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1.  In Vitro and In Vivo Activity of a Novel Locked Nucleic Acid (LNA)-Inhibitor-miR-221 against Multiple Myeloma Cells 
PLoS ONE  2014;9(2):e89659.
Background & Aim
The miR-221/222 cluster is upregulated in malignant plasma cells from multiple myeloma (MM) patients harboring the t(4;14) translocation. We previously reported that silencing of miR-221/222 by an antisense oligonucleotide induces anti-MM activity and upregulates canonical miR-221/222 targets. The in vivo anti-tumor activity occurred when miR-221/222 inhibitors were delivered directly into MM xenografts. The aim of the present study was to evaluate the anti-MM activity of a novel phosphorothioate modified backbone 13-mer locked nucleic acid (LNA)-Inhibitor-miR-221 (LNA-i-miR-221) specifically designed for systemic delivery.
Methods
In vitro anti-MM activity of LNA-i-miR-221 was evaluated by cell proliferation and BrdU uptake assays. In vivo studies were performed with non-obese diabetic/severe combined immunodeficient (NOD.SCID) mice bearing t(4;14) MM xenografts, which were intraperitoneally or intravenously treated with naked LNA-i-miR-221. RNA extracts from retrieved tumors were analyzed for miR-221 levels and modulation of canonical targets expression. H&E staining and immunohistochemistry were performed on retrieved tumors and mouse vital organs.
Results
In vitro, LNA-i-miR-221 exerted strong antagonistic activity against miR-221 and induced upregulation of the endogenous target p27Kip1. It had a marked anti-proliferative effect on t(4;14)-translocated MM cells but not on MM cells not carrying the translocation and not overexpressing miR-221. In vivo, systemic treatment with LNA-i-miR-221 triggered significant anti-tumor activity against t(4;14) MM xenografts; it also induced miR-221 downregulation, upregulated p27Kip1 and reduced Ki-67. No behavioral changes or organ-related toxicity were observed in mice as a consequence of treatments.
Conclusions
LNA-i-miR-221 is a highly stable, effective agent against t(4;14) MM cells, and is suitable for systemic use. These data provide the rationale for the clinical development of LNA-i-miR-221 for the treatment of MM.
doi:10.1371/journal.pone.0089659
PMCID: PMC3931823  PMID: 24586944
2.  microRNA-122 as a regulator of mitochondrial metabolic gene network in hepatocellular carcinoma 
A moderate loss of miR-122 function correlates with up-regulation of seed-matched genes and down-regulation of mitochondrially localized genes in both human hepatocellular carcinoma and in normal mice treated with anti-miR-122 antagomir.Putative direct targets up-regulated with loss of miR-122 and secondary targets down-regulated with loss of miR-122 are conserved between human beings and mice and are rapidly regulated in vitro in response to miR-122 over- and under-expression.Loss of miR-122 secondary target expression in either tumorous or adjacent non-tumorous tissue predicts poor survival of heptatocellular carcinoma patients.
Hepatocellular carcinoma (HCC) is one of the most aggressive human malignancies, common in Asia, Africa, and in areas with endemic infections of hepatitis-B or -C viruses (HBV or HCV) (But et al, 2008). Globally, the 5-year survival rate of HCC is <5% and about 600 000 HCC patients die each year. The high mortality associated with this disease is mainly attributed to the failure to diagnose HCC patients at an early stage and a lack of effective therapies for patients with advanced stage HCC. Understanding the relationships between phenotypic and molecular changes in HCC is, therefore, of paramount importance for the development of improved HCC diagnosis and treatment methods.
In this study, we examined mRNA and microRNA (miRNA)-expression profiles of tumor and adjacent non-tumor liver tissue from HCC patients. The patient population was selected from a region of endemic HBV infection, and HBV infection appears to contribute to the etiology of HCC in these patients. A total of 96 HCC patients were included in the study, of which about 88% tested positive for HBV antigen; patients testing positive for HCV antigen were excluded. Among the 220 miRNAs profiled, miR-122 was the most highly expressed miRNA in liver, and its expression was decreased almost two-fold in HCC tissue relative to adjacent non-tumor tissue, confirming earlier observations (Lagos-Quintana et al, 2002; Kutay et al, 2006; Budhu et al, 2008).
