitor cells) recapitulated features of 5q– syndrome, such as bone marrow dysplasia, anemia and thrombocytosis. We present preliminary results of changes in miRNA expression in IPSS lower-risk MDS with del(5q) during treatment with lenalidomide. Methods:A prospective single-arm trial investigating the efficacy and safety of lenalidomide in 46 patients with MDS with del(5q) with/without additional cytogenetic abnormalities and Hb < 10 g/dL. Lenalidomide was administered orally at a starting dose of 10 mg/day for a maximum of 12 months. When necessary, dosing was reduced to 5 mg/day or 5 mg on alternate days. Bone marrow assessments were performed at baseline and every 3 months, thereafter. For the evaluation of miRNA-145 and miRNA-146a in patient samples, 300 ng/μl of miRNAs were isolated in each purified BM sample by using mirVana™ miRNA Isolation Kit-Ambion and TaqMan miRNA Array Analysis was performed to determine the expression of miRNAs (7900HT Sequence Detection System Applied Biosystems). Patient BM-miRNAs were calibrated with miRNAs from BM of healthy volunteer donors. It was assumed that BM expression value of each calibrator miRNA was 1 unit. RPS14 gene assays were performed using TaqMan® Low Density Array Fluidic card (TaqMan® Human Array, Applied Biosystems, Foster City, CA, USA) based on Applied Biosystems PRISM® 7900HT comparative ddCT method, according to the manufacturer's instructions. Target gene expression levels were measured in triplicate and normalized against the expression of the 18S housekeeping gene from a BM pool of normal, healthy subjects at all timepoints. Median relative gene expression values in MDS patients were compared to healthy subjects, set as a value of 1. Results:Four patients have been evaluated (1 M, 3 F; ages 65, 66, 73 and 76 years, respectively) at baseline and after 12 weeks. At baseline, 2 patients were RBC-transfusion dependent. One patient had one additional cytogenetic abnormality (+8 in 15% metaphases). All patients obtained an erythroid response by week 12: mean Hb values significantly increased from 8.4 ± 0.9 at baseline to 11.6 ± 0.9 g/dL (p=0.01). All patients obtained a cytogenetic response, 2 of which were complete. miRNA-145 and miRNA-146a expression were both low at baseline and significantly increased by week 12 (Table). Conclusions:Preliminary results confirm that, in IPSS lower-risk MDS with del(5q), miRNA-145 and miRNA-146a expression is low. Lenalidomide treatment is associated with erythroid responses and cytogenetic remissions concurrent with significant increases in miRNA-145 and miRNA-146a expression.Table 1miRNA 145 and 146a and RPS14 expression before and during lenalidomide treatment.CasesmiRNA 145miRNA 146aRPS14Baseline12 weeksBaseline12 weeksBaseline12 weeks10.0004.8630.9361.7000.3057.62520.8363.8470.0871.6540.7198.83530.9276.9470.0342.7800.7352.03340.0672.8300.0740.8970.6376.164Mean ± SD0.458 ± 0.4924.622 ± 1.7580.283 ± 0.4361.758 ± 0.7750.599 ± 0.2016.164 ± 2.963p-value0.0130.0490.035 Disclosures:Oliva:Celgene: Consultancy.

Abstract 4050: Normalization of TaqMan human microRNA array data using average expression of controls

Abstract The growing interest on microRNA (miRNA) expression in cancer motivates the need for improvement of technical aspects. TaqMan Human MicroRNA Arrays with comprehensive coverage of 667 unique miRNAs from Sanger miRBase are now available (Applied Biosystems, Foster city, CA) to quantitatively assay miRNA expression. A recent study reported that these qRT-PCR arrays perform better than miRNA microarrays which utilizes U6 RNA as an endogenous control for normalization. However, the choice of control for normalization varies depending on the tissue under examination. The TaqMan Low Density Arrays (TLDA) arrays A and B include six different controls with at least quadruplet assays for each. A recent study indicated that the average expression normalization including all miRNAs on array performed better than other normalization methods indicating a further need to examine normalization of TaqMan miRNA array data. The choice of a suitable control other than an average expression over the entire array is a more general approach for qRT-PCR data since constant average expression may not be valid for these arrays. Hence, we combined both concepts to determine a normalization constant. We examined miRNA expressions of 5 endometrial cancer cell lines assayed by these TaqMan Low Density Arrays for miRNA. Each cell line was assayed in triplicate. The cycle threshold (Ct) values of positive controls U6 (MammU6), RNU48, RNU44, RNU24, RNU6B, RNU43 in these were found to be in the range of 15 to 39 where RNU6B and RNU43 had low expressions. In some cases, we observed failures in endogenous control U6 and differences between arrays A and B. Therefore multiple controls were included for normalization but with appropriate weighting for highly expressed controls. This was done by calculating the average of 2−Ct values for controls that had Ct-values below a threshold level (Ct &amp;lt; 25) and transforming this average back to logarithmic scale to the base 2. We then compared the normalized Ct-values by this method with the normalization using geometric average method. This weighted average normalization method resulted in smaller replicate variances of controls than the geometric average method. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4050.

