Role Of miR-125b Research Articles

Role of miR-125b and miR-203 expressions in the pathogenesis of BCR-ABL+ Acute Lymphoblastic Leukemia (ALL)

BackgroundThe BCR-ABL+ Acute Lymphoblastic Leukemia (ALL) has been implicated with poor prognosis and drug resistance due to aberrant miRNA expression. Recent evidences also indicate that dysregulated miRNAs interfere with the normal epigenetic and genetic pathways to cause leukemogenesis in BCR-ABL+ ALL. Thereby, the current study aims at investigating the dysregulated miRNAs and their role in pathogenesis of BCR-ABL+ ALL. MethodsThe ALL cases were diagnosed by cytochemistry, flow cytometry and the BCR-ABL+ translocation was determined using Qualitative Multiplex RT-PCR. The miRNA and mRNA expression levels in BCR-ABL positive cases were studied using Quantitative RT–PCR analysis. ResultsOne hundred and twenty cases were diagnosed for Acute Leukemia (AL). Among them, 85 cases were positive for ALL. The B-ALL precursor markers CD19, CD10 and CD22 were predominantly present in 70 cases, with twelve cases of ALL being positive for BCR-ABL+ translocation. By qRT-PCR analysis in BCR-ABL+ ALL cases, we identified that miR-203 was downregulated and miR-125b was upregulated with decreased p53 expression and increased survivin expression. ConclusionHence, the present work suggests a molecular based relationship between miRNA expression and leukemogenesis in BCR-ABL+ ALL.

Abstract 3393: miR-34b: A regulator of VEGF-A, Notch1 and Bcl-2 in thyroid carcinoma

Abstract Background: Thyroid cancer is the most common endocrine malignancy accounting for >91% of endocrine malignancy and 1.8% of all recently distinguished cancer reports, with an incidence continuing to rise globally in recent decades and current treatment strategies are not potent enough for patients. Therefore, novel and more effective treatment are outstandingly required. New strategy for treatment is target therapy, which not only hold cancer-specific expression but also limits side effects. miR-34b as part of p53 tumor suppressor network, plays crucial role in many physiological and pathological processes including cancer initiation, tumor progression and cancer angiogenesis process. Objectives: In this study, we evaluate the functional roles of miR-34b in 2 thyroid cancer cell lines for its potential in modulating angiogenesis and proliferation in thyroid malignancies. Methods: Expression levels of miR-34b was determined in metastasizing human papillary thyroid carcinoma (B-CPAP) and human undifferentiated thyroid carcinomas (MB-1) cell lines by qPCR. Exogenous miR-34b was transfected to thyroid cancer cell lines to investigate its effect on predominant genes involved in angiogenesis, cell cycle and apoptosis regulation including VEGF-A and Bcl-2 and Notch1. Confocal laser scanning microscopy (CLSM) and western blot techniques were performed to illustrate the protein expression changes. To demonstrate the perturbation of cell cycle and apoptosis pathways through exogenous miR-34b, fluorescence-activated cell sorting (FACS) was performed. Results: Significant underexpression of miR-34b was established in B-CPAP and MB-1 cell lines while outstanding overexpression of VEGF-A, Bcl-2 and Notch1 were detected. After transfection with miR-34b, significant drop in concentration of VEGF-A, Bcl-2 and Notch1 were noticed compared with controls in CLSM and western blot analysis (p< 0.05). Cell cycle analysis demonstrated that 48 hours after ectopic induction of miR-34b, significant impairment in proliferation of B-CPAP and MB-1 was induced through arresting cancer cell proliferation in G0-G1 phase (14.27% ±3.50 for B-CPAP and 13.61% ±0.16 for MB-1) (p<0.05). Apoptosis assay also revealed that miR-34b, induced cell death by increasing early and late apoptosis events, compared with controls (2.20% ±0.16 for B-CPAP and 7.87% ±0.92 for MB-1) (p<0.05). Conclusion: These results pave the way that miR-34b may be involved in balancing tumour angiogenesis, cell proliferation and apoptosis in thyroid cancer. These could potentially occur via direct modulation of downstream targets such as VEGF-A, Bcl-2 and Notch1 for their role in those events. The future of this research will investigate the regulatory role of miR-34b in Papillary and undifferentiated thyroid carcinoma through larger scale in vivo and clinical trial studies which shall potentially improve the clinical presentation of aggressive malignancies. Citation Format: Hamidreza Maroof, Ali Salajegheh, Alfred KY Lam. miR-34b: A regulator of VEGF-A, Notch1 and Bcl-2 in thyroid carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3393.