Over 1000 transcripts were correlated and over 1000 transcripts were anti-correlated with miR-122 expression. Consistent with the idea that transcripts anti-correlated with miR-122 are potential miR-122 targets, the most highly anti-correlated transcripts were highly enriched for the presence of the miR-122 central seed hexamer, CACTCC, in the 3′UTR. Although the complete set of negatively correlated genes was enriched for cell-cycle genes, the subset of seed-matched genes had no significant KEGG Pathway annotation, suggesting that miR-122 is unlikely to directly regulate the cell cycle in these patients. In contrast, transcripts positively correlated with miR-122 were not enriched for 3′UTR seed matches to miR-122. Interestingly, these 1042 transcripts were enriched for genes coding for mitochondrially localized proteins and for metabolic functions.
To analyze the impact of loss of miR-122 in vivo, silencing of miR-122 was performed by antisense inhibition (anti-miR-122) in wild-type mice (Figure 3). As with the genes negatively correlated with miR-122 in HCC patients, no significant biological annotation was associated with the seed-matched genes up-regulated by anti-miR-122 in mouse livers. The most significantly enriched biological annotation for anti-miR-122 down-regulated genes, as for positively correlated genes in HCC, was mitochondrial localization; the down-regulated mitochondrial genes were enriched for metabolic functions. Putative direct and downstream targets with orthologs on both the human and mouse microarrays showed significant overlap for regulations in the same direction. These overlaps defined sets of putative miR-122 primary and secondary targets. The results were further extended in the analysis of a separate dataset from 180 HCC, 40 cirrhotic, and 6 normal liver tissue samples (Figure 4), showing anti-correlation of proposed primary and secondary targets in non-healthy tissues.
To validate the direct correlation between miR-122 and some of the primary and secondary targets, we determined the expression of putative targets after transfection of miR-122 mimetic into PLC/PRF/5 HCC cells, including the putative direct targets SMARCD1 and MAP3K3 (MEKK3), a target described in the literature, CAT-1 (SLC7A1), and three putative secondary targets, PPARGC1A (PGC-1α) and succinate dehydrogenase subunits A and B. As expected, the putative direct targets showed reduced expression, whereas the putative secondary target genes showed increased expression in cells over-expressing miR-122 (Figure 4).
Functional classification of genes using the total ancestry method (Yu et al, 2007) identified PPARGC1A (PGC-1α) as the most connected secondary target. PPARGC1A has been proposed to function as a master regulator of mitochondrial biogenesis (Ventura-Clapier et al, 2008), suggesting that loss of PPARGC1A expression may contribute to the loss of mitochondrial gene expression correlated with loss of miR-122 expression. To further validate the link of miR-122 and PGC-1α protein, we transfected PLC/PRF/5 cells with miR-122-expression vector, and observed an increase in PGC-1α protein levels. Importantly, transfection of both miR-122 mimetic and miR-122-expression vector significantly reduced the lactate content of PLC/PRF/5 cells, whereas anti-miR-122 treatment increased lactate production. Together, the data support the function of miR-122 in mitochondrial metabolic functions.
Patient survival was not directly associated with miR-122-expression levels. However, miR-122 secondary targets were expressed at significantly higher levels in both tumor and adjacent non-tumor tissues among survivors as compared with deceased patients, providing supporting evidence for the potential relevance of loss of miR-122 function in HCC patient morbidity and mortality.
Overall, our findings reveal potentially new biological functions for miR-122 in liver physiology. We observed decreased expression of miR-122, a liver-specific miRNA, in HBV-associated HCC, and loss of miR-122 seemed to correlate with the decrease of mitochondrion-related metabolic pathway gene expression in HCC and in non-tumor liver tissues, a result that is consistent with the outcome of treatment of mice with anti-miR-122 and is of prognostic significance for HCC patients. Further investigation will be conducted to dissect the regulatory function of miR-122 on mitochondrial metabolism in HCC and to test whether increasing miR-122 expression can improve mitochondrial function in liver and perhaps in liver tumor tissues. Moreover, these results support the idea that primary targets of a given miRNA may be distributed over a variety of functional categories while resulting in a coordinated secondary response, potentially through synergistic action (Linsley et al, 2007).
