Downregulation Of microRNAs Research Articles

Disruption of microRNA Biogenesis Confers Resistance to ER Stress-Induced Cell Death Upstream of the Mitochondrion

Global downregulation of microRNAs (miRNAs) is a common feature of human tumors and has been shown to enhance cancer progression. Several components of the miRNA biogenesis machinery (XPO5, DICER and TRBP) have been shown to act as haploinsufficient tumor suppressors. How the deregulation of miRNA biogenesis promotes tumor development is not clearly understood. Here we show that loss of miRNA biogenesis increased resistance to endoplasmic reticulum (ER) stress-induced cell death. We observed that HCT116 cells with a DICER hypomorphic mutation (Exn5/Exn5) or where DICER or DROSHA were knocked down were resistant to ER stress-induced cell death. Extensive analysis revealed little difference in the unfolded protein response (UPR) of WT compared to Exn5/Exn5 HCT116 cells upon ER stress treatment. However, analysis of the intrinsic apoptotic pathway showed that resistance occurred upstream of the mitochondria. In particular, BAX activation and dissipation of mitochondrial membrane potential was attenuated, and there was altered expression of BCL-2 family proteins. These observations demonstrate a key role for miRNAs as critical modulators of the ER stress response. In our model, downregulation of miRNA biogenesis delays ER stress-induced apoptosis. This suggests that disrupted miRNA biogenesis may contribute to cancer progression by inhibiting ER stress-induced cell death.

Regulation of the Hif-system by micro-RNA 17 and 20a – Role during monocyte-to-macrophage differentiation

MiRNAs are a class of endogenous tiny RNAs that act as inhibitors of translation or promote RNA degradation by duplex-formation within the 3′-UTR of target mRNAs. They play an important role during a wide range of cellular processes by fine-tuning of gene expression. The differentiation of monocytes to macrophages plays a pivotal role in physiological as well as pathophysiological processes such as atherosclerosis. Monocytes which can be found in well-oxygenated blood migrate into areas with a high inflammation, such as the atherosclerotic plaque. There, they differentiate into macrophages. Interestingly, macrophages were found mainly at hypoxic sites of the plaque. Key regulators for the adaptation to hypoxia are the hypoxia-inducible factors (Hif). Therefore the aim of the present study was to investigate the regulation of the Hif-system by miRNAs during the process of monocyte differentiation. The present study shows that during the differentiation of monocytes into macrophages a dramatically change in the expression pattern of Hif-1α and Hif-2α took place. This was associated with a downregulation of microRNAs encoded by the miR-17-92 cluster. An in silico analysis of the 3′-UTR of Hif-α subunits for binding sites of miRNAs was performed using different miRNA databases in concert with a secondary structure prediction algorithm. This analysis revealed that both 3′-UTRs contain binding sites for miRNAs of the miR-17-92 cluster. Transfection of HeLa cells with miR-17 and miR-20a led to an inhibition of Hif-1α and -2α mRNA and protein expression and a lowered Hif DNA binding activity. Using a Luciferase-Reporter assay, it could be shown, that both Hif-α subunits are targeted by miR-17 and miR-20a. Furthermore, miR-overexpression in primary human macrophages demonstrates the important role of this microRNA-mediated regulation of the Hif-system for adaption of macrophages to hypoxia.In conclusion, the present study shows that the Hif-system is activated during monocyte-to-macrophage differentiation. This activation is in part mediated by a miRNA-dependent mechanism, which seems to be crucial for the adaption of macrophages to hypoxia.

Estrogen Regulation of microRNAs, Target Genes, and microRNA Expression Associated with Vitellogenesis in the Zebrafish

Estrogen is a steroid hormone that has been implicated in a variety of cellular and physiological processes and in the development of diseases such as cancer. Here we show a remarkable widespread microRNA (miRNA) downregulation in the zebrafish (Danio rerio) liver following 17β-estradiol (E2) treatment. This unique miRNA expression signature in the fish liver was further supported by a combination of computational predictions with gene expression microarray data, showing a significant bias toward upregulation of miRNA target genes after E2 treatment. Using pathway analysis of target genes, their involvement in the processes of cell cycle, DNA replication, and proteasome was observed, suggesting that miRNAs are incorporated into robust regulatory networks controlled by estrogen. In oviparous vertebrates, including fish, the formation of yolky eggs during a process known as vitellogenesis is regulated by estrogen. Microarrays were used to compare miRNA expression profiles between the livers of vitellogenic and nonvitellogenic zebrafish females. Among the upregulated miRNAs in vitellogenic females, were five members of the miR-17-92, a polycistronic miRNA cluster with a role in cell proliferation and cancer. Furthermore, a number of miRNA target genes related to fish vitellogenesis were revealed, including vtg3, a putative target of miR-122; the most abundant miRNA in the liver. Moreover, several of the differentially expressed miRNAs were only conserved in oviparous animals, which suggest an additional novel level of regulation during vitellogenesis by miRNAs and consequently, improves our knowledge of the process of oocyte growth in egg-laying animals.