Abstract 43: MicroRNA-130b mediates a metabolic switch to promote cutaneous squamous cell carcinoma development

Abstract Metabolic reprogramming has been emerging as a hallmark of cancer. Cutaneous squamous cell carcinoma (cSCC) is the second most common skin cancer, with about 700,000 new cases annually in the U.S. We aim to investigate the role of miR-130b in reprogramming metabolism as a driver of cSCC development. Since skin cancers have the highest mutational loads among any human cancers, we used well-controlled comparisons across tissues and across species to narrow down the number of important candidate drivers. Using the approach of matched isogenic human samples and cross-species analysis using our UV-driven hairless mouse model, we identified aberrantly expressed microRNAs (miRNAs) and their target mRNAs in order to unravel additional mechanisms behind cSCC progression. We focus on miRNAs because they regulate numerous mRNA targets and can be manipulated for cancer therapy. We observed that miR-130b is increased in both human and mouse cSCC by 3.3-fold, and 2.6-fold, respectively. In adipocytes, miR-130b is known to reduce fat deposition and cell differentiation through targeting PPARγ. Our data and the TargetScan algorithm suggested that in keratinocytes, miR-130b suppresses PPARγ, FBP1, PGC-1α and PDK4. These genes are down-regulated in SCC tumors (compared to normal skin). Also, they are regulators of glycolysis, mitochondrial activity and lipid biosynthesis. Specifically, PPARγ promotes lipid biosynthesis; PGC-1α and PDK4 control mitochondrial activity; FBP1 suppresses glycolysis. Thus, we hypothesize that the cSCC metabolic phenotype is driven mostly by glycolysis and that miR-130b is a regulator of this metabolic switch. We found that in cSCC cell lines, proliferation is reduced by up to 30% when miR-130b was inhibited. Also in the same setting, less glucose was consumed and less lactate was produced, suggesting that miR-130b promotes glycolysis. Realtime-PCR shows that miR-130b levels are up-regulated in SCC tumors and in SCC cell lines (compared to normal skin and primary keratinocytes, respectively). On the other hand, PPARγ and PGC-1α gene expression decreases during SCC development. To study the long-term effects of miR-130b depletion, we have also developed an inducible CRISPRi system that suppresses microRNA expression. Finally, rosiglitazone, a PPARγ agonist, suppresses the proliferation of several SCC cell lines and is under several trials for breast cancer and liposarcoma, thus suggesting that this pathway may be targetable in SCC as well. Our current studies are focused on the precise roles of PPARγ transcriptional targets, which may include FBH1, PGC-1α and PDK4 in cSCC. Citation Format: Tran N. Nguyen, Sydney Moyer, Charles H. Adelmann, Vida Chitsazzadeh, Kenneth Y. Tsai. MicroRNA-130b mediates a metabolic switch to promote cutaneous squamous cell carcinoma development. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 43.

MiR-125b Functions as a Tumor Suppressor and Enhances Chemosensitivity to Cisplatin in Osteosarcoma.

MicroRNAs are highly conserved noncoding RNA that negatively modulate protein expression at a posttranscriptional and/or translational level and are deeply involved in the pathogenesis of several types of cancers. To date, the potential microRNAs regulating the growth and progression of osteosarcoma are not fully identified yet. Previous reports have shown differentially expressed miR-125b in osteosarcoma. However, the role of miR-125b in human osteosarcoma has not been totally illuminated. In this study, we have shown that miR-125b was downregulated in human osteosarcoma tissues compared to the adjacent tissues and effects as a tumor suppressor in vitro We found that stable overexpression of miR-125b in osteosarcoma cell lines U2OS and MG-63 inhibited cell proliferation, migration, and invasion. Our data also verified that Bcl-2 is the target of miR-125b. Meanwhile, we showed that Bcl-2 was inversely correlated with miR-125b in osteosarcoma tissues. More importantly, we proved that miR-125b increased the chemosensitivity of osteosarcoma cell lines to cisplatin by targeting Bcl-2. In conclusion, our data demonstrate that miR-125b is a tumor suppressor and support its potential application for the treatment of osteosarcoma in the future.