Tumorigenesis involves multistep genetic alterations. To elucidate the microRNA (miRNA)–gene interaction network in carcinogenesis, we examined their genome-wide expression profiles in 96 pairs of tumor/non-tumor tissues from hepatocellular carcinoma (HCC). Comprehensive analysis of the coordinate expression of miRNAs and mRNAs reveals that miR-122 is under-expressed in HCC and that increased expression of miR-122 seed-matched genes leads to a loss of mitochondrial metabolic function. Furthermore, the miR-122 secondary targets, which decrease in expression, are good prognostic markers for HCC. Transcriptome profiling data from additional 180 HCC and 40 liver cirrhotic patients in the same cohort were used to confirm the anti-correlation of miR-122 primary and secondary target gene sets. The HCC findings can be recapitulated in mouse liver by silencing miR-122 with antagomir treatment followed by gene-expression microarray analysis. In vitro miR-122 data further provided a direct link between induction of miR-122-controlled genes and impairment of mitochondrial metabolism. In conclusion, miR-122 regulates mitochondrial metabolism and its loss may be detrimental to sustaining critical liver function and contribute to morbidity and mortality of liver cancer patients.
doi:10.1038/msb.2010.58
PMCID: PMC2950084  PMID: 20739924
hepatocellular carcinoma; microarray; miR-122; mitochondrial; survival
3.  Efficient gene silencing by delivery of locked nucleic acid antisense oligonucleotides, unassisted by transfection reagents 
Nucleic Acids Research  2009;38(1):e3.
For the past 15–20 years, the intracellular delivery and silencing activity of oligodeoxynucleotides have been essentially completely dependent on the use of a delivery technology (e.g. lipofection). We have developed a method (called ‘gymnosis’) that does not require the use of any transfection reagent or any additives to serum whatsoever, but rather takes advantage of the normal growth properties of cells in tissue culture in order to promote productive oligonucleotide uptake. This robust method permits the sequence-specific silencing of multiple targets in a large number of cell types in tissue culture, both at the protein and mRNA level, at concentrations in the low micromolar range. Optimum results were obtained with locked nucleic acid (LNA) phosphorothioate gap-mers. By appropriate manipulation of oligonucleotide dosing, this silencing can be continuously maintained with little or no toxicity for >240 days. High levels of oligonucleotide in the cell nucleus are not a requirement for gene silencing, contrary to long accepted dogma. In addition, gymnotic delivery can efficiently deliver oligonucleotides to suspension cells that are known to be very difficult to transfect. Finally, the pattern of gene silencing of in vitro gymnotically delivered oligonucleotides correlates particularly well with in vivo silencing. The establishment of this link is of particular significance to those in the academic research and drug discovery and development communities.
doi:10.1093/nar/gkp841
PMCID: PMC2800216  PMID: 19854938
4.  Inhibition of miR-15 Protects Against Cardiac Ischemic Injury 
Circulation Research  2011;110(1):71-81.
Rationale
Myocardial infarction (MI) is a leading cause of death worldwide. Because endogenous cardiac repair mechanisms are not sufficient for meaningful tissue regeneration, MI results in loss of cardiac tissue and detrimental remodeling events. MicroRNAs (miRNAs) are small, noncoding RNAs that regulate gene expression in a sequence dependent manner. Our previous data indicate that miRNAs are dysregulated in response to ischemic injury of the heart and actively contribute to cardiac remodeling after MI.
Objective
This study was designed to determine whether miRNAs are dysregulated on ischemic damage in porcine cardiac tissues and whether locked nucleic acid (LNA)-modified anti-miR chemistries can target cardiac expressed miRNAs to therapeutically inhibit miR-15 on ischemic injury.
Methods and Results
Our data indicate that the miR-15 family, which includes 6 closely related miRNAs, is regulated in the infarcted region of the heart in response to ischemia-reperfusion injury in mice and pigs. LNA-modified chemistries can effectively silence miR-15 family members in vitro and render cardiomyocytes resistant to hypoxia-induced cardiomyocyte cell death. Correspondingly, systemic delivery of miR-15 anti-miRs dose-dependently represses miR-15 in cardiac tissue of both mice and pigs, whereas therapeutic targeting of miR-15 in mice reduces infarct size and cardiac remodeling and enhances cardiac function in response to MI.