Abstract 3088: Combinations of lapatinib and all-trans retinoic acid control the coordinate expression of microRNA modules and target-mRNAs in ERBB2+/RARA+ breast carcinoma cells: Significance for the antitumor activity.

Abstract In a recent report (Paroni et al, Oncogene, 31:3431-43, 2012), we demonstrated that 25% of all ERBB2+ breast cancers present with a co-amplification of the RARA locus, encoding the nuclear retinoic acid receptor, RARα. ERBB2+/RARA+ breast cancer cell lines, like SKBR3 and AU565, are sensitive to the anti-proliferative and cyto-differentiating effects of all-trans retinoic acid (ATRA), whereas the ERBB2+/RARA− counterparts are refractory. Simultaneous targeting of RARα with ATRA and ERBB2 with lapatinib in SKBR3 and AU565 cells leads to synergistic interactions as to growth inhibition and cyto-differentiation. In addition, combinations of ATRA and lapatinib exert an apoptotic effect, which is not observed with the single components. This suggests that ERBB2+/RARA+ breast cancer patients may benefit from lapatinib+ATRA regimens and represent the rationale of an ongoing clinical trial. The use of lapatinib and ATRA for the stratified therapy of ERBB2+/RARA+ patients requires knowledge of the molecular mechanisms underlying the cross-talk between the two compounds. We evaluated the effects of lapatinib and/or ATRA on the expression profiles of more than 1,000 microRNAs in SKBR3 cells using an appropriate microarray platform. Both lapatinib and ATRA caused up- and down-regulation of numerous microRNAs. The combination of the two compounds enhanced the action of the single components of the mixture and resulted in the up- and down-regulation of novel target microRNAs as well. We integrated these results with microRNA-target-gene prediction algorithms and whole-genome gene-expression data looking for significant correlations between microRNA and potential/established target mRNA levels. Network analysis led to the identification of two highly interconnected microRNA/target-mRNA modules controlled in opposite directions by lapatinib+ATRA. The first one consists of six microRNAs (miR-1207-5-p, miR-874, miR-134, miR-762, miR-1181 and miR-320c) which are up-regulated by lapatinib+ATRA and control a common set of down-regulated target mRNAs in a coordinate fashion. Most of the mRNAs of the module control cell proliferation in a positive fashion. The second network consists of seven microRNAs (miR-203, miR-125a-5p, miR-193a-5p, miR-210, miR-362-5p, miR-532-5p and miR-342-3p) which are down-regulated by lapatinib+ATRA and control a series of up-regulated target-mRNAs. In this case, the majority of regulated genes is involved in negative regulation of cell proliferation. Coordinated regulation of microRNA sets controlling a common group of genes and functions represents a new concept that deserves further studies. Currently, we are in the process of establishing the functional significance of the identified microRNA modules for the anti-tumor effects of lapatinib and ATRA. Citation Format: Enrico Garattini, James N. Fisher, Gabriela Paroni, Floriana Centritto, Gregory J. Goodall, Anna Tsykin, Maddalena Fratelli, Mineko Terao. Combinations of lapatinib and all-trans retinoic acid control the coordinate expression of microRNA modules and target-mRNAs in ERBB2+/RARA+ breast carcinoma cells: Significance for the antitumor activity. [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 3088. doi:10.1158/1538-7445.AM2013-3088

Abstract 2866: Functional impairment of microRNAs induced by chronic inflammation is the cause of inflammation-associated colon tumorigenesis.

