MiR‐221 and MiR‐222 alterations in sporadic ovarian carcinoma: Relationship to CDKN1B,CDKNIC and overall survival

MicroRNAs are often aberrantly expressed in human neoplasms and are postulated to play a role in neoplastic initiation and progression. miR‐221 and miR‐222 negatively regulate expression of CDKN1B (p27) and CDKN1C (p57), two cell cycle regulators expressed in ovarian surface epithelium and down‐regulated in ovarian carcinomas. We characterized miR‐221 and miR‐222 expression in 49 sporadic high grade ovarian carcinomas and determined whether somatic mutation or epigenetic alterations explained the differences in expression of these miRNAs. We correlated these findings with protein expression of CDKN1B and CDKN1C as assessed by immunohistochemistry. Expression of miR‐221 and miR‐222 were closely correlated with each other (P = 0.0001). Interestingly, a lower ratio of miR‐221 to miR‐222 expression was significantly correlated with worse overall survival (P = 0.01) and remained a significant predictor of overall survival in multivariate analysis using the covariate adequacy of surgical cytoreduction (P = 0.03). Higher miR‐222 and miR‐221 expression were significantly associated with decreased CDKN1C expression (P = 0.009 and 0.01). In contrast, CDKN1B expression was not associated with miR‐221 or miR‐222 expression. Neither somatic mutations nor methylation of the studied region explained the alterations in miR‐221 and miR‐222 expression in most carcinomas. © 2010 Wiley‐Liss, Inc.     

miR‐221 redirects precursor B cells to the BM and regulates their residence

Pluripotent hematopoietic stem cells and multipotent myeloid/lymphoid progenitors express miR‐221 and miR‐222. When Pax5 expression commits these progenitors to monopotent pre‐B lymphocytes the two microRNAs (miRNAs) are downregulated. Upon transplantation, stem cells and progenitors can reside in the BM, while pre‐B cells, after their commitment, no longer do so. Retrovirally transduced, doxycycline‐induced overexpression of either miR‐221 or miR‐222 in pre‐B‐I cells does not revert their monopotency to multipotency. However, upon transplantation miR‐221, but not miR‐222, transduced pre‐B‐I cells regain the capacity to reside in the BM. Upon subsequent termination of miR‐221‐expression by removal of doxycycline, the transplanted cells leave the BM again. Microarray analyses identified 25 downregulated miR‐221‐target genes, which could function to localize phases of B‐lymphocyte development in BM before and after commitment.     

Expression of miR‐100 and miR‐138 as prognostic biomarkers in non‐muscle‐invasive bladder cancer

microRNA alterations are involved in bladder cancer tumorigenesis. The aim of the current study was to evaluate the potential role of miR‐100 and miR‐138 as prognostic biomarkers in Ta/T1 non‐muscle‐invasive bladder cancer (NMIBC). We assessed a quantitative RT‐PCR analysis of miR‐100 and miR‐138 in 50 bladder tumor samples (stage Ta/T1) and four healthy adjacent tissues. Western blot analysis was used to measure protein expression of FGFR3 and cyclin D3 in order to know whether these targets can be regulated by miR‐100 and miR‐138, respectively. The statistical analysis included non‐parametric tests (Mann–Whitney U and Kruskal–Wallis) and univariate survival analysis by Kaplan–Meier method and the log‐rank test. Low expression of miR‐138 characterized recurrent tumors (p = 0.043), and higher expression levels were associated with longer recurrence‐free survival (p = 0.012). However, low miR‐100 expression correlated with longer progression‐free survival (marginal significance; p = 0.053) and cancer‐specific overall survival (p = 0.006). Additionally, higher levels of miR‐100 were associated with negative FGFR3 protein expression (p = 0.032) and higher levels of miR‐138 were associated with positive cyclin D3 protein expression (p = 0.037). Our results support miR‐138 and miR‐100 as prognostic biomarkers in patients with NMIBC.     

