UTR Luciferase Assays Research Articles

MiR-18b and miR-518b TargetFOXN1During Epithelial Lineage Differentiation in Pluripotent Cells

MicroRNAs (miRNAs) regulate myriad biological processes; however, their role in cell fate choice is relatively unexplored. Pluripotent NT2/D1 embryonal carcinoma cells differentiate into an epithelial/smooth muscle phenotype when treated with bone morphogenetic protein-2 (BMP-2). To identify miRNAs involved in epithelial cell development, we performed miRNA profiling of NT2/D1 cells treated with BMP-2 at 6, 12, and 24 h, and on days 6 and 10. Integration of the miRNA profiling data with previously obtained gene expression profiling (GEP) data of NT2/D1 cells treated with BMP-2 at the same time points identified miR-18b and miR-518b as the top two miRNAs with the highest number of up-regulated predicted targets with known functions in epithelial lineage development. Silencing of miR-18b and miR-518b in NT2/D1 cells revealed several up-regulated TFs with functions in epithelial lineage development; among these, target prediction programs identified FOXN1 as the only direct target of both miRNAs. FOXN1 has previously been shown to play an important role in keratinocyte differentiation and epithelial cell proliferation. NT2/D1 and H9 human embryonic stem cells with silenced miR-18b and miR-518b showed up-regulation of FOXN1 and the epithelial markers CDH1, EPCAM, KRT19, and KRT7. A 3'UTR luciferase assay confirmed FOXN1 to be a target of the two miRNAs, and up-regulation of FOXN1 in NT2/D1 cells led to the expression of epithelial markers. Overexpression of the two miRNAs in BMP-2-treated NT2/D1 cells led to down-regulation of FOXN1 and epithelial lineage markers. These results show that miR-18b and miR-518b are upstream controllers of FOXN1-directed epithelial lineage development.

Identifying microRNAs regulating B7-H3 in breast cancer: the clinical impact of microRNA-29c

Background:B7-H3, an immunoregulatory protein, is overexpressed in several cancers and is often associated with metastasis and poor prognosis. Here, our aim was to identify microRNAs (miRNAs) regulating B7-H3 and assess their potential prognostic implications in breast cancer.Methods:MicroRNAs targeting B7-H3 were identified by transfecting two breast cancer cell lines with a library of 810 miRNA mimics and quantifying changes of B7-H3 protein levels using protein lysate microarrays. For validations we used western immunoblotting and 3′-UTR luciferase assays. Clinical significance of the miRNAs was assayed by analysing whether their expression levels correlated with outcome in two cohorts of breast cancer patients (142 and 81 patients).Results:We identified nearly 50 miRNAs that downregulated B7-H3 protein levels. Western immunoblotting validated the impact of the 20 most effective miRNAs. Thirteen miRNAs (miR-214, miR-363*, miR-326, miR-940, miR-29c, miR-665, miR-34b*, miR-708, miR-601, miR-124a, miR-380-5p, miR-885-3p, and miR-593) targeted B7-H3 directly by binding to its 3′-UTR region. Finally, high expression of miR-29c was associated with a significant reduced risk of dying from breast cancer in both cohorts.Conclusions:We identified miRNAs efficiently downregulating B7-H3 expression. The expression of miR-29c correlated with survival in breast cancer patients, suggesting a tumour suppressive role for this miRNA.

Genistein downregulates onco-miR-1260b and upregulates sFRP1 and Smad4 via demethylation and histone modification in prostate cancer cells.

Background:Recently several microRNAs (miRNAs) have been found to be regulated by genistein in cancer cells. In this study, we focused on the gene regulatory effect of genistein on microRNA and its target genes in prostate cancer (PC).Methods:Initially, we investigated the effect of genistein on prostate cancer cells and identified that the expression of miRNA-1260b was decreased by genistein. We performed functional analyses and investigated the relationship between miRNA-1260b expression and prostate cancer patient outcomes. Two target genes (sFRP1 and Smad4) of miR-1260b were identified based on computer algorithm and 3′UTR luciferase assay was carried out to determine direct miRNA regulation of the genes.Results:Genistein promoted apoptosis while inhibiting prostate cancer cell proliferation, invasion and TCF reporter activity in PC cells. MiR-1260b was highly expressed in prostate cancer tissues and significantly downregulated by genistein in PC cells. After knocking down miR-1260b, cell proliferation, invasion, migration and TCF reporter activity were decreased in PC cells. Western analysis and 3′UTR luciferase assay showed that the two target genes (sFRP1 and Smad4) were directly regulated by miR-1260b. The expression of sFRP1 and Smad4 was significantly decreased in prostate cancer tissues. Genistein also increased expression of these two genes via DNA demethylation and histone modifications.Conclusions:Our data suggest that genistein exerts its anti-tumour effect via downregulation of miR-1260b that targeted sRRP1 and Smad4 genes in prostate cancer cells. The expression of sFRP1 and Smad4 was also modulated by genistein via DNA methylation or histone modifications in PC cell lines.