Conclusions
Oligonucleotide-based therapies using LNA-modified chemistries for modulating cardiac miRNAs in the setting of heart disease are efficacious and validate miR-15 as a potential therapeutic target for the manipulation of cardiac remodeling and function in the setting of ischemic injury.
doi:10.1161/CIRCRESAHA.111.244442
PMCID: PMC3354618  PMID: 22052914
microRNA; ischemia reperfusion; miR-15 family; anti-miR therapy
5.  Chemical structure requirements and cellular targeting of microRNA-122 by peptide nucleic acids anti-miRs 
Nucleic Acids Research  2011;40(5):2152-2167.
Anti-miRs are oligonucleotide inhibitors complementary to miRNAs that have been used extensively as tools to gain understanding of specific miRNA functions and as potential therapeutics. We showed previously that peptide nucleic acid (PNA) anti-miRs containing a few attached Lys residues were potent miRNA inhibitors. Using miR-122 as an example, we report here the PNA sequence and attached amino acid requirements for efficient miRNA targeting and show that anti-miR activity is enhanced substantially by the presence of a terminal-free thiol group, such as a Cys residue, primarily due to better cellular uptake. We show that anti-miR activity of a Cys-containing PNA is achieved by cell uptake through both clathrin-dependent and independent routes. With the aid of two PNA analogues having intrinsic fluorescence, thiazole orange (TO)-PNA and [bis-o-(aminoethoxy)phenyl]pyrrolocytosine (BoPhpC)-PNA, we explored the subcellular localization of PNA anti-miRs and our data suggest that anti-miR targeting of miR-122 may take place in or associated with endosomal compartments. Our findings are valuable for further design of PNAs and other oligonucleotides as potent anti-miR agents.
doi:10.1093/nar/gkr885
PMCID: PMC3300011  PMID: 22070883
6.  Antisense 2′-Deoxy, 2′-Fluoroarabino Nucleic Acid (2′F-ANA) Oligonucleotides: In Vitro Gymnotic Silencers of Gene Expression Whose Potency Is Enhanced by Fatty Acids 
Gymnosis is the process of the delivery of antisense oligodeoxynucleotides to cells, in the absence of any carriers or conjugation, that produces sequence-specific gene silencing. While gymnosis was originally demonstrated using locked nucleic acid (LNA) gapmers, 2′-deoxy-2′fluoroarabino nucleic acid (2′F-ANA) phosphorothioate gapmer oligonucleotides (oligos) when targeted to the Bcl-2 and androgen receptor (AR) mRNAs in multiple cell lines in tissue culture, are approximately as effective at silencing of Bcl-2 expression as the iso-sequential LNA congeners. In LNCaP prostate cancer cells, gymnotic silencing of the AR by a 2′F-ANA phosphorothioate gapmer oligo led to downstream silencing of cellular prostate-specific antigen (PSA) expression even in the presence of the androgenic steroid R1881 (metribolone), which stabilizes cytoplasmic levels of the AR. Furthermore, gymnotic silencing occurs in the absence of serum, and silencing by both LNA and 2′F-ANA oligos is augmented in serum-free (SF) media in some cell lines when they are treated with oleic acid and a variety of ω-6 polyunsaturated fatty acids (ω-6 PUFAs), but not by an aliphatic (palmitic) fatty acid. These results significantly expand our understanding of and ability to successfully manipulate the cellular delivery of single-stranded oligos in vitro.
doi:10.1038/mtna.2012.35
PMCID: PMC3499694  PMID: 23344235
2′F-ANA; endocytosis; gymnosis; LNA; oleic acid; phosphorothioate oligonucleotides; polyunsaturated fatty acids
7.  Inhibition of MicroRNA miR-92a Inhibits Cell Proliferation in Human Acute Promyelocytic Leukemia 
Turkish Journal of Hematology  2013;30(2):157-162.
Objective: MicroRNAs (miRNAs) are endogenous non-coding RNAs, 19-25 nucleotides in length involved in post-transcriptional regulation of gene expression in a considerable majority of mRNAs. In many tumors, up- or down-regulation of different miRNAs has been reported. In acute myeloid leukemia up-regulation of miR-92a has been reported in humans in vitro studies. In this study it is mainly aimed to assess the effect of inhibition of miR-92a in viability of acute promyelocytic leukemia (APL).