A five‐miRNA expression signature predicts survival in hepatocellular carcinoma

The aim of this study was to identify a microRNA (miRNA) expression signature for predicting HCC (hepatocellular carcinoma) survival. A total of 322 HCC patients in The Cancer Genome Atlas (TCGA) database were randomly divided into training and testing set. miRNAs, associated with survival time in the training set, were identified by using univariate Cox regression analysis. The risk score was formulated based on the expression levels of these miRNAs. Then the miRNA signature was validated in testing set through Kaplan–Meier analysis and log‐rank test. hsa‐miR‐301a, hsa‐miR‐132, hsa‐miR‐212, hsa‐miR‐489, and hsa‐miR‐1468 were identified to formulate risk score in training set and used to calculate the risk score of each patients in testing set. About 161 patients in testing set were segregated into high‐ and low‐risk group according to the median risk score. The survival time of high‐risk group was significantly shorter (p = 0.0248) than low‐risk group in testing test. The target genes of five miRNAs were significantly enriched in valine, leucine, and isoleucine degradation pathway and PPAR signaling pathway. hsa‐miR‐1468 had an up‐regulated tendency in HCC tissues compared to adjacent tumor tissues. The expression of hsa‐miR‐301a, hsa‐miR‐132, hsa‐miR‐212, hsa‐miR‐489, and hsa‐miR‐1468, which might be potential biomarkers to evaluate HCC patients' prognosis.     

Epigenetic regulation of SMARCB1 By miR‐206, ‐381 and ‐671‐5p is evident in a variety of SMARCB1 immunonegative soft tissue sarcomas,while miR‐765 appears specific for epithelioid sarcoma. A miRNA study of 223 soft tissue sarcomas

Complete/partial loss of SMARCB1 nuclear‐immunopositivity is characteristic of a certain subset of soft tissue sarcomas (STSs). Our previous work showed that oncomiRs‐206,‐381, and 671‐5p could silence the SMARCB1 mRNA and protein expression and that they display significant overexpression in epithelioid sarcomas (ESs). MiR‐765 was overexpressed too, but functionally was inactive in the silencing. In the current work, using quantitative PCR, we conducted a miRNA study of 51 ESs, 20 rhabdoid tumors (RTs), 20 synovial sarcomas (SSs), 15 malignant peripheral nerve sheath tumors (MPNSTs), 11 myoepithelial carcinomas (MECs), and 10 extraskeletal myxoid chondrosarcomas (EMCSs) with complete/partial loss of SMARCB1 nuclear immunostain, in contrast to controls (SMARCB1‐immunopositive) of 96 STSs, 13 melanomas and 10 sarcomatoid carcinomas. The SMARCB1 genetic status of ESs was determined by MLPA and FISH. A subset of ESs (5/51) showed biallelic deletion of SMARCB1 with no overexpression of any miRNA, suggesting these tumors could be the counterpart of pediatric RT, at least genetically. Another subset (5/51) was genetically either intact or monoallelic deleted with at least threefold overexpression of one of miR‐206,‐381,‐671‐5p, suggesting epigenetic regulation only. 39/51 ESs had a biallelic deletion (>20% by FISH and/or by MLPA) but with overexpressed miR‐206,‐381, and 671‐5p, suggesting intratumoral heterogeneity, i.e., both genetic and epigenetic regulation. At least threefold overexpression of one of miR‐206,‐381, and 671‐5p was detected in all MPNSTs, EMCSs, SSs and 7 MCs. Except for ESs, four SSs and one MPNST, there was no event above threefold overexpression of miR‐765 among all 195 tested tumors. Our results suggest a general role of miR‐206,‐381, and 671‐5p in SMARCB1 gene silencing of ES, MC, EMCS, MPNST and SS. In the future, miR‐765 could possibly be a diagnostic tool for ES because of its 97% specificity and 80% sensitivity. © 2016 Wiley Periodicals, Inc.     