MiR-138 Induces Renal Carcinoma Cell Senescence by Targeting EZH2 and Is Downregulated in Human Clear Cell Renal Cell Carcinoma

MiR-138 has been shown to be downregulated in various cancers, including head and neck squamous cell carcinoma (HNSCC) and clear cell renal carcinoma (ccRCC). In the present study, we aimed to reveal the mechanism of miR-138 induction of senescence in renal carcinoma cells and identify its specific target genes. We used qRT-PCR to analyze miR-138 expression levels in renal carcinoma cell lines and ccRCC samples. The activity of β-galactosidase was measured for functional analysis after miR-138 mimic transfection. To identify the targets of miR-138, we used three types of target prediction software to determine three candidate target genes. Furthermore, a 3'UTR luciferase assay was performed. Western blotting was used to detect the protein expression levels of candidate target genes. Additionally, knockdown of EZH2 by its siRNA was performed. The expression of miR-138 was downregulated in RCC cells lines and in tumor samples compared with their controls. Transfection of miR-138 mimic induced SN-12 cell senescence, decreased the protein expression of EZH2, and increased the protein expression of P16. Furthermore, miR-138 decreased the 3'UTR luciferase activity of EZH2. The knockdown of EZH2 by siRNA induced SN-12 cell senescence, decreased the protein expression level of EZH2, and increased the protein expression of P16. MiR-138 is a tumor-suppressor miRNA in ccRCC that induces SN-12 cell senescence by downregulating EZH2 expression and upregulating P16 expression.

Abstract C36: miR-22 represses Wnt Signaling in human colon cancer cells

Abstract Background: Wnt signaling is an evolutionarily conserved pathway that plays an important role in processes like development, differentiation and metastasis. Aberrant activation of Wnt signaling has been implicated in various tumors, predominantly of breast and colon. MicroRNAs are short non-coding RNAs that are frequently deregulated in cancer. Expression of the microRNA miR-22 was reported to be significantly lower in colorectal cancer tissues than that in normal adjacent mucosa. Based on bioinformatics predictions, we identified several Wnt signaling intermediates as potential targets for miR-22. As Wnt signalling is constitutively active in colon cancer cell lines SW480 and SW620, we hypothesize that miR-22 might be a potential regulator of Wnt signaling in colon cancer. Methods: Wnt signaling was stimulated in HEK293 cells by treating them with 25mM Lithium Chloride (LiCl). Wnt signaling activation was assayed using TOPFlash/FOPFlash luciferase activity in HEK293, SW480 and its metastatic derivative SW620, after overexpressing miR-22 using a plasmid construct. CyclinD1 promoter luciferase assay, Western blotting and quantitative RT-PCR were used to study the expression of downstream targets of Wnt signaling. Direct targets of miR-22 were validated by performing 3'-UTR luciferase assays. Plasmid constructs were used to overexpress or knockdown c-Myc. Results: Overexpression of miR-22 resulted in decreased TOP-Flash luciferase activity in LiCl-treated HEK293 cells and the colon cancer cells SW480 and SW620. miR-22 repressed Wnt signaling target genes CyclinD1, c-Myc and TCF7L2, at both mRNA and protein levels. It also decreased the mesenchymal markers (Snail and Vimentin) at the protein level. Overexpression of miR-22 down-regulated BCL9L protein levels and also targeted the BCL9L 3' UTR, as demonstrated by luciferase reporter assay. In HEK293 cells, overexpression of c-MYC repressed miR-22 levels whereas knockdown of endogenous c-Myc by shRNA up-regulated miR-22 levels. Conclusions: miR-22 inhibits Wnt signaling and potentially exerts this effect by targeting the Wnt activator BCL9L. The repression of mesenchymal markers by miR-22 suggests a role for it in Epithelial to Mesenchymal Transition. Further, the Wnt target gene c-Myc regulates the expression of miR-22, suggesting the existence of a positive feedback loop involving miR-22/Wnt Signaling/c-Myc by which miR-22 may indirectly regulate its own expression. Our findings therefore demonstrate a novel role for miR-22 in the regulation of Wnt signaling in colon cancer cells. Citation Format: Chetlangia Neha, Srinivasa Rao Rao, Devarajan Karunagaran. miR-22 represses Wnt Signaling in human colon cancer cells. [abstract]. In: Proceedings of the Twelfth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2013 Oct 27-30; National Harbor, MD. Philadelphia (PA): AACR; Can Prev Res 2013;6(11 Suppl): Abstract nr C36.