Abstract Background: A widespread downregulation of microRNAs is commonly observed in human cancers and is involved in cellular transformation and tumorigenesis. Because we previously reported that microRNA functions are inhibited under various kinds of cellular stresses including inflammatory cytokines in vitro, we hypothesized that chronic inflammation in vivo may also cause chronic functional impairment of microRNAs, which mimics a global reduction of microRNA abundance, resulting in a causal role in inflammation-associated tumorigenesis. Methods & Results: 1) To examine this hypothesis, we first confirmed the functional impairment of microRNAs under inflammatory stimuli in vitro using artificial reporter constructs. The derepression of microRNA-targeting reporter activities by inflammatory stimuli could also be observed using reporter constructs containing natural 3’UTR sequences of c-myc and Lin28B genes, which are targeted by endogenous let-7. 2) Next, we constructed reporter transgenic mice bearing CMV-promoter driven GFP construct carrying target sequences of microRNAs in its 3’UTR, and monitored the changes of microRNA functions in vivo during the course of a Dextran Sulfate Sodium (DSS) induced colitis-associated colon tumor model. The expression levels of GFP were significantly increased during colitis, suggesting that microRNA functions were impaired during the chronic inflammation in vivo. The expression levels of microRNA-target oncogenes such as c-myc and Lin28B in the colonic mucosa were indeed upregulated. 3) Lastly, to determine the consequences of the global functional impairment of microRNAs during the colitis, we examined Dicer-deficient mice, which have globally low levels of microRNA expression and may mimic the functional impairment of global microRNAs induced by chronic inflammation described above. As expected, Dicer deficient mice were more liable to have the colitis-associated tumors. This indicates that the global decrease of microRNAs leads to oncogenesis, suggesting that the functional impairment of global miRNAs during chronic inflammation can also be a cause of the inflammation-associated carcinogenesis. Conclusion: In conclusion, we propose a novel concept that the attenuation of global microRNA functions by chronic inflammation is the cause of inflammation-associated tumorigenesis. Citation Format: Takeshi Yoshikawa. Functional impairment of microRNAs induced by chronic inflammation is the cause of inflammation-associated colon tumorigenesis. [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 2866. doi:10.1158/1538-7445.AM2013-2866

Down-regulation of specific miRNAs enhances the expression of the gene Smoothened and contributes to T-cell lymphoblastic lymphoma development

Inappropriate activation of the GLI/hedgehog (GLI/Hh) signalling occurs in several human cancers, including haematological neoplasms. However, little is known about its relevance in precursor T-cell lymphoblastic lymphomas (T-LBL) development. Moreover, the mechanisms whereby GLI/Hh signalling is activated in haematological malignancies are not fully clear. Here, we show that the gene Smoothened (SMO), the only non-redundant gene of this pathway, is up-regulated in mouse and human T-LBL. Interestingly, down-regulation of micro-RNAs mmu-miR-30a and mmu-miR-141 as well as hsa-miR-193b clearly contributes to enhance the expression of this gene in mouse and human lymphomas and, subsequently, to activate the GLI/Hh signalling. Activation of the GLI/Hh signalling in T-LBL promotes cell survival and proliferation, since inhibition of the pathway using short-hairpin-RNA-mediated SMO knockdown, or cyclopamine as a specific antagonist, significantly reduces these cellular processes. These findings suggest that sustained SMO up-regulation may contribute to T-LBL development and advocate the use of specific SMO inhibitors or microRNAs-based therapies as an attractive possibility to treat an important subset of T-LBL.

Downregulation and Prognostic Performance of MicroRNA 224 Expression in Prostate Cancer

The extensive use of prostate-specific antigen as a general prostate cancer biomarker has introduced the hazards of overdiagnosis and overtreatment. Recent studies have revealed the immense biomarker capacity of microRNAs (miRNAs) in prostate cancer. The aim of this study was to analyze the expression pattern of miR-224, a cancer-related miRNA, in prostate tumors and investigate its clinical utility. Total RNA was isolated from 139 prostate tissue samples. After the polyadenylation of total RNA by poly(A) polymerase, cDNA was synthesized with a suitable poly(T) adapter. miR-224 expression was assessed by quantitative real-time PCR and analyzed with the comparative quantification cycle method, C(q)(2(-ΔΔCq)). We performed comprehensive biostatistical analyses to explore the clinical value of miR-224 in prostate cancer. miR-224 expression was significantly downregulated in malignant samples compared with benign samples (P < 0.001). Higher miR-224 expression levels were found in prostate tumors that were less aggressive (P = 0.017) and in an earlier disease stage (P = 0.018). Patients with prostate cancer who were positive for miR-224 had significantly enhanced progression-free survival intervals compared with miR-224-negative patients (P = 0.021). Univariate bootstrap Cox regression confirmed that miR-224 was associated with favorable prognosis (hazard ratio 0.314, P = 0.013); nonetheless, multivariate analysis, adjusted for conventional markers, did not identify miR-224 as an independent prognostic indicator. miR-224 is aberrantly expressed in prostate cancer. Its assessment by cost-effective quantitative molecular methodologies could provide a useful biomarker for prostate cancer.