Localization‐ and mutation‐dependent microRNA (miRNA) expression signatures in gastrointestinal stromal tumours (GISTs), with a cluster of co‐expressed miRNAs located at 14q32.31

The molecular biology and clinical behaviour of gastrointestinal stromal tumours (GISTs) are associated with their anatomical localization (stomach or intestine), and also with the mutation status of the receptor tyrosine kinases KIT and PDGFRA. Twelve GISTs were evaluated for differential miRNA expression signatures by use of microarrays representing 734 human miRNAs. Thirty‐two miRNAs were found to be differentially expressed according to localization and mutation status. Differential expression was further analysed and confirmed for four miRNAs (miR‐132, miR‐221, miR‐222, and miR‐504) by qRT‐PCR in 49 additional GISTs. Differentially expressed miRNAs were functionally mapped to KIT/PDGFRA signalling and G1/S‐phase transition of the cell cycle, revealing 22 predicted miRNA/mRNA interactions for ten gene targets from KIT/PDGFRA signalling, and 12 interactions for 12 gene targets of G1/S‐phase transition. Moreover, the expression of 44 miRNAs clustered in a genetically imprinted region at 14q32.31 was found to be strongly correlated in the microarray analysis. This was confirmed for two selected miRNAs (miR‐134 and miR‐370) from the 14q32.31 cluster by qRT‐PCR in 49 additional GISTs, and the expression of these two miRNAs was significantly lower in GISTs with 14q loss, and also in GISTs with tumour progress. miRNA profiling may prove to be a key determinant of the biology and clinical features of GISTs Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Prognostic relevance of miR‐124‐3p and its target TP53INP1 in pediatric ependymoma

Ependymoma is a malignant pediatric brain tumor, often incurable under the current treatment regimen. We aimed to evaluate the expression of microRNAs (miRs) in pediatric ependymoma tumors in an attempt to identify prognostic molecular markers which would lead to potential therapeutic targets. Following miR‐array expression analysis, we focused on 9 miRs that correlated with relapse which were further validated by quantitative real‐time PCR (qRT‐PCR) in a cohort of 67 patients. Western blotting and immunohistochemistry were used to measure target protein expression in 20 and 34 tumor samples, respectively. High expression of miR‐124‐3p significantly correlated with the lower progression‐free survival (PFS) of 16% compared to 67% in those expressing low levels (P = .002). Interestingly, in the group of patients with local disease (n = 56) expression levels of this miR distinguished 2 subgroups with a significantly different outcome (P = .001). miR‐124‐3p was identified as an independent prognostic factor of relapse in the multivariate analysis performed in the whole cohort and in the group with localized disease. In the localized group, a patient expressing high levels of miR‐124‐3p had a 4.1‐fold increased risk for relapse (P = .005). We demonstrated the direct binding of miR‐124‐3p to its target TP53INP1. Negative TP53INP1 protein levels correlated with a poor outcome (P = .034). We propose miR‐124‐3p and TP53INP1 as new biomarkers for prognostic stratification that may be possible therapeutic targets for ependymoma.     

Human papillomavirus‐stratified analysis of the prognostic role of miR‐21 in oral cavity and oropharyngeal squamous cell carcinoma

Human papillomavirus (HPV) infection plays a significant role in the development and progression of head and neck squamous cell carcinoma (HNSCC). Expression of miR‐21 has a prognostic role in a wide variety of cancers. The upregulation of miR‐21 suppresses a number of target genes, including phosphatase tensin homologue (PTEN) and programmed cell death 4 (PDCD4). We investigated the association between the expression of miR‐21 and the clinical features of HNSCC using stratified analysis based on HPV infection status. HPV status and miR‐21 expression in HNSCC tissues from 167 patients were evaluated using in situ hybridization. The expression of PDCD4 and PTEN was examined by immunohistochemistry. The up‐regulation of stromal miR‐21 expression occurred in 40.6% of HPV‐negative samples and 28.3% of the HPV‐positive group. In HPV‐stratified multivariate analysis, high miR‐21 expression was associated with poor cancer‐specific survival in HPV‐negative tumors, but not in HPV‐positive tumors. There was a significant association between miR‐21 and cytoplasmic PDCD4 overexpression in HPV‐negative HNSCCs. We suggest that stromal miR‐21 expression is an independent prognostic factor in HPV‐negative tumors and miR‐21 may play different roles depending on HPV infection status.     