Genistein downregulates onco-miR-1260b and inhibits Wnt-signalling in renal cancer cells

Background:Wnt-signalling has an important role in renal cancer and it is modulated by genistein in other cancers. Recently, microRNAs (miRNAs) have emerged as new regulators of gene expression. Thus, we focused on miRNAs to examine the regulatory mechanism of genistein on the Wnt-signalling pathway in renal cell carcinoma (RCC).Methods:Initially, we investigated the effect of genistein on Wnt-signalling (TOPflash reporter assay (TCF reporter assays)) in renal cancer cells, and using microarray identified candidate miRNAs whose expression was decreased by genistein. We performed functional analyses and investigated the relationship between miRNA expression and renal cancer patient outcomes. We also did 3′UTR luciferase assays to look at direct miRNA regulation of Wnt-signalling-related genes.Results:Genistein promoted apoptosis while inhibiting RCC cell proliferation and invasion. Genistein also decreased TCF reporter activity in RCC cells. We found that miR-1260b was highly expressed and significantly downregulated by genistein in RCC cells. The expression of miR-1260b was significantly higher in renal cancer tissues compared with normal, and significantly related to overall shorter survival. In addition, miR-1260b promoted renal cancer cell proliferation and invasion in RCC cells. The 3′UTR luciferase activity of target genes (sFRP1, Dkk2, Smad4) was significantly decreased and their protein expression significantly upregulated in miR-1260b inhibitor-transfected renal cancer cells.Conclusion:Our data suggest that genistein inhibited Wnt-signalling by regulating miR-1260b expression in renal cancer cells.

Abstract 1959: Identification of microRNAs that target Id-1 in esophageal cancer.

Abstract Inhibitor of DNA binding-1 (Id-1) belongs to a family of helix-loop-helix (HLH) proteins that lack the fundamental domain for DNA binding. Since Id-1 is overexpressed in esophageal squamous cell carcinoma (ESCC), and its expression level is associated with cancer invasion and metastasis, suppression of Id-1 may have therapeutic significance. MicroRNAs (miRNAs), which constitute non-coding small RNAs of around 22-nucleotides and negatively regulate gene expression as post-transcriptional regulator, were reported to be involved in carcinogenesis. However, there is limited information about the miRNAs that regulate Id-1. This project aims to identify miRNAs that can target Id-1 and exert tumor suppressive functions. In this study, several bioinformatics software programs, including TargetScan, RegRNA, MicroRNA and EMBL-EBI, were used to predict miRNAs that can directly bind to the 3’UTR of Id-1 mRNA. The miRNAs which have high scores and are predicted to target Id-1 by at least 3 different target prediction algorithms were chosen as candidates for this study. These miRNA candidates are: miR-29 family (miR-29a, miR-29b and miR-39c), miR-330, miR-300, miR-593, miR-654, miR-338, miR-346, miR-632. Plasmids which can over-express these miRNAs were designed and constructed using BLOCK-iT™ Pol II miR RNAi Expression system (Invitrogen®). Successful over-expression of the miRNAs in ESCC cell lines were verified by qRT-PCR. The transient and stable miRNA over-expressing ESCC cells were harvested and subjected to western blot analysis for Id-1 protein expression level. Direct binding of miRNAs to the 3’UTR of Id-1 was analyzed by 3’UTR luciferase assay. The pGL3-Id-1 3’UTR luciferase reporter vector was constructed by cloning the Id-1 3’ UTR downstream of the firefly luciferase open reading frame and the luciferase activities were measured using the Dual Luciferase Assay System. The cell invasion ability of miRNA over-expressing ESCC cells was analyzed by using BD BioCoat™ Matrigel™ Invasion Chamber. Cell proliferation and colony formation assays were performed to investigate the anti-proliferation effect of these miRNAs. In this study, ten miRNA over-expressing plasmids were constructed and over-expression of several miRNAs was achieved. We found that miR-29c could directly target the 3’UTR of Id-1 and down-regulate Id-1 protein level. miR-29c exerts tumor suppressing effects by reducing the ESCC cell invasion ability. Further study using miR-29c inhibitors in western blot assay, Id-1 3’UTR luciferase assay and cell invasion assay will be conducted. In summary, this study shows that miR-29c can suppress Id-1 and inhibit cell invasion in ESCC cells. This study is supported by General Research Funds from Research Grants Council of the Hong Kong SAR (Project Nos. HKU762610M and HKU763111M) Citation Format: L Han, B Li, SW Tsao, ALM Cheung. Identification of microRNAs that target Id-1 in esophageal 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 1959. doi:10.1158/1538-7445.AM2013-1959

Abstract 4181: Oncogenic microRNA-183 induces cell growth and motility through down-regulation of Dkk-3 and SMAD4 in bladder cancer.