SP1 mediates the link between methylation of the tumour suppressor miR‐149 and outcome in colorectal cancer

Although recent studies indicate that DNA methylation contributes to the down-regulation of microRNAs (miRNAs) in colorectal cancer (CRC), this field remains largely unexplored. To identify methylation-silenced miRNAs and clarify their role in CRC, we performed a microarray analysis and screened for miRNAs that were induced in CRC cells by 5-aza-2'-deoxycytidine treatment or by the knockdown of DNA methyltransferases. The DNA methylation status of the candidate miRNA was analysed by bisulphite sequencing PCR and methylation-specific PCR. We found that miRNA-149 (miR-149) was epigenetically silenced in CRC and down-regulation of miR-149 was associated with hypermethylation of the neighbouring CpG island (CGI). Quantitative RT-PCR analysis demonstrated that the miR-149 level was markedly reduced in 51.6% of the CRC tissues compared with matched non-cancerous tissues. In addition, low expression of miR-149 was associated with a greater depth of invasion (p = 0.012), lower 5-year survival rate (p = 0.025), and was found to be an independent prognostic factor for overall survival (p = 0.016) in a multivariate analysis. Moreover, transfection of miR-149 inhibited cell growth and invasion of CRC cells in vitro. We also identified mRNA for Specificity Protein 1 (SP1, Sp1), a potential oncogenic protein, as a target of miR-149. Our data suggest that, as a methylation-sensitive miRNA, miR-149 may play an important role as a tumour suppressor in CRC, which has prognostic and therapeutic implications.

MicroRNA-155 as a therapeutic target for inflammatory diseases

MicroRNAs are short non-coding molecules expressed in different tissues and regulate the transcription of different genes. They are highly specific in their action. Upregulation or downregulation of specific microRNAs has been observed during different diseases like cancers, embryogenesis, organogenesis, apoptosis and arthritis. They are also known to be involved in autoimmune diseases. MicroRNAs are also found to be stable and easy to validate. Differential expression of microRNA-155 has been studied by different groups in inflammatory diseases including arthritis along with other miRNAs. This suggests that it can be used as a potential biomarker or therapeutic in the autoimmune diseases, especially rheumatoid arthritis. Experimental studies are needed to explore their role as biomarker or therapeutic.

Curcumin and synthetic analogs induce reactive oxygen species and decreases specificity protein (Sp) transcription factors by targeting microRNAs

BackgroundCurcumin inhibits growth of several cancer cell lines, and studies in this laboratory in bladder and pancreatic cancer cells show that curcumin downregulates specificity protein (Sp) transcription factors Sp1, Sp3 and Sp4 and pro-oncogenic Sp-regulated genes. In this study, we investigated the anticancer activity of curcumin and several synthetic cyclohexanone and piperidine analogs in colon cancer cells.MethodsThe effects of curcumin and synthetic analogs on colon cancer cell proliferation and apoptosis were determined using standardized assays. The changes in Sp proteins and Sp-regulated gene products were analysed by western blots, and real time PCR was used to determine microRNA-27a (miR-27a), miR-20a, miR-17-5p and ZBTB10 and ZBTB4 mRNA expression.ResultsThe IC50 (half-maximal) values for growth inhibition (24 hr) of colon cancer cells by curcumin and synthetic cyclohexanone and piperidine analogs of curcumin varied from 10 μM for curcumin to 0.7 μM for the most active synthetic piperidine analog RL197, which was used along with curcumin as model agents in this study. Curcumin and RL197 inhibited RKO and SW480 colon cancer cell growth and induced apoptosis, and this was accompanied by downregulation of specificity protein (Sp) transcription factors Sp1, Sp3 and Sp4 and Sp-regulated genes including the epidermal growth factor receptor (EGFR), hepatocyte growth factor receptor (c-MET), survivin, bcl-2, cyclin D1 and NFκB (p65 and p50). Curcumin and RL197 also induced reactive oxygen species (ROS), and cotreatment with the antioxidant glutathione significantly attenuated curcumin- and RL197-induced growth inhibition and downregulation of Sp1, Sp3, Sp4 and Sp-regulated genes. The mechanism of curcumin-/RL197-induced repression of Sp transcription factors was ROS-dependent and due to induction of the Sp repressors ZBTB10 and ZBTB4 and downregulation of microRNAs (miR)-27a, miR-20a and miR-17-5p that regulate these repressors.ConclusionsThese results identify a new and highly potent curcumin derivative and demonstrate that in cells where curcumin and RL197 induce ROS, an important underlying mechanism of action involves perturbation of miR-ZBTB10/ZBTB4, resulting in the induction of these repressors which downregulate Sp transcription factors and Sp-regulated genes.