Materials and Methods: We performed inhibition of miR-92a in an acute promyelocytic leukemia (APL) cell line (HL-60) using locked nucleic acid (LNA) antagomir. At different time points after LNA-anti-miR92a transfection, miR-92a quantitation and cell viability were assessed by qRT-real-time-polymerase chain reaction (PCR) and MTT assays. The data was processed using the ANOVA test.
Results: Down-regulation of miR-92a in APL cell line (HL-60) by LNA antagomir extensively decreased cell viability in APL. Cell viability gradually decreased over time as the viability of LNA-anti-miR transfected cells was less than 50% of untreated cells at 72 h post-transfection. The difference of cell viability between LNA-anti-miR and control groups was statistically significant (p<0.024).
Conclusion: Based on our findings, it is concluded that inhibition of miR-92a may represent a potential novel therapeutic approach for treatment of APL.
Conflict of interest:None declared.
doi:10.4274/Tjh.2012.0171
PMCID: PMC3878471  PMID: 24385779
microRNA; miR-92a; Acute promyelocytic leukemia; Locked Nucleic Acid
8.  Miravirsen (SPC3649) can inhibit the biogenesis of miR-122 
Nucleic Acids Research  2013;42(1):609-621.
MicroRNAs (miRNAs) are short noncoding RNAs, which bind to messenger RNAs and regulate protein expression. The biosynthesis of miRNAs includes two precursors, a primary miRNA transcript (pri-miRNA) and a shorter pre-miRNA, both of which carry a common stem-loop bearing the mature miRNA. MiR-122 is a liver-specific miRNA with an important role in the life cycle of hepatitis C virus (HCV). It is the target of miravirsen (SPC3649), an antimiR drug candidate currently in clinical testing for treatment of HCV infections. Miravirsen is composed of locked nucleic acid (LNAs) ribonucleotides interspaced throughout a DNA phosphorothioate sequence complementary to mature miR-122. The LNA modifications endow the drug with high affinity for its target and provide resistance to nuclease degradation. While miravirsen is thought to work mainly by hybridizing to mature miR-122 and blocking its interaction with HCV RNA, its target sequence is also present in pri- and pre-miR-122. Using new in vitro and cellular assays specifically developed to discover ligands that suppress biogenesis of miR-122, we show that miravirsen binds to the stem-loop structure of pri- and pre-miR-122 with nanomolar affinity, and inhibits both Dicer- and Drosha-mediated processing of miR-122 precursors. This inhibition may contribute to the pharmacological activity of the drug in man.
doi:10.1093/nar/gkt852
PMCID: PMC3874169  PMID: 24068553
9.  Antagonism of microRNA-122 in mice by systemically administered LNA-antimiR leads to up-regulation of a large set of predicted target mRNAs in the liver 
Nucleic Acids Research  2007;36(4):1153-1162.
MicroRNA-122 (miR-122) is an abundant liver-specific miRNA, implicated in fatty acid and cholesterol metabolism as well as hepatitis C viral replication. Here, we report that a systemically administered 16-nt, unconjugated LNA (locked nucleic acid)-antimiR oligonucleotide complementary to the 5′ end of miR-122 leads to specific, dose-dependent silencing of miR-122 and shows no hepatotoxicity in mice. Antagonism of miR-122 is due to formation of stable heteroduplexes between the LNA-antimiR and miR-122 as detected by northern analysis. Fluorescence in situ hybridization demonstrated uptake of the LNA-antimiR in mouse liver cells, which was accompanied by markedly reduced hybridization signals for mature miR-122 in treated mice. Functional antagonism of miR-122 was inferred from a low cholesterol phenotype and de-repression within 24 h of 199 liver mRNAs showing significant enrichment for miR-122 seed matches in their 3′ UTRs. Expression profiling extended to 3 weeks after the last LNA-antimiR dose revealed that most of the changes in liver gene expression were normalized to saline control levels coinciding with normalized miR-122 and plasma cholesterol levels. Combined, these data suggest that miRNA antagonists comprised of LNA are valuable tools for identifying miRNA targets in vivo and for studying the biological role of miRNAs and miRNA-associated gene-regulatory networks in a physiological context.
doi:10.1093/nar/gkm1113
PMCID: PMC2275095  PMID: 18158304
10.  Knockdown of miR-21 in human breast cancer cell lines inhibits proliferation, in vitro migration and in vivo tumor growth 
Introduction
MicroRNAs (miRNAs) are a class of small non-coding RNAs (20 to 24 nucleotides) that post-transcriptionally modulate gene expression. A key oncomir in carcinogenesis is miR-21, which is consistently up-regulated in a wide range of cancers. However, few functional studies are available for miR-21, and few targets have been identified. In this study, we explored the role of miR-21 in human breast cancer cells and tissues, and searched for miR-21 targets.