SMARCB1 expression in epithelioid sarcoma is regulated by miR‐206, miR‐381, and miR‐671‐5p on Both mRNA and protein levels

Proximal type epithelioid sarcoma shares similarities with malignant rhabdoid tumor, including the lack of nuclear immunoreactivity of SMARCB1. Biallelic mutation of SMARCB1 has been convincingly established as the cause of loss of protein expression in rhabdoid tumor, but the cause in epithelioid sarcoma remains unknown. In our previous work, we demonstrated that DNA hypermethylation and post‐translational modification mechanisms were not involved. In this current work, we explored the hypothesis that miRNAs regulate SMARCB1 gene expression in epithelioid sarcomas. In silico target prediction analysis revealed eight candidate miRNAs, and quantitative PCR—in 32 formalin‐fixed, paraffin‐embedded tumor samples comprising 30 epithelioid sarcomas and two malignant rhabdoid tumors—demonstrated significant (P < 0.001) overexpression of four miRNAs in epithelioid sarcomas: miR‐206, miR‐381, miR‐671‐5p, and miR‐765. Two human tumors (fibrosarcoma and colon adenocarcinoma) and a normal cell line (human dermal fibroblast) with retained SMARCB1 expression were cultured for miRNA transient transfection (electroporation) experiments. SMARCB1 mRNA expression was analyzed by quantitative real‐time PCR and immunostaining of SMARCB1 was performed to examine the effect of miRNAs transfections on both RNA and protein levels. Only three of the overexpressed miRNAs (miR‐206, miR‐381, and miR‐671‐5p) could silence the SMARCB1 mRNA expression in cell cultures; most effectively miR‐206. Transfection of miR‐206, miR‐381, miR‐671‐5p, and some combination of them also eliminated SMARCB1 nuclear staining, demonstrating a strong effect on not only mRNA but also protein levels. Our results suggest loss of SMARCB1 protein expression in epithelioid sarcoma is due to the epigenetic mechanism of gene silencing by oncomiRs. © 2013 Wiley Periodicals, Inc.     

miR expression profiling at diagnosis predicts relapse in pediatric precursor B‐cell acute lymphoblastic leukemia

Our aim was to identify miRNAs that can predict risk of relapse in pediatric patients with acute lymphoblastic leukemia (ALL). Following high‐throughput miRNA expression analysis (48 samples), five miRs were selected for further confirmation performed by real time quantitative PCR on a cohort of precursor B‐cell ALL patients (n = 138). The results were correlated with clinical parameters and outcome. Low expression of miR‐151‐5p, and miR‐451, and high expression of miR‐1290 or a combination of all three predicted inferior relapse free survival (P = 0.007, 0.042, 0.025, and <0.0001, respectively). Cox regression analysis identified aberrant expression of the three miRs as an independent prognostic marker with a 10.5‐fold increased risk of relapse (P = 0.041) in PCR‐MRD non‐high risk patients. Furthermore, following exclusion of patients harboring IKZF1 deletion, the aberrant expression of all three miRs could identify patients with a 24.5‐fold increased risk to relapse (P < 0.0001). The prognostic relevance of the three miRNAs was evaluated in a non‐BFM treated precursor B‐cell ALL cohort (n = 33). A significant correlation between an aberrant expression of at least one of the three miRs and poor outcome was maintained (P < 0.0001). Our results identify an expression profile of miR‐151‐5p, miR‐451, and miR‐1290 as a novel biomarker for outcome in pediatric precursor B‐cell ALL patients, regardless of treatment protocol. The use of these markers may lead to improved risk stratification at diagnosis and allow early therapeutic interventions in an attempt to improve survival of high risk patients. © 2015 Wiley Periodicals, Inc.     