Abstract Introduction and Objective : The purpose of this study was to carry out functional analysis of oncogenic microRNA-183 and identify their specific target genes in bladder cancer. Methods : Expression levels of miR-183 in bladder normal/cancer tissues and bladder cancer cell lines were confirmed using real-time PCR. To examine the function of miR-183, cell growth, and migration were performed using UM-UC-3 cells because the miR-183 expression level was highest among bladder cancer cell lines. To search for target genes of miR-183, we used a target scan algorithm and performed Dkk-3 and SMAD4 3’UTR luciferase assays. To analyze whether Dkk-3 and SMAD4 affect cell growth and motility, Dkk-3 and SMAD4 expressing plasmids were transfected into UM-UC-3 cells and cell growth and motility were performed. Results: Expression of miR-183 in cancer bladder tissues was significantly up-regulated compared to normal bladder tissues. MiR-183 expression was significantly higher in the pT3+4 tissues than in pT1+2 tissues. Higher miR-183 expression was significantly associated with shorter survival. MiR-183 increased cell growth and migration abilities in UM-UC-3 cells. MiR-183 decreased 3’UTR luciferase activity of Dkk-3 and SMAD4. Cell growth and motility was significantly decreased in Dkk-3 and SMAD4 transfected cells compared with control cells. Conclusions : MiR-183 is a oncogenic microRNA in bladder cancer through down-regulation of Dkk-3 and SMAD4. Citation Format: Koji Ueno, Hiroshi Hirata, Varahram Shahryari, Yuichiro Tanaka, Z. Laura Tabatabai, Yuji Hinoda, Rajvir Dahiya. Oncogenic microRNA-183 induces cell growth and motility through down-regulation of Dkk-3 and SMAD4 in bladder 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 4181. doi:10.1158/1538-7445.AM2013-4181

MicroRNA-182-5p Promotes Cell Invasion and Proliferation by Down Regulating FOXF2, RECK and MTSS1 Genes in Human Prostate Cancer

Recently miR-182 has been reported to be over-expressed in prostate cancer (PC) tissues, however detailed functional analysis of miR-182-5p has not been carried out. The purpose of this study was to: 1. analyze the function of miR-182-5p in prostate cancer, 2. assess its usefulness as a tumor marker, 3. identify miR-182-5p target genes in PC, 4. investigate the potential for miR-182-5p inhibitor to be used in PC treatment. Initially we found that miR-182-5p expression was significantly higher in prostate cancer tissues and cell lines compared to normal prostate tissues and cells. Moreover high miR-182-5p expression was associated with shorter overall survival in PC patients. To study the functional significance of miR-182-5p, we knocked down miR-182-5p with miR-182-5p inhibitor. After miR-182-5p knock-down, prostate cancer cell proliferation, migration and invasion were decreased. We identified FOXF2, RECK and MTSS1 as potential target genes of miR-182-5p using several algorithms which was confirmed by 3’UTR luciferase assay and Western analysis. Knock-down of miR-182-5p also significantly decreased in vivo prostate tumor growth. In conclusion this is the first report documenting that over-expression of miR-182-5p is associated with prostate cancer progression and potentially useful as a prognostic biomarker. Also knock down of miR-182-5p in order to increase expression of tumor suppressor genes FOXF2, RECK and MTSS1 may be of therapeutic benefit in prostate cancer treatment.

Cardiovascular dysregulation of miR‐17‐92 causes a lethal hypertrophic cardiomyopathy and arrhythmogenesis

MicroRNA cluster miR-17-92 has been implicated in cardiovascular development and function, yet its precise mechanisms of action in these contexts are uncertain. This study aimed to investigate the role of miR-17-92 in morphogenesis and function of cardiac and smooth muscle tissues. To do so, a mouse model of conditional overexpression of miR-17-92 in cardiac and smooth muscle tissues was generated. Extensive cardiac functional studies identified a dose-dependent induction of dilated, hypertrophic cardiomyopathy, and arrhythmia inducibility in transgenic animals, which correlated with premature mortality (98.3 ± 42.5 d, P<0.0001). Expression analyses revealed the abundance of Pten transcript, a known miR-17-92 target, to be inversely correlated with miR-17-92 expression levels and heart size. In addition, we demonstrated through 3'-UTR luciferase assays and expression analyses that Connexin43 (Cx43) is a novel direct target of miR-19a/b and its expression is suppressed in transgenic hearts. Taken together, these data demonstrate that dysregulated expression of miR-17-92 during cardiovascular morphogenesis results in a lethal cardiomyopathy, possibly in part through direct repression of Pten and Cx43. This study highlights the importance of miR-17-92 in both normal and pathological functions of the heart, and provides a model that may serve as a useful platform to test novel antiarrhythmic therapeutics.