Bone Morphogenetic Protein‐2 Decreases MicroRNA‐30b and MicroRNA‐30c to Promote Vascular Smooth Muscle Cell Calcification

BackgroundVascular calcification resembles bone formation and involves vascular smooth muscle cell (SMC) transition to an osteoblast‐like phenotype to express Runx2, a master osteoblast transcription factor. One possible mechanism by which Runx2 protein expression is induced is downregulation of inhibitory microRNAs (miR).Methods and ResultsHuman coronary artery SMCs (CASMCs) treated with bone morphogenetic protein‐2 (BMP‐2; 100 ng/mL) demonstrated a 1.7‐fold (P<0.02) increase in Runx2 protein expression at 24 hours. A miR microarray and target prediction database analysis independently identified miR‐30b and miR‐30c (miR‐30b‐c) as miRs that regulate Runx2 expression. Real‐time–polymerase chain reaction confirmed that BMP‐2 decreased miR‐30b and miR‐30c expression. A luciferase reporter assay verified that both miR‐30b and miR‐30c bind to the 3′‐untranslated region of Runx2 mRNA to regulate its expression. CASMCs transfected with antagomirs to downregulate miR‐30b‐c demonstrated significantly increased Runx2, intracellular calcium deposition, and mineralization. Conversely, forced expression of miR‐30b‐c by transfection with pre–miR‐30b‐c prevented the increase in Runx2 expression and mineralization of SMCs. Calcified human coronary arteries demonstrated higher levels of BMP‐2 and lower levels of miR‐30b than did noncalcified donor coronary arteries.ConclusionsBMP‐2 downregulates miR‐30b and miR‐30c to increase Runx2 expression in CASMCs and promote mineralization. Strategies that modulate expression of miR‐30b and miR‐30c may influence vascular calcification.

A global downregulation of microRNAs occurs in human quiescent satellite cells during myogenesis

During myogenesis, human satellite cells differentiate and form multinucleated myotubes, while a fraction of the human satellite cells enter quiescence. These quiescent satellite cells are able to activate, proliferate and contribute to muscle regeneration. Post-transcriptional regulation of myogenesis occurs through specific myogenic microRNAs, also known as myomiRs. Although many microRNAs are involved in myotube formation, little is known on the involvement of microRNAs in satellite cells entering quiescence. This current study aims to investigate microRNA involvement during differentiation of human satellite cells, specifically proliferating satellite cells entering quiescence.For this, clonally expanded human satellite cells were differentiated for 5 days, after which myotubes and quiescent satellite cells were separated through FACS sorting. Next, a microRNA microarray comparison of proliferating satellite cells, myotubes and quiescent satellite cells was performed and verified through qRT-PCR.We show that during human satellite cell differentiation, microRNAs are globally downregulated in quiescent satellite cells compared to proliferating satellite cells, in particular microRNA-106b, microRNA-25, microRNA-29c and microRNA-320c. Furthermore, we show that during myogenesis microRNA-1, microRNA-133, microRNA-206 and microRNA-486 are involved in myotube formation rather than satellite cells entering quiescence. Finally, we show an overall decrease in total mRNA in quiescent satellite cells, and an indication that RNaseL regulation plays a role in promoting and maintaining quiescence. Given the importance of quiescent satellite cells in skeletal muscle development and regenerative medicine, it is imperative to distinguish between myotubes and quiescent satellite cells when investigating skeletal muscle development, especially in microRNA studies, since we show that microRNAs are globally downregulated in quiescent human satellite cells.