Abstract 616: MicroRNA miR-29b is a Mediator of Aortic Stiffness and Hypertension in a Murine Model of Type 2 Diabetes Mellitus

Background: Accelerated arterial stiffening is a complication of diabetes mellitus and associated with the development of hypertension. Arterial stiffening results from extensive extracellular matrix remodeling (elastin breakdown, collagen accumulation). MicroRNA miR-29b directly regulates the expression of genes governing fibrosis (such as COL1A1, COL3A1) and elastin breakdown ( MMP2, MMP9 ). However, its impact on aortic stiffness is unclear. Objective: This study was designed to investigate the role of miR-29b as potential mediator of diabetic aortic stiffening. Methods and Results: Serial ex vivo mechanical testing of the thoracic aorta and volume-pressure recording (VPR) based tail-cuff blood pressure measurements revealed that aortic stiffening precedes blood (pulse) pressure elevations in diabetic db/db mice. Vascular stiffening was accompanied by increased elastin fragmentation and collagen deposition (EvG and Picrosirius Red staining). qRT-PCR, in-situ hybridization and immunohistochemistry revealed decreased expression of miR-29b and de-repression of target genes ( Col1A1, COL3A1, MMP2, MMP9 ) in db/db mice compared to controls. Investigating the mechanistic significance of miR-29b for arterial stiffening, forced downregulation of miR-29b (via systemic LNA-miR-29b inhibitor application) results in enhanced elastin fragmentation, increased medial collagen deposition, aortic stiffness and augmented pulse pressure. Conclusions: In conclusion this study identifies miR-29b as a regulator and potential therapeutic target of diabetic aortic stiffening.

MicroRNA-130b promotes cell proliferation and invasion by inhibiting peroxisome proliferator-activated receptor-γ in human glioma cells.

MicroRNA-130b (miR-130b) is a novel tumor-related miRNA that has been found to be involved in several biological processes. However, there is limited evidence regarding the role of miR-130b in the tumorigenesis of human gliomas. In the present study, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assays were used to quantify miR-130b expression levels in human glioma tissues and glioma cell lines (U251, U87, SNB19 and LN229). The expression level of miR-130b was found to be markedly higher in human glioma tissues than in non-neoplastic brain specimens. Specifically, higher expression levels of miR-130b were observed in the glioma cell lines, compared with those in normal human astrocytes (NHA). We also confirmed that miR-130b interacted with the 3′-untranslated region of peroxisome proliferator-activated receptor-γ (PPAR-γ), which negatively affected the protein levels of E-cadherin. Furthermore, its effects on cell proliferation and invasion were examined using CCK8, colony formation, cell cycle and Transwell assays. We found that the upregulation of miR-130b induced cell proliferation, decreased the percentage of cells in the G0/G1 phase and enhanced the invasiveness of U251 glioma cells whereas the downregulation of miR-130b exerted opposing effects. Moreover, it was demonstrated that the downregulation of miR-130b in U251 glioma cells restored the expression of PPAR-γ and E-cadherin, and inhibited the expression of β-catenin. Notably, PPAR-γ knockdown abolished the inhibitory effect of miR-130b inhibitor on the proliferation and invasivness of U251 cells. Taken together, these findings suggest that miR-130b promotes the proliferation and invasion of U251 glioma cells by inhibiting PPAR-γ.

MiR-133b ameliorates axon degeneration induced by MPP+ via targeting RhoA

Increasing evidence suggests that microRNAs (miRs) play a significant role in the pathogenesis of Parkinson’s disease (PD). MiR-133b, which is significantly decreased in the PD midbrain, has recently been shown to promote neurite outgrowth and enhance neural functional recovery. However, the role of miR-133b in PD has not been clearly established. Here, using a well-established PD model culture based on the neurotoxin 1-methyl-4-phenyl-pyridinium (MPP+), we demonstrated that miR-133b could promote axon outgrowth in dopaminergic neurons (DNs) and ameliorated MPP+-induced axon degeneration. Additional experiments suggested that the mechanisms of this miR-133b-mediated effect might rely on RhoA inhibition. We demonstrated that RhoA, an inhibitor of axonal growth, was increased in DNs under MPP+ treatment, and this increase could be attenuated by miR-133b overexpression. Moreover, we demonstrated that the induced expression of miR-133b could inhibit α-synuclein, which is critically involved in the pathological process of PD. Furthermore, we found that overexpression of miR-133b abrogated the MPP+-induced decrease in the Bcl-2/Bax ratio and upregulated phosphorylated Akt (p-Akt), which is a pro-survival kinase. Together these findings reveal novel roles for miR-133b in the pathogenesis of PD and provide new therapeutic avenues for the treatment of the disease.

MiR-26b inhibits autophagy by targeting ULK2 in prostate cancer cells.