The Mir-17∼92 Cluster Activates mTORC1 Signaling in MCL by Targeting Multiple Regulators of the LKB1/AMPK/mTOR Pathway

Abstract 5118Mantle cell lymphoma (MCL) is an aggressive hematological malignancy with a median survival ranging between 3 and 5 years. Novel therapeutic strategies are urgently needed to improve the outcome. Mammalian target of rapamycin (mTOR) pathway which plays a central role in controlling cell growth, proliferation and metabolism has been shown to be deregulated in MCL. mTOR inhibitors, such as rapamycin and its analogues, have been approved for treatment of relapsed/refractory MCL. However, the molecular mechanism of mTOR activation in MCL has yet to be defined. MiRNA (miR)-17∼92 is a cluster of six miRNAs which are frequently overexpressed in MCL cases and overexpression of the miR-17∼92 cluster in MCL predicts poor prognosis. Our previous study demonstrated that miR-17∼92 activated the PI3K/AKT pathway by directly targeting PTEN and PHLPP2; and knock-down of miR-17∼92 expression inhibited MCL tumor growth in a xenograft/SCID MCL mouse model. In the present study, we further demonstrated that knock-down miR-17∼92 decreased the cell size, similar effect as seen in cells treated with mTORC1 inhibitors. Knockdown of miR-17∼92 expression also decreased the glycolysis, protein synthesis and glucose uptake in MCL cells. We found that knockdown miR-17∼92 activated AMPK signaling as demonstrated by increased phosphorylation of AMPK at Thr172, especially under low glucose condition. Activated AMPK further phosphorylated TSC2 and Raptor at S1387 and S792, respectively, thereby inhibiting mTORC1 signaling as evidenced by decreased phosphorylation of RPS6 and 4E-BP1. Using TargetScan and other prediction algorithms, we found that several factors in the LKB1/AMPK/mTOR pathway, such as LKB1, CAB39, PRKAA1 and TSC1, are predicted the targets of miR-17∼92. In this study, we validated these factors as direct targets of the miR-17∼92 by 3'UTR luciferase assays using reporter plasmids containing the 3'UTR of the targets or the 3'UTR with mutations in the predicted miRNA binding sites. The protein levels of these targets decreased in MCL cells with the miR-17∼92 overexpression. Conversely, the levels of these factors were increased upon knockdown of miR-17∼92 cluster. Our results indicate that overexpression of miR-17∼92 in MCL plays important role in mTORC1 activation by inactivating the LKB1/AMPK signaling, in addition to its effect on the PI3K/AKT pathway activation. Disclosures:No relevant conflicts of interest to declare.

Targeting of syndecan‐1 by microRNA miR‐10b promotes breast cancer cell motility and invasiveness via a Rho‐GTPase‐ and E‐cadherin‐dependent mechanism

microRNAs are small endogenous noncoding RNAs, which post-transcriptionally regulate gene expression. In breast cancer, overexpression of the transmembrane heparan sulfate proteoglycan syndecan-1, a predicted target of the oncomiR miR-10b, correlates with poor clinical outcome. To investigate the potential functional relationship of miR-10b and syndecan-1, MDA-MB-231 and MCF-7 breast cancer cells were transiently transfected with pre-miR-10b, syndecan-1 siRNA or control reagents, respectively. Altered cell behavior was monitored by proliferation, migration and invasion chamber assays, and time-lapse video microscopy. miR-10b overexpression induced post-transcriptional downregulation of syndecan-1, as demonstrated by quantitative real-time PCR (qPCR), flow cytometry, and 3'UTR luciferase assays, resulting in increased cancer cell migration and matrigel invasiveness. Syndecan-1 silencing generated a copy of this phenotype. Adhesion to fibronectin and laminin and basal cell proliferation was increased. Syndecan-1 coimmunoprecipitated with focal adhesion kinase, which showed increased activation upon syndecan-1 depletion. Affymetrix screening and confirmatory qPCR and Western blotting analysis of syndecan-1-deficient cells revealed upregulation of ATF-2, COX-2, cadherin-11, vinculin, actin γ 2, MYL9, transgelin-1, RhoA/C, matrix metalloproteinase 2 (MMP2) and heparanase, and downregulation of AML1/RUNX1, E-cadherin, CLDN1, p21WAF/CIP, cyclin-dependent kinase 6, TLR-4, PAI1/2, Collagen1alpha1, JHDM1D, Mpp4, MMP9, matrilin-2 and ANXA3/A10. Video microscopy demonstrated massively increased Rho kinase-dependent motility of syndecan-1-depleted cells, which displayed increased filopodia formation. We conclude that syndecan-1 is a novel target of the oncomiR miR-10b. Rho-GTPase-dependent modulation of cytoskeletal function and downregulation of E-cadherin expression are identified as relevant effectors of the miR-10b-syndecan-1 axis, which emerges as a promising target for the development of new therapeutic approaches for breast cancer.

