Target Of miR-125b Research Articles

A Risk Variant in an miR-125b Binding Site in BMPR1B Is Associated with Breast Cancer Pathogenesis

MicroRNAs regulate diverse cellular processes and play an integral role in cancer pathogenesis. Genomic variation within miRNA target sites may therefore be important sources for genetic differences in cancer risk. To investigate this possibility, we mapped HapMap single nucleotide polymorphisms (SNP) to putative miRNA recognition sites within genes dysregulated in estrogen receptor-stratified breast tumors and used local linkage disequilibrium patterns to identify high-ranking SNPs in the Cancer Genetic Markers of Susceptibility (CGEMS) breast cancer genome-wide association study for further testing. Two SNPs, rs1970801 and rs11097457, scoring in the top 100 from the CGEMS study, were in strong linkage disequilibrium with rs1434536, an SNP that resides within a miR-125b target site in the 3' untranslated region of the bone morphogenic receptor type 1B (BMPR1B) gene encoding a transmembrane serine/threonine kinase. We validated the CGEMS association findings for rs1970801 in an independent cohort of admixture-corrected cases identified from families with multiple case histories. Subsequent association testing of rs1434536 for these cases and CGEMS controls with imputed genotypes supported the association. Furthermore, luciferase reporter assays and overexpression of miR-125b-mimics combined with quantitative reverse transcription-PCR showed that BMPR1B transcript is a direct target of miR-125b and that miR-125b differentially regulates the C and T alleles of rs1434536. These results suggest that allele-specific regulation of BMPR1B by miR-125b explains the observed disease risk. Our approach is general and can help identify and explain the mechanisms behind disease association for alleles that affect miRNA regulation.

Potential role of miR-29b in modulation ofDnmt3aandDnmt3bexpression in primordial germ cells of female mouse embryos

MicroRNAs (miRNAs) are a recently discovered class of small noncoding RNAs and are implicated in an increasing number of biological processes. To examine whether miRNAs might contribute to sexual differentiation, we performed expression profiling of miRNAs in mouse embryonic gonads with the use of a highly sensitive cloning method, mRAP. Our profiling data revealed substantial differences in the miRNA repertoire between male and female gonads at embryonic (E) day 13.5 (E13.5), suggesting that such differentially expressed miRNAs may function in sexual differentiation. Female-specific miRNAs included miR-29b, which also has been known to be expressed in a similar sex-dependent manner in the gonads of chicken embryos, suggestive of a conserved role in gonadogenesis. Transcripts of the human genes for the de novo methyltransferases DNMT3A and DNMT3B have been identified as targets of miR-29b, and we found that mouse miR-29b also negatively regulates Dnmt3a and Dnmt3b expression in luciferase reporter assays. We also found that miR-29b is expressed in mouse primordial germ cells (PGCs) at E13.5 and that its expression is up-regulated in a female-specific manner between E13.5 and E17.5, when male-specific de novo methylation of the PGC genome is known to occur. Our data thus suggest that miR-29b may play an important role in female gonadal development by targeting Dnmt3a and Dnmt3b and thereby modulating methylation of genomic DNA in PGCs.

Identification of proteins implicated in Kit receptor signalling

Excessive collagen synthesis and deposit during skin wound healing results in scar formation. MicroRNAs (miRNAs) are endogenous noncoding RNA regulators that mediate diverse biological functions through repressing target genes and hold great potentials for clinical therapeutic applications. The aim of the present study was to identify miRNAs as post-transcriptional regulators of collagen 1 in skin fibroblasts.miRNA candidates that potentially target collagen 1 were predicted by computational algorithms PicTar and TargetScan. The expression changes of collagen subunits 1α1 and 1α2 were measured by real-time reverse transcription–polymerase chain reaction and western blot after the primary skin fibroblasts were transfected with miR-29b mimics or inhibitor, respectively. A luciferase reporter assay was performed to further determine whether both collagen 1 subunits were probably direct targets of miR-29b.Computational predictions identified several miRNAs as possible regulators for collagen 1 synthesis, including miR-29b. Enforced overexpression of miR-29b resulted in remarkable decrease of collagen 1α1 and 1α2, whereas knockdown of endogenous miR-29b induced pronounced increase of collagen 1α1 and 1α2 at both the messenger RNA and the protein levels. The luciferase activities were significantly inhibited when cells were cotransfected with reporter constructs and miR-29 mimics in vitro. Moreover, miR-29b transcriptional abundance inversely related to the levels of both collagen 1 subunits in skin scar as compared with normal skin.Our data indicate that miR-29b is a potent post-transcriptional repressor of collagen 1 in skin fibroblasts and its deregulation might be implicated in scar formation, suggesting that miR-29b might represent a potential therapeutic target for scar reduction.