Autophagy is a catabolic process widely conserved among eukaryotes that permits the rapid degradation of unwanted proteins and organelles through the lysosomal pathway. The Serine/threonine protein kinase ULK2 (unc-51 like kinase 2) plays an important regulatory role in autophagy thanks to its involvement in mTOR-regulated-initiation and downstream ATG protein-related progression of this catabolic process. An increasing number of miRNAs have been found to modulate autophagy by targeting some ATG genes. In this study, we focus on the role of mir-26b in autophagy in prostate cancer (PCa) cells. We found that miR-26b inhibited autophagy in PC-3 and C4-2 cells, through down-regulation of ULK2 expression. Dual luciferase reporter assays showed that miR-26b binds the 3'UTR of ULK2, suggesting that ULK2 is a direct target of miR-26b. Real-time PCR and Western blot analysis confirmed that over-expression of miR-26b reduced ULK2 mRNA and protein levels. Our results showed also that miR-26b was down-regulated in LNCaP, DU145, C4-2 and PC-3 cells compared to the two normal prostate cells RWPE-1 and WPMY-1 except DU145 cells. This inversely correlates with ULK2 level in the same cell lines. Expression level of ULK2 in tissues microarray (TMA) of prostate cancer derived from 96 patients positively correlated with the pathologic stage of the patients (∗P < 0.05). Over-expression of ULK2 significantly reversed miR-26b-mediated autophagy inhibition. Taken together, our findings indicate that mir-26b inhibits autophagy through targeting ULK2 which is up-regulated in PCa.

MicroRNA-130b promotes proliferation and EMT-induced metastasis via PTEN/p-AKT/HIF-1α signaling.

Hepatocellular carcinoma (HCC) is a major cause of cancer-related deaths owing to its high rate of postoperative recurrence and metastasis. New research is continuously identifying novel metastasis-associated oncogenes and tumor suppressor genes. miRNAs are noncoding RNAs that regulate protein synthesis post-translationally. miR-130b is one of several miRNAs involved in tumor metastasis. However, the role of miR-130b in HCC remains controversial. Here, we demonstrate that miR-130b is highly expressed in HCC and that it correlates with tumor number, vascular invasion, and TNM stage-important predictors of postoperative recurrence and metastases. Moreover, high levels of miR-130b predicted poor overall and disease-free survival of HCC patients, and in vitro and in vivo research revealed that knockdown or overexpression of miR-130b inhibited and promoted proliferation and metastasis of HCC cells, respectively. We identified PTEN as a direct functional target of miR-130b using miRNA databases and a dual luciferase report assay. Next, using a gain and loss assay and epithelial-mesenchymal transition (EMT) relative assays, we show that miR-130b may promote proliferation and EMT-induced metastasis via PTEN/p-AKT/HIF-1α signaling. Collectively, our data suggests that miR-130b may have prognostic value in HCC. Additionally, the miR-130b/PTEN/p-AKT/HIF-1α axis identified in this study provides novel insight into the mechanisms of HCC metastasis, which may facilitate the development of new therapeutics against HCC.

MicroRNA-106b is involved in transforming growth factor β1-induced cell migration by targeting disabled homolog 2 in cervical carcinoma.

BackgroundMicroRNA-106b (miR-106b) was recently identified as an oncogene participating in cancer progression. Transforming growth factor β1(TGF-β1) is an indispensable cytokine regulating the local microenvironment, thereby promoting cervical cancer progression. However, the roles of miR-106b in cervical carcinoma progression and TGF-β1-involvement in the tumorigenesis of cervical cancer remain unknown.MethodsThe expression of miR-106b in human cervical specimens was detected by real-time PCR analysis and in situ hybridization assay. The effect of miR-106b on cell migration was analyzed by scratch and transwell assays. TGF-β1 was used to induce cell migration. The expression of the miR-106b target gene DAB2 in human cervical tissues and cell lines were measured by qRT-PCR, western blot and immunohistochemistry. Dual-luciferase reporter assay was used to identify DAB2 as a miR-106b-directed target gene.ResultsmiR-106b was frequently up-regulated in human cervical carcinoma specimens and cervical cancer cell lines. Over-expression of miR-106b significantly promoted HeLa and SiHa cells migration. Likewise, inhibition of miR-106b decreased HeLa and SiHa cells migration. The multifunctional cytokine TGF-β facilitates metastasis in cervical carcinoma. miR-106b inhibitor treatment decreased the TGF-β1-stimulated migration of HeLa and SiHa cells. DAB2, a predicted target gene of miR-106b, was inhibited by TGF-β1 partly through miR-106b and was involved in TGF-β1-induced cervical cancer cell migration. The expression of DAB2 was low in cervical cancer tissues, and negatively correlated with miR-106b expression. Finally, DAB2 was identified as a miR-106b-directed target gene by dual-luciferase reporter assay.ConclusionOur data suggest that the TGF-β1/miR-106b/DAB2 axis may be involved in the pathogenesis of cervical carcinoma.