Overexpression of miR‐17 in gastric cancer is correlated with proliferation‐associated oncogene amplification

The molecular mechanism underlying microRNA (miR)‐17 overexpression has not been clearly evaluated in gastric cancer. We aimed to evaluate the functional roles of miR‐17 in gastric cancer and test its viability as a therapeutic target. We conducted comparative genomic hybridization and expression array analyses on human gastric cancer tissue samples, as well as evaluating the functional roles of miR‐17 in gastric cancer cell lines and transgenic mice. miR‐17 overexpression in gastric cancer patients was associated with copy number gain of proliferation‐associated oncogenes such as MYC, CCNE1, ERBB2, and FGFR2. Copy number gain of MIR17HG gene (13q31.3) was rare, with an overall frequency of 2% in gastric cancers (1 of 51). miR‐17 knockdown suppressed the monolayer and anchorage‐independent growth of FGFR2‐amplified KATO‐III gastric cancer cells. mir‐17–92 TG/TG mice overexpressing the mir‐17–92 cluster under the villin promoter developed spontaneous benign tumors in the intestinal tract (log‐rank P for tumor‐free survival = 0.069). Taken together, miR‐17 overexpression in gastric cancer was rarely associated with MIR17HG gene amplification, but correlated with proliferation‐associated oncogene amplification. Therefore, miR‐17‐targeting approach may benefit patients with gastric cancers harboring proliferation‐associated oncogene amplification.     

MiR221 promotes precursor B‐cell retention in the bone marrow by amplifying the PI3K‐signaling pathway in mice

Hematopoietic stem cells and lineage‐uncommitted progenitors are able to home to the bone marrow upon transplantation and reconstitute the host with hematopoietic progeny. Expression of miR221 in B‐lineage committed preBI‐cells induces their capacity to home to the bone marrow. However, the molecular mechanisms underlying miR221‐controlled bone marrow homing and retention remain poorly understood. Here, we demonstrate, that miR221 regulates bone marrow retention of such B‐cell precursors by targeting PTEN, thus enhancing PI3K signaling in response to the chemokine CXCL12. MiR221‐enhanced PI3K signaling leads to increased expression of the anti‐apoptotic protein Bcl2 and VLA4 integrin‐mediated adhesion to VCAM1 in response to CXCL12 in vitro. Ablation of elevated PI3K activity abolishes the retention of miR221 expressing preBI‐cells in the bone marrow. These results suggest that amplification of PI3K signaling by miR221 could be a general mechanism for bone marrow residence, shared by miR221‐expressing hematopoietic cells.     

Characterization of B‐ and T‐lineage acute lymphoblastic leukemia by integrated analysis of MicroRNA and mRNA expression profiles

Acute lymphoblastic leukemia (ALL) is an heterogeneous disease comprising several subentities that differ for both immunophenotypic and molecular characteristics. Over the years, the biological understanding of this neoplasm has largely increased. Gene expression profiling has allowed to identify specific signatures for the different ALL subsets and permitted the identification of pathways deregulated by a given lesion. MicroRNAs (miRNAs) are small noncoding RNAs, which play a pivotal role in several cellular functions. In this study, we investigated miRNAs expression profiles in a series of adult ALL cases by microarray analysis. Unsupervised hierarchical clustering largely recapitulated ALL subgroups. Furthermore, we identified miR‐148, miR‐151, and miR‐424 as discriminative of T‐lineage versus B‐lineage ALL; ANOVA highlighted a set of six miRNAs—namely miR‐425‐5p, miR‐191, miR‐146b, miR‐128, miR‐629, and miR‐126—that can discriminate B‐lineage ALL subgroups harboring specific molecular lesions. These results were confirmed and extended by quantitative‐PCR on a further cohort of cases. Finally, we used Pearson correlation analysis to combine miRNA and gene expression profiles. The distribution of correlation coefficients generated by comparing the expression of every miRNA/gene pair in our data set shows enrichment of both positively and negatively correlated pairs over background distributions obtained using randomized data. Moreover, a clear enrichment for predicted miRNA:target pairs is observed at negative correlation coefficient intervals. Signal‐to‐noise ratio highlighted several miRNA/gene pairs with a possible role in the disease. In fact, gene set enrichment analysis of genes composing the selected miRNA/gene pairs displays over‐representation of functional categories related to cancer and cell‐cycle regulation. © 2009 Wiley‐Liss, Inc.     