HETEROGENEOUS NUCLEAR RIBONUCLEOPROTEIN K IS A NOVEL REGULATOR OF ANDROGEN RECEPTOR TRANSLATION IN PROSTATE CANCER

Since the association of miR-125b with CaP is totally unknown, the purpose of this study was to understand the role of this miRNA in CaP. METHODS: LNCaP and cds1 cells cultured in androgendepleted medium were transfected with synthetic miR-125b and antimiR-125b, respectively, and cell proliferation was assessed using the WST-1 assay. Chromatin immunoprecipitation (ChIP) assay was used to determine the loading of androgen receptor (AR) onto the 5’-DNA region of miR-125b. To test whether BAK1 mRNA is a direct target of miR-125b, a luciferase assay was performed in AR-negative DU145 cells to evaluate the effects of miR-125b on the 3’-untranslated region (3’-UTR) of the BAK1 gene. RESULTS: Synthetic miR-125b stimulated the growth of LNCaP cells in the absence of androgens. These cells retained typical morphological features. In contrast, in cds cells, inhibition of miR125b activity using synthetic anti-miR-125b inhibited proliferation of

MiRNA-29b Targets MCL-1 and Is Down-Regulated in Chemotherapy-Resistant Acute Myeloid Leukemia (AML).

AML is a heterogeneous disorder that includes several differently biologic entities. Patients with intermediate and poor-risk cytogenetics represent the majority of AML and chemotherapy-based regimens fail to cure most of these patients that are either resistant to or relapse shortly after treatment. Therefore, it is crucial to understand the biology of chemotherapy resistance in AML. Here, we proposed to investigate the relation between chemoresistance and miRNA expression in AML. First, using a miRNA microarray platform, we analyzed the miRNA profiles in pretreatment samples from a group of 24 patients with newly diagnosed AML treated with idarubicin 12mg/m2/day (days 1–3) and cytarabine 1500 mg/m2/d (days 1–4). After background subtraction and normalization to a set of housekeeping genes, Significance Analysis of Microarrays (SAM) was performed. Among others miRNAs, miR-29b was found significantly down-regulated in pts with resistant disease (n=13) compared to those who achieved CR (n=11)(SAM score −2.92). To validate these results, we measured miR-29b levels by quantitative(q)RT-PCR in an independent set of 36 AML patients treated with the same chemotherapy regimen as described above and with no statistical differences in baseline pretreatment characteristics. We found that miR-29b was down-regulated in pts with resistant disease compared with pts who achieved complete remission (p=0.015, t-test). Interestingly miR-29b is predicted to target MCL1, an oncogene whose over-expression has been associated with resistance to chemotherapy. An inverse correlation was indeed observed between MCL1 protein levels measured by immunoblotting and miR-29b expression measured by miRNA microarray in AML patients (n=9). Next, we inserted the 3′ untranslated region (3′UTR) of the MCL1 gene containing the putative interaction sites of miR-29b into a luciferase reporter. When the miR-29b precursor oligonucleotide was transfected into MEG-O1 cells along with the MCL1 reporter construct, a 70% reduction of luciferase activity was observed. In contrast, no changes in the luciferase activity was observed when co-transfection was performed with controls inckluding a construct containing a mutated 3′UTR sequence of MCL1. To validate MCL1 as a target of miR-29b, a synthetic miR-29b oligonucleotide was transfected by nucleoporation into K562 cells, which overexpress MCL1 and lack endogenous miR-29b. This resulted in a marked reduction of MCL1 protein levels compared with controls. Finally, to explore the functional impact of restoring miR-29b and downregulating MCL1 in AML cells, we measure the apoptosis rates in K562 cells transfected with miR-29b or scrambled oligonucleotides by annexin V staining. A 3-fold increase in apoptosis was found in miR-29b transfected cells compared with scrambled oligonucleotides-treated controls. Consistent with these results, miR-29b-treated K562 cells exhibited increased chemosensitivity to ARA-C compared with scrambled oligonucleotides-treated cells. In conclusion, we demonstrate here that miR-29b down-regulation is associated with chemotherapy resistance, which might occur as the results of MCL1 overexpression. Restoring miR-29b expression levels down modulates MCL1, induces apoptosis and enhances chemosensitivity in AML cells, thereby suggesting miRNA-based therapy as a novel pro-apoptotic approach to increase response in high-risk AML.

MicroRNAs: A new class of regulatory genes affecting metabolism

MicroRNAs (miRNAs) are short noncoding RNAs that regulate gene expression by binding to target mRNAs, which leads to reduced protein synthesis and sometimes decreased steady-state mRNA levels. Although hundreds of miRNAs have been identified, much less is known about their biological function. Several studies have provided evidence that miRNAs affect pathways that are fundamental for metabolic control in higher organisms such as adipocyte and skeletal muscle differentiation. Furthermore, some miRNAs have been implicated in lipid, amino acid, and glucose homeostasis. These studies open the possibility that miRNAs may contribute to common metabolic diseases and point to novel therapeutic opportunities based on targeting of miRNAs.