Abstract C17: Role of miR-135b in gemcitabine sensitivity for metastatic breast cancer patients

Abstract Background: MicroRNAs are a class of small non-coding RNAs that modulate gene expression at post-transcriptional level, resulting crucial in many physiological and pathological processes. Their involvement in cancer initiation and tumor progression has been clearly established, as well as their linkage with chemoresistance. Gemcitabine is a nucleoside analog used in the treatment of metastatic breast cancer (MBC), characterized by a favorable safety profile. Only a small percentage of pts show a strong and prolonged response to this drug, thus suggesting the need of predictive biomarkers. Here, we aimed to identify microRNAs whose expression could be related to gemcitabine sensitivity in MBC. Methods: 24 MBC pts were treated with gemcitabine as single-agent therapy from 1999 to 2011 at Fondazione IRCCS Istituto Nazionale Tumori (Milan). They were selected and classified by the response to the treatment (11 gemcitabine sensitive, stable or partial response after &amp;gt;24 weeks of treatment, and 13 gemcitabine resistant, progressive disease within 16 weeks from the first drug administration). MicroRNA expression profiling was performed by Nanostring nCounter Technology using RNA from FFPE metastatic lesions. Biological normalization was executed to correct for differences in sample abundances. Each sample was normalized to the geometric mean of the top 100 most highly expressed microRNAs. Student's t test was used to calculate statistical significances of pairwise comparisons. The nCounter analysis was validated on a wider population of 50 MBC pts (17 sensitive and 33 resistant) by qRT-PCR. The correlation between microRNA expression levels and gemcitabine half maximal inhibitory concentration (IC50) was evaluated using different BC cell lines. Finally, gemcitabine sensitivity was assessed on these cell lines by cell survival assays following exogenous modulation of microRNA expression levels. Results: Median age at diagnosis was 47 yrs (range 21-74 yrs). Hormone receptors were positive in thirty-one pts (62%), while Her2 in 15 cases (30%). Soft tissues were the most frequent site of metastasis (84%), followed by bone (64%), liver (36%), lung (34%) and CNS (16%). Sixteen pts (32%) had local recurrences. Median number of chemotherapies and endocrine therapies received prior to gemcitabine was 3 (range 1-6) and 2 (range 1-5) respectively. From the nCounter analysis, a subset of 20 microRNAs was significantly deregulated (p&amp;lt;0.001) between sensitive and resistant pts. Among them, the three most relevant (miR-135b, miR-146b and miR-155) were selected for further investigation. RT-PCR validation confirmed microRNA expression profiling data on the wider cohort of 50 pts (p&amp;lt;0.05). In vitro studies confirmed the correlation between miR-135b levels and gemcitabine sensitivity in different BC cell lines. Moreover, the exogenous upregulation of this microRNA increased the response to the drug. Conclusions: We identified miR-135b, miR-146b and miR-155 as potentially predictive for gemcitabine sensitivity in MBC pts. In particular, miR-135b appears to be causally involved in BC cells response to gemcitabine. These results pave the way to a better understanding of the molecular mechanisms underlying gemcitabine resistance in BC, and may have a clinical impact to identify those patients expected to obtain strong and prolonged benefits from this well-tolerated treatment. Citation Format: Anna Tessari, Dario Palmieri, Giovanni Nigita, Dario Veneziano, Sara Cresta, Biagio Paolini, Maria Silvia Cona, Taylor Vargo, Erika Reese, Tyler Sheetz, Vincenzo Coppola, Filippo De Braud, Carlo M. Croce. Role of miR-135b in gemcitabine sensitivity for metastatic breast cancer patients. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C17.

Different expression of miR-29b and VEGFA in glioma

Glioma is one of the most common carcinomas in terms of both incidence and mortality worldwide. This is a case–control study with 240 cases and age- and gender-matched controls in the rate of 1:1. The results of this present word indicated that the expressions of both miR-29b and VEGFA in blood were significantly different compared with the control group, and thus may help to differentiate glioma cases from the controls. In addition, the diagnostic role of miR-29b and VEGFA was important for the clinical application. In conclusion, circulating miR-29b and VEGFA could be used as diagnosis biomarkers.