Combinatorial Treatment of DNA and Chromatin-Modifying Drugs Cause Cell Death in Human and Canine Osteosarcoma Cell Lines

Downregulation of microRNAs (miRNAs) at the 14q32 locus stabilizes the expression of cMYC, thus significantly contributing to osteosarcoma (OS) pathobiology. Here, we show that downregulation of 14q32 miRNAs is epigenetically regulated. The predicted promoter regions of miRNA clusters at 14q32 locus showed no recurrent patterns of differential methylation, but Saos2 cells showed elevated histone deacetylase (HDAC) activity. Treatment with 4-phenylbutyrate increased acetylation of histones associated with 14q32 miRNAs, but interestingly, robust restoration of 14q32 miRNA expression, attenuation of cMYC expression, and induction of apoptosis required concomitant treatment with 5-Azacytidine, an inhibitor of DNA methylation. These events were associated with genome-wide gene expression changes including induction of pro-apoptotic genes and downregulation of cell cycle genes. Comparable effects were achieved in human and canine OS cells using the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA/Vorinostat) and the DNA methylation inhibitor Zebularine (Zeb), with significantly more pronounced cytotoxicity in cells whose molecular phenotypes were indicative of aggressive biological behavior. These results suggested that the combination of these chromatin-modifying drugs may be a useful adjuvant in the treatment of rapidly progressive OS.

Sequence-Dependent Synergistic Inhibition of Human Glioma Cell Lines by Combined Temozolomide and miR-21 Inhibitor Gene Therapy

Down-regulation of microRNA-21 (miR-21) can induce cell apoptosis and reverse drug resistance in cancer treatments. In this study, we explored the most effective schedule of the miR-21 inhibitor (miR-21i) and Temozolomide (TMZ) combined treatment in human glioma cells. Three tumor cell lines, U251 phosphatase and tensin homologue (PTEN) mutant, LN229 (PTEN wild-type), and U87 (PTEN loss of function), were subjected to evaluate the antitumor effects of deigned treatments (a predose of miR-21i for 4/8 h and then a subsequent TMZ treatment, a predose of TMZ for 4/8 h and then a subsequent miR-21i treatment, or a concomitant treatment) in vitro. A synergistic antiproliferative and proapoptotic activity was only obtained in U251 and U87 cells when a predose was administered for 4 h before the treatment of the other therapeutic agent, while the best antitumor effect in LN229 cells was achieved by using the concomitant treatment. Our data indicate that the effect of sequence and timing of administration is dependent on the PTEN status of cell lines. The best suppression effect was achieved by a maximal inhibition of STAT3 and phosphorylated STAT3, in PTEN loss of function cells. Our results reveal that both the sequence and the timing of administration are crucial in glioma combination therapy.

ZEB1 drives prometastatic actin cytoskeletal remodeling by downregulating miR-34a expression

Metastatic cancer is extremely difficult to treat, and the presence of metastases greatly reduces a cancer patient's likelihood of long-term survival. The ZEB1 transcriptional repressor promotes metastasis through downregulation of microRNAs (miRs) that are strong inducers of epithelial differentiation and inhibitors of stem cell factors. Given that each miR can target multiple genes with diverse functions, we posited that the prometastatic network controlled by ZEB1 extends beyond these processes. We tested this hypothesis using a mouse model of human lung adenocarcinoma metastasis driven by ZEB1, human lung carcinoma cells, and human breast carcinoma cells. Transcriptional profiling studies revealed that ZEB1 controls the expression of numerous oncogenic and tumor-suppressive miRs, including miR-34a. Ectopic expression of miR-34a decreased tumor cell invasion and metastasis, inhibited the formation of promigratory cytoskeletal structures, suppressed activation of the RHO GTPase family, and regulated a gene expression signature enriched in cytoskeletal functions and predictive of outcome in human lung adenocarcinomas. We identified several miR-34a target genes, including Arhgap1, which encodes a RHO GTPase activating protein that was required for tumor cell invasion. These findings demonstrate that ZEB1 drives prometastatic actin cytoskeletal remodeling by downregulating miR-34a expression and provide a compelling rationale to develop miR-34a as a therapeutic agent in lung cancer patients.