MicroRNA Expression Analysis in Multiple Myeloma.

Introduction: The analysis of what goes awry in a malignant cell has focused primarily on mutations of protein-encoding genes and their regulatory sequences. However, recent work on microRNA (miRNA) has shed light on the possible involvement of miRNA genes in human disease. miRNA are small “noncoding” or “non-messenger” RNAs of about 21–25 nucleotides in length that function as regulators of gene expression essentially by pairing to the mRNA of protein-coding genes to initiate mRNA degradation or repression of translation. We are interested in characterizing miRNA expression profiles in human myeloma cell lines (HMCLs) and myeloma patient samples. Multiple myeloma (MM) is a plasma cell tumor characterized by frequent chromosomal translocations.Materials and methods: The expression of miRNA in HMCLs, primary MM cells (hyperdiploid samples) and normal plasma cells was determined using human microRNA chips. The GenoExporerTM Human microRNA chips were developed by GenoSensor (Tempe, AZ) and the microarray contains 226 human microRNA sequences in addition to control sequences. The DNA oligo probes are synthesized and immobilized on the chips (1”x 3” standard glass microslide). The probes are designed based on the active mature miRNA sequences and some of their flanking sequences. Our analysis included seven myeloma patients (hyperdiploid MM), eight different HMCLs (MM1, 8226 with t(14;16); SKMM2, U266, INA-6 with t(11;14) and H929, KMS11, UTMC-2 with t(4;14)), and eight normal human plasma cells. The normal human plasma cells were collected at Mayo Clinic Rochester from bone samples of patients undergoing orthopedic surgery. For the assay 5–10 μg of total RNA per sample were used at a concentration of 1 μg/μL. The RNA is directly labeled with biotin and used as a target for the on-chip hybridization assays. A streptavidin-Alexa dye is used to stain the hybridized targets and the fluorescent signals are captured and analyzed. The gene signal intensities were normalized to tRNA signal intensity.Results: The primary MM cells and HMCLs displayed a distinctive miRNA expression profile compared with normal plasma cells. Using the GeneSpring 7 (Agilent Technologies, Palo Alto, CA) for gene expression analysis we have identified miRNA genes with significant variation in expression levels between tumor and normal samples including miR-125b, miR-133a, miR1, and miR-124a (p<0.01). Recently defined algorithms were used to identify putative targets for the miRNA genes of interest such as leukemia inhibitory factor (LIF) as targets of miR-125b and Stat3 and angiopoietin-1 precursor as targets of miR-124a (Lewis et al. 2003). Of note, miR-15 and miR-16, previously identified to be downregulated in CLL, were expressed at low levels in some MM patients and HMCLs, but not in the normal plasma cells. Further analysis will be carried out to validate the data from miRNA profiling using northern blot analysis or real-time RT-PCR to measure expression levels of miRNA genes and that of their target genes.Conclusion: Analysis of miRNA expression pattern (along with aCGH and gene expression profiling studies) will undoubtedly refine our understanding of the various genotypic subtypes of multiple myeloma.

Micro-RNA Regulation of the Mammalian lin-28 Gene during Neuronal Differentiation of Embryonal Carcinoma Cells

Vertebrate genomes each encode hundreds of micro-RNAs (miRNAs), yet for few of these miRNAs is there empirical evidence as to which mRNA(s) they regulate. Here we report the identification of human lin-28 mRNA as a regulatory target of human miR-125b and its homolog miR-125a. Studies of miR-125b function in mouse P19 embryonal carcinoma cells induced to develop into neurons suggest a role for this regulatory miRNA in mammalian neuronal differentiation, since its increased concentration in these cells contributes to lin-28 downregulation. Within the lin-28 3' untranslated region (UTR) are two conserved miRNA responsive elements (miREs) that mediate repression by miR-125b and miR-125a. Simultaneous deletion of both miREs renders the lin-28 3' UTR almost completely insensitive to these miRNAs, indicating that these two miREs are the principal elements in the lin-28 3' UTR that respond to miR-125. At the 3' end of each element is an adenosine residue that makes a significant contribution to function irrespective of its complementarity to the 5'-terminal nucleotide of miR-125. By contrast to most earlier reports of gene repression by other miRNAs that are imperfectly complementary to their targets, lin-28 downregulation by miR-125 involves reductions in both translational efficiency and mRNA abundance. The decrease in the mRNA concentration is achieved by a posttranscriptional mechanism that is independent of the inhibitory effect on translation.