Methods
We used in vitro and in vivo assays to explore the role of miR-21 in the malignant progression of human breast cancer, using miR-21 knockdown. Using LNA silencing combined to microarray technology and target prediction, we screened for potential targets of miR-21 and validated direct targets by using luciferase reporter assay and Western blot. Two candidate target genes (EIF4A2 and ANKRD46) were selected for analysis of correlation with clinicopathological characteristics and prognosis using immunohistochemistry on cancer tissue microrrays.
Results
Anti-miR-21 inhibited growth and migration of MCF-7 and MDA-MB-231 cells in vitro, and tumor growth in nude mice. Knockdown of miR-21 significantly increased the expression of ANKRD46 at both mRNA and protein levels. Luciferase assays using a reporter carrying a putative target site in the 3' untranslated region of ANKRD46 revealed that miR-21 directly targeted ANKRD46. miR-21 and EIF4A2 protein were inversely expressed in breast cancers (rs = -0.283, P = 0.005, Spearman's correlation analysis).
Conclusions
Knockdown of miR-21 in MCF-7 and MDA-MB-231 cells inhibits in vitro and in vivo growth as well as in vitro migration. ANKRD46 is newly identified as a direct target of miR-21 in BC. These results suggest that inhibitory strategies against miR-21 using peptide nucleic acids (PNAs)-antimiR-21 may provide potential therapeutic applications in breast cancer treatment.
doi:10.1186/bcr2803
PMCID: PMC3109565  PMID: 21219636
11.  MicroRNA 21 Blocks Apoptosis in Mouse Periovulatory Granulosa Cells1 
Biology of Reproduction  2010;83(2):286-295.
MicroRNAs (miRNAs) play important roles in many developmental processes, including cell differentiation and apoptosis. Transition of proliferative ovarian granulosa cells to terminally differentiated luteal cells in response to the ovulatory surge of luteinizing hormone (LH) involves rapid and pronounced changes in cellular morphology and function. MicroRNA 21 (miR-21, official symbol Mir21) is one of three highly LH-induced miRNAs in murine granulosa cells, and here we examine the function and temporal expression of Mir21 within granulosa cells as they transition to luteal cells. Granulosa cells were transfected with blocking (2′-O-methyl) and locked nucleic acid (LNA-21) oligonucleotides, and mature Mir21 expression decreased to one ninth and one twenty-seventh of its basal expression, respectively. LNA-21 depletion of Mir21 activity in cultured granulosa cells induced apoptosis. In vivo, follicular granulosa cells exhibit a decrease in cleaved caspase 3, a hallmark of apoptosis, 6 h after the LH/human chorionic gonadotropin surge, coincident with the highest expression of mature Mir21. To examine whether Mir21 is involved in regulation of apoptosis in vivo, mice were treated with a phospho thioate-modified LNA-21 oligonucleotide, and granulosa cell apoptosis was examined. Apoptosis increased in LNA-21-treated ovaries, and ovulation rate decreased in LNA-21-treated ovaries, compared with their contralateral controls. We have examined a number of Mir21 apoptotic target transcripts identified in other systems; currently, none of these appear to play a role in the induction of ovarian granulosa cell apoptosis. This study is the first to implicate the antiapoptotic Mir21 (an oncogenic miRNA) as playing a clear physiologic role in normal tissue function.
In vivo and in vitro loss of microRNA 21, an LH-induced microRNA, results in mouse granulosa cell apoptosis.
doi:10.1095/biolreprod.109.081448
PMCID: PMC2907287  PMID: 20357270
apoptosis,; granulosa cells,; luteinizing hormone,; microRNA,; ovary
12.  Mechanism of chemoresistance mediated by miR-140 in human osteosarcoma and colon cancer cells 
Oncogene  2009;28(46):4065-4074.