CBIO-27TUMOR SUPPRESSION EFFECT OF mir-29b IN GLIOBLASTOMA

Glioblastoma (GBM) is known as one of the most fatal forms of cancer. MicroRNAs have been widely implicated in the regulation of mammalian development and its pathogenesis. The brain-enriched miR-29 subfamilies are known to be exclusively expressed in the developing brain, while, they are aberrantly down-regulated in brain tumors. Besides, the mechanisms by which miR-29 mediated signal components in brain neoplasms remain undetermined. This study aims to elucidate the role of miR-29b in GBM development and the feasibility of therapeutic target using conjugated nanoparticles. We have discovered that increase in apoptotic cell populations with introduction of miR-29b in A172, a PTEN-deficient GBM cell line. An established human-derived GBM tissue slice culture system confirmed the anticancer effect of the miR-29b-conjugated-nanoparticle. Using PCR array, we found that exogenous miR-29b inhibit SPARC (secreted protein acidic and rich in cysteine) expression which mediates anticancer effect. The suppression of miR-29b regulates SPARC and contributes tumor development in GBM. Introduction of miR-29b using nanoparticle-delivery system can be considered as putative therapeutic strategy in GBM.

Role of miR-10b in non-small cell lung cancer (NSCLC) cells by targeting Klotho

MiR-10b was overexpressed in various cancers, including NSCLC. In early study, miR-10b was described as a carcinoma enhancer in NSCLC. Our recent study showed that silencing of miR-10b could promote NSCLC cells apoptosis and decrease proliferation, and regulation of these cell behaviors might target Klotho via miR-10b. In addition, we also explored the correlation between miR-10b and Klotho or other underlying targets. Our investigation indicated that miR-10b could play a critical role as a helpful prognosis marker and underlying target in terms of therapy of NSCLC.

Abstract 3060: The role of miR-135b in clinical outcome and metastasis in colorectal cancer

Abstract Colorectal cancer (CRC) is one of the leading causes of cancer death in both the Western world and Taiwan. Age, gender, and genes are important risk factors for colorectal cancer. Activation of Wnt signaling pathway is an important step involved in the pathogenesis of colorectal cancer. Approximately 70-80% of sporadic colorectal carcinomas have somatic mutations that inactivate APC. But, only 30% CRC patients in Taiwan have APC gene mutation that was significantly lower than the other countries. Thus, the other mechanism involved in APC gene inactivation may contributed to CRC progression in Taiwan. Here, we focus on the Wnt signaling associate microRNAs. Four wnt signaling-associated microRNAs were selected in this study, including miR-21, miR-200a, miR-135a and miR-135b. A total of 156 colorectal cancer patients were enrolled in this study. The expression of miR-21, miR-200a, miR-135a, miR-135b and wnt signaling association genes were analyzed by quantitative RT-PCR and immunohistochemistry. The expression levels of miR-21, miR-200a and miR-135a were not correlated with clinical parameters of CRC patients. The expression of miR-135b were positive correlated with gender, tumor stage, and lymph node metastasis. No any significantly correlation were found between miR-135b, Wnt, β-catenin and APC. But the expression levels of miR-135b were positive correlated with the upstream gene STAT3, β-catenin and C-Myc. Thus, we suggested that upregulation of miR-135b may through β-catenin mediated STAT3 activation. Citation Format: Wei-Ran Wang, Yawen Cheng, Nan-Yung Hsu Hsu. The role of miR-135b in clinical outcome and metastasis in colorectal cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3060. doi:10.1158/1538-7445.AM2015-3060

Abstract 179: Regulation of stromal miR-125b on normal colonic epithelial cell renewal and its putative role in tumorigenesis