Effect of targeting of syndecan-1 by microRNA miR-10b on breast cancer cell motility and invasiveness via a rho-GTPase- and E-cadherin-dependent mechanism.

e21041 Background: microRNAs are small endogenous non-coding RNAs, which posttranscriptionally regulate gene expression. In breast cancer, overexpression of the transmembrane heparan sulfate proteoglycan syndecan-1, a predicted target of the oncomiR miR-10b, correlates with poor clinical outcome. Here, we investigatet the potential functional relationship of miR-10b and syndecan-1 in an in vitro study. Methods: MDA-MB-231 and MCF-7 breast cancer cells were transiently transfected with pre-miR-10b, syndecan-1 siRNA or control reagents, respectively. Altered cell behaviour was monitored by proliferation, migration and invasion chamber assays, and time-lapse video microscopy. Results: miR-10b overexpression induced posttranscriptional downregulation of syndecan-1, as demonstrated by qPCR, flow cytometry, and 3’UTR luciferase assays, resulting in increased cancer cell migration and matrigel invasiveness. Syndecan-1 silencing generated a copy of this phenotype. Adhesion to fibronectin and laminin and basal cell proliferation were increased. Syndecan-1 co-immunoprecipitated with focal adhesion kinase, which showed increased activation upon syndecan-1 depletion. Affymetrix screening and confirmatory qPCR and Western blotting analysis of syndecan-1 deficient cells revealed upregulation of ATF-2, COX- 2, cadherin-11, vinculin, ACTG2, MYL9, transgelin-1, RhoA/C, MMP2 and heparanase, and downregulation of AML1/RUNX1, E-cadherin, CLDN1, p21WAF/CIP, Cdk6, TLR-4, PAI1/2, Collagen1alpha1, JHDM1D, Mpp4, MMP9, matrilin-2 and ANXA3/A10. Video microscopy demonstrated massively increased Rho kinase-dependent motility of syndecan-1-depleted cells, which displayed increased filopodia formation. Conclusions: We conclude that syndecan-1 is a novel target of the oncomiR miR-10b. Rho-GTPase dependent modulation of cytoskeletal function and downregulation of E-cadherin expression are identified as relevant effectors of the miR-10b-syndecan-1 axis, which emerges as a promising target for the development of new therapeutic approaches for breast cancer.

Aromatase inhibitor treatment of breast cancer cells increases the expression of let‐7f, a microRNA targeting CYP19A1

Aromatase inhibitors (AIs) are considered the gold standard of endocrine therapy for oestrogen receptor-positive postmenopausal breast cancer patients. AI treatment was reported to result in marked alterations of genetic profiles in cancer tissues but its detailed molecular mechanisms have not been elucidated. Therefore, we profiled miRNA expression before and after treatment with letrozole in MCF-7 co-cultured with primary breast cancer stromal cells. Letrozole significantly altered the expression profiles of cancer miRNAs in vitro. Among the elevated miRNAs following letrozole treatment, computational analysis identified let-7f, a tumour-suppressor miRNA which targeted the aromatase gene (CYP19A1) expression. Quantitative real-time PCR assay using MCF-7 and SK-BR-3 cells as well as clinical specimens of a neoadjuvant study demonstrated a significant inverse correlation between aromatase mRNA and let-7f expression. In addition, high let-7f expression was significantly correlated with low aromatase protein levels evaluated by both immunohistochemistry and the western blotting method in breast cancer cases. Results of 3'UTR luciferase assay also demonstrated the actual let-7f binding sites in CYP19A1, indicating that let-7f directly targets the aromatase gene. Subsequent WST-8 and migration assays performed in let-7f-transfected MCF-7 and SK-BR-3 cells revealed a significant decrement of their proliferation and migration. These findings all demonstrated that let-7f, a tumour suppressor miRNA in breast cancer, directly targeted the aromatase gene and was restored by AI treatment. Therefore, AIs may exert tumour-suppressing effects upon breast cancer cells by suppressing aromatase gene expression via restoration of let-7f.