In this study, our high throughput microRNA (miRNA) expression analysis revealed that the expression of miR-140 was associated with chemosensitivity in osteosarcoma tumor xenografts. Tumor cells ectopically transfected with miR-140 were more resistant to methotrexate (MTX) and 5-fluorouracil (5-FU). Overexpression of miR-140 inhibited cell proliferation in both osteosarcoma U-2 OS (wt-p53) and colon cancer HCT 116 (wt-p53) cell lines, but less so in osteosarcoma MG63 (mut-p53) and colon cancer HCT 116 (null-p53) cell lines. miR-140 induced p53 and p21 expression accompanied with G1 and G2 phase arrest only in cell lines containing wild type of p53. Histone deacetylase 4 (HDAC4) was confirmed to be one of the important targets of miR-140. The expression of endogenous miR-140 was significantly elevated in CD133+hiCD44+hi colon cancer stem-like cells which exhibit slow proliferating rate and chemoresistance. Blocking endogenous miR-140 by locked nucleic acid (LNA) modified anti-miR partially sensitized resistant colon cancer stem-like cells to 5-FU treatment. Taken together, our findings indicate that miR-140 is involved in the chemoresistance by reduced cell proliferation via G1 and G2 phase arrest mediated in part, through the suppression of HDAC4. miR-140 might be a candidate target to develop novel therapeutic strategy to overcome drug resistance.
doi:10.1038/onc.2009.274
PMCID: PMC2783211  PMID: 19734943
miR-140; chemosensitivity; histone deacetylase 4; cancer stem cells
13.  Cellular Uptakes, Biostabilities and Anti-miR-210 Activities of Chiral Arginine-PNAs in Leukaemic K562 Cells 
Chembiochem  2012;13(9):1327-1337.
A series of 18-mer peptide nucleic acids (PNAs) targeted against micro-RNA miR-210 was synthesised and tested in a cellular system. Unmodified PNAs, R8-conjugated PNAs and modified PNAs containing eight arginine residues on the backbone, either as C2-modified (R) or C5-modified (S) monomers, all with the same sequence, were compared. Two different models were used for the modified PNAs: one with alternated chiral and achiral monomers and one with a stretch of chiral monomers at the N terminus. The melting temperatures of these derivatives were found to be extremely high and 5 m urea was used to assess differences between the different structures. FACS analysis and qRT-PCR on K562 chronic myelogenous leukaemic cells indicated that arginine-conjugated and backbone-modified PNAs display good cellular uptake, with best performances for the C2-modified series. Resistance to enzymatic degradation was found to be higher for the backbone-modified PNAs, thus enhancing the advantage of using these derivatives rather than conjugated PNAs in the cells in serum, and this effect is magnified in the presence of peptidases such as trypsin. Inhibition of miR-210 activity led to changes in the erythroid differentiation pathway, which were more evident in mithramycin-treated cells. Interestingly, the anti-miR activities differed with use of different PNAs, thus suggesting a role of the substituents not only in the cellular uptake, but also in the mechanism of miR recognition and inactivation. This is the first report relating to the use of backbone-modified PNAs as anti-miR agents. The results clearly indicate that backbone-modified PNAs are good candidates for the development of very efficient drugs based on anti-miR activity, due to their enhanced bioavailabilities, and that overall anti-miR performance is a combination of cellular uptake and RNA binding.
doi:10.1002/cbic.201100745
PMCID: PMC3401907  PMID: 22639449
cell permeation; cellular differentiation; chiral PNA; microRNA; peptide nucleic acids; RNA
14.  Efficient inhibition of miR-155 function in vivo by peptide nucleic acids 
Nucleic Acids Research  2010;38(13):4466-4475.
MicroRNAs (miRNAs) play an important role in diverse physiological processes and are potential therapeutic agents. Synthetic oligonucleotides (ONs) of different chemistries have proven successful for blocking miRNA expression. However, their specificity and efficiency have not been fully evaluated. Here, we show that peptide nucleic acids (PNAs) efficiently block a key inducible miRNA expressed in the haematopoietic system, miR-155, in cultured B cells as well as in mice. Remarkably, miR-155 inhibition by PNA in primary B cells was achieved in the absence of any transfection agent. In mice, the high efficiency of the treatment was demonstrated by a strong overlap in global gene expression between B cells isolated from anti-miR-155 PNA-treated and miR-155-deficient mice. Interestingly, PNA also induced additional changes in gene expression. Our analysis provides a useful platform to aid the design of efficient and specific anti-miRNA ONs for in vivo use.
doi:10.1093/nar/gkq160
PMCID: PMC2910044  PMID: 20223773
15.  The liver-specific microRNA miR-122 controls systemic iron homeostasis in mice 
The Journal of Clinical Investigation  2011;121(4):1386-1396.