Abstract MicroRNAs (miRNAs) are small non-coding RNAs which exert their effects by post-transcriptionally silencing target mRNAs. Deregulation of miRNA expression is a frequent event in tumorigenesis. MiR-125b is a highly conserved miRNA among various species and is composed of three homologs: hsa-miR-125a, hsa-miR-125b-1 and hsa-miR-125-2. The tumorigenic roles of miR-125b have been studied in various cancers including prostate, colon, glioma etc, and studies have demonstrated that it can act as a tumor suppressor or an oncogene depending on the cellular context. It was characterized as an oncogene in prostate cancer and glioma through down-regulation of pro-apoptotic regulators BAK1 and Bcl-2 modifying factor (BMF), respectively. In colon cancer, a recent clinico-pathological study showed that high expression of miR-125b was associated with tumor invasion and poor prognosis. However, the localization of miR125b in normal colon and tumors is currently unknown. Therefore, we aimed to address the precise expression and the functional role of miR-125b in normal colon, which may provide insight on its potential oncogenic effects during carcinogenesis. We performed gene expression analysis of miR-125b in normal colon tissues from 16 pairs of colon top versus basal crypts and 4 pairs of crypts versus stroma by real-time RT-PCR. Colon top and basal crypts were microdissected from frozen sections, whereas pure normal crypts and stromal fractions were isolated from freshly resected human colon specimens. We found that miR-125b was significantly enriched in the basal crypts (p&amp;lt;0.001) and the stromal compartment (p = 0.027). Functionally, knockdown of miR-125b by a specific miRCURY antisense oligonucleotide was able to dose dependently inhibit the growth of a normal colon myofibroblast cell line, CCD18, and significantly increase the mRNA level of several key Hedgehog signaling components, including PTCH (p&amp;lt;0.001), SMO (p&amp;lt;0.001), GLI1 (p&amp;lt;0.001) and its downstream target BMP4 (p = 0.017). Finally, we observed a drastic decrease in both the number and size of normal colon organoids when they were co-cultured with myofibroblasts transfected with miR-125b antisense oligonucleotide, as compared with negative control. Our results suggest that expression of miR-125b in the colonic stromal basal crypt compartment functions to inhibit Hedgehog signaling, leading to paracrine enhancement of colon crypt proliferation and stem cell renewal. Thus, given the well-known importance of hedgehog signaling in colon cancer, our findings suggest that dysregulated miR-125b expression may contribute to carcinogenesis through disruption of this pathway. Note: This abstract was not presented at the meeting. Citation Format: Helen H N Yan, Jackie K Y Lau, Annie S Y Chan, Wai Yin Tsui, Tsun Leung Chan, Suet Yi Leung. Regulation of stromal miR-125b on normal colonic epithelial cell renewal and its putative role in tumorigenesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 179. doi:10.1158/1538-7445.AM2015-179

Abstract 3977: Evaluation of miR10b and miR9 expression in breast cancer and correlations with distant metastases development

Abstract We have recently demonstrated that the evaluation of miR10b expression relative to its normal counterparts (Relative Expression Ratio, RER) is able to predict patients risk to develop distant metastases. To confirm these results and evaluate whether other microRNAs could be predictive of outcome in breast cancer we evaluated the expression of miR10b and miR9 in paired tumors and apparently normal breast tissues 2 cm distant from tumour (NBT) on an extended cohort (n = 127) with a longer follow up (median 42 months). As control normal breast tissues from 10 healthy subjects (HBT) treated by reductive mammoplasty were analyzed. An increased expression was seen for the miR9 and miR10b in NBT samples as compared with HBT (P&amp;lt;0.01). Whereas the expression of each of the two miRNAs was significantly lower in tumour samples as compared to the NBT specimens with RER value of 0.15 for miR10b (P = 0.001) and 0.59 for miR9 (P = 0.05). However only higher miR10b RERs were associated with distant metastases development in multivariable analysis (HR = 1.12; P = 0.04). Our results confirm the role of miR10b as predictive biomarkers of metastases development in breast cancer. In addition our data suggest a deregulation of miRNAs expression in the normal tissue surrounding tumor as compared to normal breast of healthy individuals. Citation Format: Raffaela Barbano, Barbara Pasculli, Massimiliano Copetti, Andrea Fontana, Michelina Coco, Lorenzo Vitulano, Maria Luana Poeta, Francesca Picardo, Gianfranco Marangi, Roberto Murgo, Luigi Ciuffreda, Vanna Maria Valori, Vanna Maria Valori, Maria Morritti, Evaristo Maiello, Vito Michele Fazio, Paola Parrella. Evaluation of miR10b and miR9 expression in breast cancer and correlations with distant metastases development. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3977. doi:10.1158/1538-7445.AM2015-3977

Abstract 189: KRAS-induced microRNA-29b attenuates apoptosis in non-small cell lung cancer