A Distinct Expression Pattern of the Long 3′-Untranslated Region Dicer mRNA and Its Implications for Posttranscriptional Regulation in Colorectal Cancer

OBJECTIVES:Reduced expression of Dicer is associated with global downregulation of microRNAs. Primary Dicer transcripts can be processed at two alternative polyadenylation sites, generating two pools of messenger RNAs (mRNAs) that carry either a long or a short 3′-untranslated regions (3′UTRs), that both encode the same Dicer protein. The short 3′UTR Dicer mRNA is not regulated by miR-103/107. The aim of this study was to investigate the expression of total Dicer mRNA, long 3′UTR Dicer mRNA, and miR-103 in colorectal cancer (CRC).METHODS:Paired tumor and normal mucosal specimens were obtained from 66 patients with CRC. Real-time reverse transcription PCR of long 3′UTR Dicer mRNA, total Dicer mRNA and miR-103 was carried out using the TaqMan Expression assay and the TaqMan MicroRNA assay.RESULTS:The median expression level of coding Dicer mRNA in the tumors was significantly lower than that in normal mucosa (P<0.001). There was no significant difference in expression levels of long 3′UTR Dicer mRNA between the tumors and the normal mucosa (P=0.90). The median expression ratio of long 3′UTR Dicer mRNA to total Dicer mRNA in tumors was significantly higher than in normal mucosa (P<0.001). There was no significant difference in expression levels of miR-103 between the tumors and normal mucosa (P=0.17). There was no significant correlation between clinicopathological findings, such as stage, tumor location, and histological grade and expression levels of total Dicer mRNA, long 3′UTR Dicer mRNA, or expression ratio of long 3′UTR Dicer mRNA to total Dicer mRNA.CONCLUSIONS:These results suggest that both transcriptional and posttranscriptional dysregulation of Dicer expression may be involved in colon carcinogenesis.

The roles of microRNAs in sarcomas

MicroRNAs are a class of small regulatory RNAs that influence the stabilities and translational efficiencies of target mRNAs. They have been implicated in an increasing number of biological processes, including carcinogenesis. A huge body of literature exists documenting up- or down-regulation of specific microRNAs during carcinogenesis and identifying molecular pathways by which these microRNAs influence every aspect of cancer development, including proliferation, angiogenesis, and metastasis. These studies have provided many insights into basic cancer biology as well as allowing identification of novel biomarkers and potential drug targets. However, the vast bulk of this literature concerns solid epithelial tumours, while sarcomas remain relatively under-studied. The purpose of this article is to review the roles of microRNAs in sarcomas and to highlight microRNAs or related molecular pathways that demonstrate consistent roles within individual or across sarcoma subtypes, with a view to identifying the key regulatory molecules. Further insights into sarcoma biology may be particularly valuable since sarcomas represent a tumour group with a particularly poor prognosis and rather limited treatment options.

Mo1595 Farnesoid X Receptor Expression is Inversely Correlated With Neoplastic Progression in Colitis-Associated Colon Carcinogenesis

Background: A widespread downregulation of microRNAs is commonly observed in human cancers and is involved in cellular transformation and tumorigenesis. Because we previously reported that microRNA functions are inhibited under various kinds of cellular stresses including inflammatory cytokines In Vitro, we hypothesized that chronic inflammation In Vivo may also cause chronic functional impairment of microRNAs, which mimics a global reduction of microRNA abundance, resulting in a causal role in inflammation-associated tumorigenesis. Methods & Results: 1) To examine this hypothesis, we first confirmed the functional impairment of microRNAs under inflammatory stimuli In Vitro using artificial reporter constructs. The derepression of microRNA-targeting reporter activities by inflammatory stimuli could also be observed using reporter constructs containing natural 3'UTR sequences of c-myc and Lin28B genes, which are targeted by endogenous let-7. 2) Next, we constructed reporter transgenic mice bearing CMV-promoter driven GFP construct carrying target sequences of microRNAs in its 3'UTR, and monitored the changes of microRNA functions In Vivo during the course of a Dextran Sulfate Sodium (DSS) induced colitisassociated colon tumor model. The expression levels of GFP were significantly increased during colitis, suggesting that microRNA functions were impaired during the chronic inflammation In Vivo. The expression levels of microRNA-target oncogenes such as c-myc and Lin28B in the colonic mucosa were indeed upregulated. 3) Lastly, to determine the consequences of the global functional impairment of microRNAs during the colitis, we examined Dicer-deficient mice, which have globally low levels of microRNA expression and may mimic the functional impairment of global microRNAs induced by chronic inflammation described above. As expected, Dicer deficient mice were more liable to have the colitis-associated tumors. This indicates that the global decrease of microRNAs leads to oncogenesis, suggesting that the functional impairment of global microRNAs during chronic inflammation can also be a cause of the inflammation-associated carcinogenesis. Conclusion: In conclusion, we propose a novel concept that the attenuation of global microRNA functions by chronic inflammation is the cause of inflammation-associated tumorigenesis.