Micro RNA analysis reveals novel genes in human systemic lupus erythematosus: miR-21 affects PDCD4 expression and regulates aberrant T cell responses

MicroRNAs (miRNAs) are potent negative regulators of gene expression involved in innate and adaptive immune responses. The authors examined whether miRNAs are implicated in immune deregulation and lymphocyte hyperactivity in...

Abstract A31: The role of microRNA 125a-3P in the pathogenesis of lung cancer

Abstract Tobacco exposure results in specific oncogene and tumor suppressor gene alterations in lung epithelial cells that often lead to lung cancer development. Activating point mutation of the K-ras proto-oncogene is one of the most frequently occurring genetic changes found in lung cancer both in active and former smokers. Understanding how K-ras mutations mediate the malignant phenotype may yield more effective prevention and diagnosis strategies to be applied to individuals at elevated risk for lung cancer. Micro RNAs are a class of short (19–24 nucleotides) noncoding RNAs that play a critical role in posttranscriptional gene regulation. A rapidly developing literature supports an important role for micro RNAs in the regulation of lung carcinogenesis and tumor progression. In order to better understand the role of micro RNAs in the K-ras-mutated bronchial epithelium, we utilized hTERT- and Cdk4-immortalized human bronchial epithelial cells (HBEC) expressing mutant KrasV12. We next generated the micro RNA profile (TaqMan miRNA array v.2, Applied Biosystems, Foster City, CA), gene expression profile (Affymetrix U133 plus 2 array), and protein expression profile (50-plex inflammatory marker panel) of the K-ras mutated and vector control HBECs. We found that the micro RNA miR-125a-3p was significantly suppressed in K-ras-mutated HBECs compared to the vector control cells. We then evaluated basal expression of miR-125a-3p in both HBECs and NSCLC cell lines containing mutated K-ras by real time RT-PCR. Both K-ras-mutated HBECs and NSCLC cell lines expressed low levels of miR125a-3P. In order to further evaluate the role of miR-125a-3p, we stably expressed this micro RNA in K-ras-mutated HBECs. Our results indicate that miR-125a-3p expression down-regulates several tumor-promoting factors, including Gro-α, HGF, VEGF, GM-CSF, and G-CSF. Further studies revealed that suppression of Gro-α and HGF, but not VEGF, is regulated at the level of mRNA expression. Overexpression of miR-125a-3p suppressed proliferation of the K-ras-mutated HBECs but did not alter the proliferation of the vector control HBECs. Based on these findings, we anticipate that loss of miR-125a-3p may have a broad impact in the bronchial epithelium that harbors K-ras mutations, potentially regulating cellular proliferation, angiogenesis and inflammation. Further exploration is required to evaluate the role of this micro RNA and its downstream effects as potential screening tools and targets of prevention and therapy in the setting of K-ras-mutated lung cancers. Citation Information: Cancer Prev Res 2010;3(1 Suppl):A31.

Low‐density Taqman miRNA array reveals miRNAs differentially expressed in prostatic stem cells and luminal cells

miRNAs are a class of naturally occurring small RNAs that generally repress gene expression. They have been shown to actively control diverse biological processes including stem cell differentiation and lineage commitment. Fluorescence-activated cell sorting was utilized to isolate murine prostate stem cells and differentiated luminal cells. The expression levels of Drosha and Dicer1, the two key RNAseIII enzymes for miRNA maturation, were evaluated by quantitative RT-PCR. Low-density Taqman miRNA array analyses were also performed to identify miRNAs that are differentially expressed in individual lineages. Drosha and Dicer1 are expressed at comparable transcriptional levels in murine prostate stem cells and differentiated luminal cells. Twenty-nine miRNAs were discovered to be differentially expressed in prostate stem cells and luminal cells. Many of these miRNAs are coded in clusters, suggesting a cell-specific transcriptional regulation. Some of these differentially expressed miRNAs have been reported to regulate genes relevant to the molecular and phenotypic features of each lineage. miRNAs may play a potentially critical role in fine regulation of prostatic lineage identity.