Abstract 182: Tumor suppressor microRNA-493 decreases cell motility and migration ability in human bladder cancer cells by down-regulating RhoC and FZD4

Abstract Introduction and Objective: The purpose of this study was to identify new tumor suppressor microRNAs in bladder cancer to carry out functional analysis of their suppressive role and identify their specific target genes. Methods: To identify tumor suppressor microRNAs in bladder cancer, microRNA microarray was performed using SV-HUC-1, T24, J82 and TCCSUP cells. Based on the microRNA microarray, 10 microRNAs were selected and expression levels confirmed using real-time PCR. The function of miR-493 was measured by MTS, cell cycle, migration, and wound healing assays using T24 and J82 cells. Based on the observation that miR-493 affects cell motility and migration, we searched for target genes of miR-493 related to motility and migration using a target scan algorithm, identified RhoC and FZD4 and performed 3′UTR luciferase assays. To examine whether RhoC and FZD4 affects T24 and J82 cell migration, RhoC siRNA, FZD4 siRNA or control siRNA was transfected into T24 and J82 cells and migration, and wound healing assays were performed. We also analyzed whether over-expression of miR-493 affects the binding of RhoC to Rock-1by immunoprecipitation at 72 hr after transfection in T24 cells. Results: Expression of miR-493 in bladder cancer (T24, J82 and TCCSUP) cells was down-regulated compared to normal SV-HUC-1 cells. Also the expression of miR-493 was significantly lower in human bladder cancer tissues than in their corresponding non-cancerous tissues. Transfection of miR-493 into T24 and J82 cells decreased cell growth and migration abilities. To identify potential miR-493 target genes, we used target scan algorithms to identify target oncogenes (FZD4 and RhoC) related to invasion and migration. MiR-493 decreased 3′UTR luciferase activity and protein expression of FZD4 and RhoC. Cell migration was significantly decreased by 35 to 50% in RhoC and FZD4 siRNA transfectants compared with control cells. MiR-493 also decreased binding of RhoC and Rock-1. Conclusions: MiR-493 is a new tumor suppressor microRNA in bladder cancer and inhibits cell motility through down-regulation of RhoC and FZD4. 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 182. doi:1538-7445.AM2012-182

Abstract 1652: Targetome profiling and genetic association analyses implicate miR-202 in follicular lymphomagenesis

Abstract Complex etiologic heterogeneity among non-Hodgkin's lymphoma (NHL) subtypes has made it difficult to identify risk factors for this disease. However, recent genetic association analyses, including two genome-wide association studies, suggest that genetic susceptibility to NHL may be strongest for the follicular lymphoma subtype. As recent studies have demonstrated that microRNAs (miRNAs) may be important in the etiology of many cancer types, including NHL, we performed a population-based genetic association study which identified a polymorphism in the precursor of miR-202 that is strongly associated with follicular lymphoma risk (rs12355840 in miR-202: PTREND = 0.006). An in vitro functional genetics assay demonstrated that the polymorphism is biologically active, and operates by altering the processing efficiency of the miR-202 precursor to the mature effector miRNA species. A ribonucleoprotein immunoprecipitation-microarray (RIP-Chip) assay, performed with an anti-AGO2 antibody followed by a whole-genome array, was used to characterize the miR-202 targetome, and selected targets were confirmed using a 3′UTR luciferase assay. Among the identified targets were several transcripts with relevance for tumorigenesis, including the miRNA biogenesis gene DICER1 and the lymphoma-associated cell cycle regulator and proto-oncogene SKP2. Both of these genes have been shown to be upregulated in lymphomas, which is consistent with our data, which show that the miR-202 risk allele is associated with diminished mature miR-202 levels in vitro. Taken together, these findings suggest that rs12355840 may represent a novel risk biomarker for follicular lymphoma, and implicate miR-202 in follicular lymphomagenesis. 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 1652. doi:1538-7445.AM2012-1652

Abstract 2293: MicroRNA-1826 targets VEGFC, beta-catenin (CTNNB1) and MEK1 (MAP2K1) in human bladder cancer.

Abstract Introduction and Objective: The Wnt/beta-catenin (CTNNB1) and Ras-Raf-MEK-ERK signaling pathway play an important role in bladder cancer progression. Tumor suppressive miRNAs targeting these cancer pathways may provide a new therapeutic approach for bladder cancer. Methods: We initially identified miRNA-1826 potentially targeting CTNNB1, VEGFC, MEK1 using several target scan algorithms. The expression of miR-1826 was confirmed in bladder cancer tissues and bladder cancer cell lines. Then we performed several functional analyses of miR-1826. Finally 3′UTR luciferase assay and Western blot were performed to look at relationship between miR-1826 and target genes. Results: MiR-1826 expression was significantly lower in bladder cancer tissues and bladder cancer cell lines (J82, T24, TCC-SUP) compared to normal bladder tissues and normal bladder cell lines (SV-HUC-1). The expression of miR-1826 was lower in bladder cancer tissues and inverse correlation of miR-1826 with several clinical parameters (pT, grade) was observed. The functional analyses showed that miR-1826 inhibited bladder cancer cell viability, invasion and migration. Also we also found increased apoptosis and G1 cell cycle arrest in miR-1826 transfected bladder cancer cells. 3′UTR luciferase activity and protein expression of these target genes (CTNNB1, MEK1, VEGFC) were significantly down-regulated in miR-1826 transfected bladder cancer cells (J82, T24). To examine whether the effect of miR-1826 was through CTNNB1 (beta-catenin) or MEK1 knock down, we knocked down CTNNB1/MEK1 mRNA using a si-RNA technique. We observed that CTNNB1 or MEK1 siRNA knockdown resulted in effects similar to those with miR-1826 in bladder cancer cells. Conclusions: Our data suggests that the miR-1826 plays an important role as tumor suppressor via CTNNB1/MEK1/VEGFC down-regulation in bladder cancer. 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 2293. doi:1538-7445.AM2012-2293