Abstract EGFR tyrosine kinase or downstream components of this pathway, e.g. KRAS, are frequently mutated in NSCLC patients. These mutations enable a constitutive active downstream signaling pathway and result in lung cancer development by uncontrolled proliferation and survival. MicroRNAs (miRNAs) are noncoding short nucleic acids, and are implicated in NSCLC progression. We aim to identify miRNAs that are regulated by EGFR downstream key molecules and to investigate their role in malignant processes of NSCLC. We demonstrate that viral transduction of bronchial epithelial cells (BEAS-2B) with mutated KRAS induces an activated downstream signaling pathway. Subsequently, a miRNA array profiling of KRAS-transduced cells identified upregulated miR-29b expression. We confirmed the upregulation of miR-29b expression in individual clones of KRAS-transduced cells. Moreover, the upregulated miR-29b expression was affirmed in NSCLC tissue harboring KRAS mutations by comparing it to the expression in matched normal tissue. Furthermore, to elucidate the downstream signaling pathway involved in the underlying regulation of miR-29b expression, NSCLC cell lines were treated with EGFR and MEK inhibitors which lead to downregulated miR-29b expression. Next, to study the role of miR-29b in NSCLC apoptosis, we overexpressed miR-29b in adenocarcinoma cells (A549) and performed cell death assays. Interestingly, and in contrast to other cell systems, A549 cells transfected with precursor miR-29b demonstrated attenuated TNF-α plus actinomycin D-induced apoptosis compared to control-transfected cells. Conversely, the downregulation of endogenous miR-29b levels by overexpressing antagomiR-29b, lead to increased sensitivity to apoptosis compared to scramble-transduced A549 cells. Consistently, KRASG12V-transduced BEAS-2B cells showed reduced sensitivity to TNF-α plus actinomycin D-induced apoptosis, while the effect was reversed in KRAS/antagomiR-29b co-transduced cells, indicating that KRAS attenuates apoptosis by overexpressing miR-29b. Furthermore, miR-29b is predicted to target the pro-apoptotic BH3-only protein Bim and the tumor necrosis alpha-induced protein 3 (TNFAIP3/A20), an important negative regulator of the NFκB signaling pathway and thereby may confer resistance to apoptosis. We plan to verify the predicted regulation of these genes by performing luciferase reporter assays and Western blot analysis. In addition, NFκB activation will be assessed by verifying nuclear and cytoplasmic fractions of cells transfected with precursor miR-29b. Finally, we plan to stably overexpress TNFAIP3 in NSCLC cell lines and assess apoptosis by overexpressing miR-29b. In summary, we provide a novel regulation mechanism of miR-29b by KRAS signaling, leading to attenuated apoptosis in NSCLC. Citation Format: Stephanie Langsch, Stefan Haemmig, Ulrich Baumgartner, Mario P. Tschan, Erik Vassella. KRAS-induced microRNA-29b attenuates apoptosis in non-small cell lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 189. doi:10.1158/1538-7445.AM2015-189

MiRNAs and chemoresistance of pancreatic cancer: the role of miR-125b and miR-100 in gemcitabine resistance

miRNAs and chemoresistance of pancreatic cancer: the role of miR-125b and miR-100 in gemcitabine resistance

Inhibition of Ovarian Epithelial Carcinoma Tumorigenesis and Progression by microRNA 106b Mediated through the RhoC Pathway.

Epithelial ovarian cancer (EOC) is the most lethal of the gynecological malignancies. Exploring the molecular mechanisms and major factors of invasion and metastasis could have great significance for the treatment and prognosis of EOC. Studies have demonstrated that microRNA 106b (miR-106b) may be a promising therapeutic target for inhibiting breast cancer bone metastasis, but the role of miR-106b in EOC is largely unknown. In this work, miRNA-106b expression was quantified in various ovarian tissues and tumors. Ovarian carcinoma cell lines were transfected with miR-106b, after which, cell phenotype and expression of relevant molecules was assayed. Dual-luciferase reporter assays and xenograft mouse models were also used to investigate miR-106b and its target gene. MiR-106b mRNA expression was found to be significantly higher in normal ovarian tissues and benign tumors than in ovarian carcinomas and borderline tumors (p < 0.01), and was negatively associated with differentiation (Well vs. Por & Mod) and the International Federation of Gynecology and Obstetrics (FIGO) staging (stage I/II vs. stage III/IV) in ovarian carcinoma (p < 0.05). MiR-106b transfection reduced cell proliferation; promoted G1 or S arrest and apoptosis (p < 0.05); suppressed cell migration and invasion (p < 0.05); reduced Ras homolog gene family member C (RhoC), P70 ribosomal S6 kinase (P70S6K), Bcl-xL, Matrix metallopeptidase 2 (MMP2), MMP9 mRNA and protein expression; and induced p53 expression (p < 0.05). Dual-luciferase reporter assays indicated that miR-106b directly targets RhoC by binding its 3’UTR. MiR-106b transfection also suppressed tumor development and RhoC expression in vivo in xenograft mouse models. This is the first demonstration that miR-106b may inhibit tumorigenesis and progression of EOC by targeting RhoC. The involvement of miR-106b-mediated RhoC downregulation in EOC aggression may give extended insights into molecular mechanisms underlying cancer aggression. Approaches aimed at overexpressing miR-106b may serve as promising therapeutic strategies for treating EOC patients.