MiR‐23b and miR‐199a impair epithelial‐to‐mesenchymal transition during atrioventricular endocardial cushion formation

Background: Valve development is a multistep process involving the activation of the cardiac endothelium, epithelial‐mesenchymal transition (EMT) and the progressive alignment and differentiation of distinct mesenchymal cell types. Several pathways such as Notch/delta, Tgf‐beta and/or Vegf signaling have been implicated in crucial steps of valvulogenesis. We have previously demonstrated discrete changes in microRNAs expression during cardiogenesis, which are predicted to target Bmp‐ and Tgf‐beta signaling. We now analyzed the expression profile of 20 candidate microRNAs in atrial, ventricular, and atrioventricular canal regions at four different developmental stages. Results: qRT‐PCR analyses of microRNAs demonstrated a highly dynamic and distinct expression profiles within the atrial, ventricular, and atrioventricular canal regions of the developing chick heart. miR‐23b, miR‐199a, and miR‐15a displayed increased expression during early AVC development whereas others such as miR‐130a and miR‐200a display decreased expression levels. Functional analyses of miR‐23b, miR‐199a, and miR‐15a overexpression led to in vitro EMT blockage. Molecular analyses demonstrate that distinct EMT signaling pathways are impaired after microRNA expression, including a large subset of EMT‐related genes that are predicted to be targeted by these microRNAs. Conclusions: Our data demonstrate that miR‐23b and miR‐199a over‐expression can impair atrioventricular EMT. Developmental Dynamics 244:1259–1275, 2015. © 2015 Wiley Periodicals, Inc.     

MIR‐137 Suppresses Growth and Invasion,is Downregulated in Oligodendroglial Tumors and Targets CSE1L

MicroRNA‐137 (miR‐137) expression has been reported to be decreased in astrocytic tumors in two expression profiling studies but its role in the pathogenesis of oligodendroglial tumors is still limited. In this study, we demonstrate that miR‐137 expression is significantly downregulated in a cohort of 35 oligodendroglial tumors and nine glioma cell lines compared with normal brains. Lower miR‐137 expression is associated with shorter progressive‐free survival and overall survival. Restoration of miR‐137 expression in an oligodendroglial cells TC620, and also glioblastoma cells of U87 and U373 significantly suppressed cell growth, anchorage‐independent growth, as well as invasion. Demethylation and deacetylation treatments resulted in upregulation of miR‐137 expression in TC620 cells. In silico analysis showed that CSE1 chromosome segregation 1‐like (yeast) (CSE1L) is a potential target gene of miR‐137. Luciferase reporter assay demonstrated that miR‐137 negatively regulates CSE1L by interaction between miR‐137 and complementary sequences in the 3′ UTR of CSE1L. Immunohistochemistry revealed that CSE1L is upregulated in oligodendroglial tumors. Knockdown of CSE1L resulted in similar outcomes as overexpressing miR‐137 in oligodendroglioma cells and glioblastoma cells. Overall, our data suggest that miR‐137 regulates growth of glioma cells and targets CSE1L, providing further understanding in the tumorigenesis of gliomas.