MiR‐210: an important player in the pathogenesis of preeclampsia?

MiR‐210: an important player in the pathogenesis of preeclampsia?

MiR‐199b‐5p directly targets PODXL and DDR1 and decreased levels of miR‐199b‐5p correlate with elevated expressions of PODXL and DDR1 in acute myeloid leukemia

Aberrant expression of Podocalyxin (PODXL), a CD34 orthologue, has been associated with acute myeloid leukemia (AML). Herein, via tissue microarray, we discovered elevated PODXL expression in M2, M4 and M1 FAB-subtype patients. Importantly, various investigations have linked aberrant miRNA expression with AML (1). A miRNA prediction algorithm identified PODXL as a conserved target for miR-199b, a significantly down-regulated miRNA in AML. Further prediction of miR-199b-5p targets identified Discoidin domain receptor 1 (DDR1) as another highly conserved target. For the first time, IHC analyses showed that DDR1 levels were also highly up-regulated in AML and more significantly, were elevated in the same AML cases where PODXL levels were increased. Experimental validation (via-mimics) confirmed that both PODXL and DDR1 are targets of miR-199b-5p. Furthermore, 3’UTR-luciferase assays established that miR-199b-5p targets PODXL and DDR1. Most importantly, we found significant decrease in miR-199b-5p levels in most AML patients with elevated PODXL and DDR1 expressions. Importantly, overexpression of miR-199b-5p in K562 cells caused significant decrease in collagen IV induced migration. Taken together, our studies have identified concurrent increased expression of PODXL and DDR1 in AML and directly connect decreased miR-199b-5p to these novel targets and potential antigomir-mediated therapeutic implications in AML.

Abstract A9: Androgen-regulated miR-32 targets BTG2 and is overexpressed in castration-resistant prostate cancer

Abstract The androgen receptor (AR) signaling pathway is central to the emergence of castration-resistant prostate cancer (CRPC). We set out to identify androgen-regulated microRNAs (miRNAs) that may contribute to the development of CRPC. We found miR-32 to be an androgen regulated miRNA which is differentially expressed in CRPC compared to benign prostate hyperplasia (BPH) according to microarray analyses. qRT-PCR analyses confirmed that miR-32 expression increases in CRPC compared to BPH, and that in untreated prostate cancer (PC) miR-32 expression is higher in tumors of stage pT3 compared to pT2. Transfection of pre-miR-32 to prostate cancer cell line LNCaP provided a significant growth advantage to the cells, and miR-32 was further demonstrated to reduce apoptosis. To identify target genes for miR-32, an mRNA microarray analysis was performed with LNCaP cells transfected with pre-miR-32. In this assay, B cell translocation gene 2 (BTG2) was identified as a novel putative target for miR-32. BTG2 was reduced both at mRNA and protein level in the cells transfected with pre-miR-32, and BTG2 was confirmed to be a miR-32 target by 3′-UTR-luciferase assay. Furthermore, BTG2 was able to partially rescue the growth advantage of miR-32 in LNCaP cells. Immunostainings of prostate cancer patient material showed a statistically significant (p &amp;lt; 0.0001) reduction of BTG2 protein in CRPCs compared to PC. The lack of BTG2 staining was also associated (p=0.0021) with a short progression-free time in patients who underwent prostatectomy and inversely correlated to miR-32 expression levels. In conclusion, we find that miR-32 is an androgen-regulated miRNA targeting BTG2 and overexpressed in CRPC. Thus, miR-32 is a potential marker for aggressive disease and is a putative drug target in PC. Citation Format: Sanni E. Jalava, Leena Latonen, Mauro Scaravilli, Kati K. Waltering, Janne Seppälä, Harri Lähdesmäki, Teuvo L.J. Tammela, Tapio Visakorpi. Androgen-regulated miR-32 targets BTG2 and is overexpressed in castrationresistant prostate cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr A9.