Mo1594 The Role Played by PepT1 in Colitis-Associated Cancer

Background: A widespread downregulation of microRNAs is commonly observed in human cancers and is involved in cellular transformation and tumorigenesis. Because we previously reported that microRNA functions are inhibited under various kinds of cellular stresses including inflammatory cytokines In Vitro, we hypothesized that chronic inflammation In Vivo may also cause chronic functional impairment of microRNAs, which mimics a global reduction of microRNA abundance, resulting in a causal role in inflammation-associated tumorigenesis. Methods & Results: 1) To examine this hypothesis, we first confirmed the functional impairment of microRNAs under inflammatory stimuli In Vitro using artificial reporter constructs. The derepression of microRNA-targeting reporter activities by inflammatory stimuli could also be observed using reporter constructs containing natural 3'UTR sequences of c-myc and Lin28B genes, which are targeted by endogenous let-7. 2) Next, we constructed reporter transgenic mice bearing CMV-promoter driven GFP construct carrying target sequences of microRNAs in its 3'UTR, and monitored the changes of microRNA functions In Vivo during the course of a Dextran Sulfate Sodium (DSS) induced colitisassociated colon tumor model. The expression levels of GFP were significantly increased during colitis, suggesting that microRNA functions were impaired during the chronic inflammation In Vivo. The expression levels of microRNA-target oncogenes such as c-myc and Lin28B in the colonic mucosa were indeed upregulated. 3) Lastly, to determine the consequences of the global functional impairment of microRNAs during the colitis, we examined Dicer-deficient mice, which have globally low levels of microRNA expression and may mimic the functional impairment of global microRNAs induced by chronic inflammation described above. As expected, Dicer deficient mice were more liable to have the colitis-associated tumors. This indicates that the global decrease of microRNAs leads to oncogenesis, suggesting that the functional impairment of global microRNAs during chronic inflammation can also be a cause of the inflammation-associated carcinogenesis. Conclusion: In conclusion, we propose a novel concept that the attenuation of global microRNA functions by chronic inflammation is the cause of inflammation-associated tumorigenesis.

Abstract 4042: Loss of imprinting at the 14q32 locus contributes to osteosarcoma

Abstract Osteosarcoma (OS) is the most common primary bone malignancy affecting children and young adolescents. Downregulation of microRNAs at 14q32 locus is implicated in the pathogenesis of OS. However, array comparative genome hybridization did not show any DNA copy number loss at this locus. We hypothesized that epigenetic alterations may regulate genes and microRNAs present in this locus and contribute to the development and progression of OS. Here, we show that OS is characterized by a selective loss of imprinting (LOI) at 14q32 locus. LOI at the 14q32 locus was unique to OS, compared with 10 other cancer cell lines representing 6 tissue types. Furthermore, the average demethylation at 14q32 locus was 24%, but the global demethylation as determined by LINE-1 retrotransposons was only 7%. We also found LOI in H19, MEST, and PEG3 loci in OS samples, albeit less frequently. LOI at the 14q32 locus is highly prevalent (82.6%, 19 out of 23) only in OS patients with early-onset (12-30 years of age) of the disease. Imprinted genes at 14q32 locus were downregulated in OS due to histone modifications and the locus was controlled by bivalent domain enriched with both K4-3m and K27-3m. We determined the tumor suppressor function of this locus by overexpression of 3 imprinting genes, DLK1, RTL1 or DIO3 in SaoS2 cells. We found that all 3 genes inhibited cell proliferation and enhanced apoptosis. We also tested methylation patterns using matched buccal DNA samples obtained from OS patients and their parents. Interestingly, loss of imprinting at 14q32 locus was found in most of the OS patients and their parents, suggesting that children might be predisposed to developing OS by inheriting the instability of imprinting at this genomic site. To determine if loss of imprinting at 14q32 locus is the driving mechanism, or a secondary genetic event, we evaluated tumor tissues obtained from p53LSLR270H/;Osx-Cre mice OS models. These mice developed OS at 8-12 months (potentially representing the late-onset (&amp;gt; 30yrs) of human OS). LOI was found only in 33% of mice OS samples, which is in agreement with the low rate of LOI detected in late-onset human OS. In conclusion, our findings suggest that LOI at the 14q32 genomic locus is likely a contributing event in the pathogenesis of OS. 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 4042. doi:1538-7445.AM2012-4042