Systemic iron homeostasis is mainly controlled by the liver through synthesis of the peptide hormone hepcidin (encoded by Hamp), the key regulator of duodenal iron absorption and macrophage iron release. Here we show that the liver-specific microRNA miR-122 is important for regulating Hamp mRNA expression and tissue iron levels. Efficient and specific depletion of miR-122 by injection of a locked-nucleic-acid–modified (LNA-modified) anti-miR into WT mice caused systemic iron deficiency, characterized by reduced plasma and liver iron levels, mildly impaired hematopoiesis, and increased extramedullary erythropoiesis in the spleen. Moreover, miR-122 inhibition increased the amount of mRNA transcribed by genes that control systemic iron levels, such as hemochromatosis (Hfe), hemojuvelin (Hjv), bone morphogenetic protein receptor type 1A (Bmpr1a), and Hamp. Importantly, miR-122 directly targeted the 3′ untranslated region of 2 mRNAs that encode activators of hepcidin expression, Hfe and Hjv. These data help to explain the increased Hamp mRNA levels and subsequent iron deficiency in mice with reduced miR-122 levels and establish a direct mechanistic link between miR-122 and the regulation of systemic iron metabolism.
doi:10.1172/JCI44883
PMCID: PMC3069782  PMID: 21364282
16.  Functional Analysis of a Crustacean MicroRNA in Host-Virus Interactions 
Journal of Virology  2012;86(23):12997-13004.
Growing evidence from mammals suggests that host microRNAs (miRNAs) play important roles in the antiviral immune response. However, the roles of invertebrate miRNAs in response to virus infection remain to be investigated. Based on our previous studies, the shrimp miR-7 was found to be upregulated in response to white spot syndrome virus (WSSV) infection. In this study, the results showed that shrimp miR-7 could target the 3′-untranslated region (3′UTR) of the WSSV early gene wsv477, implying that miR-7 was involved in viral DNA replication. In insect High Five cells, the synthesized miR-7 significantly decreased the expression level of the fluorescent construct bearing the 3′UTR of wsv477 compared with the expression of the control constructs. When the activity of transfected miR-7 was blocked by locked-nucleic-acid (LNA)-modified anti-miR-7 oligonucleotide (AMO-miR-7), the repression of luciferase gene expression by miR-7 was relieved. In vivo, when the synthesized miR-7 was injected into shrimp, the numbers of WSSV genome copies/mg gills were 1,000-fold lower than those of WSSV only at 72 and 96 h postinfection. The results indicated that the blocking of endogenous miR-7 by AMO-miR-7 led to about a 10-fold increase of WSSV genome copies/mg gills in WSSV-infected shrimp compared with the control WSSV only. Further, it was revealed that the host Dicer1 was an important component for the biogenesis of miR-7, which had a large effect on virus infection. Therefore, our study revealed a novel regulatory function for an invertebrate miRNA in host-virus interactions by targeting the viral early gene.
doi:10.1128/JVI.01702-12
PMCID: PMC3497620  PMID: 23015693
17.  PEI-complexed LNA antiseeds as miRNA inhibitors 
RNA Biology  2012;9(8):1088-1098.
Antisense inhibition of oncogenic or other disease-related miRNAs and miRNA families in vivo may provide novel therapeutic strategies. However, this approach relies on the development of potent miRNA inhibitors and their efficient delivery into cells. Here, we introduce short seed-directed LNA oligonucleotides (12- or 14-mer antiseeds) with a phosphodiester backbone (PO) for efficient miRNA inhibition. We have analyzed such LNA (PO) antiseeds using a let-7a-controlled luciferase reporter assay and identified them as active miRNA inhibitors in vitro. Moreover, LNA (PO) 14-mer antiseeds against ongogenic miR-17–5p and miR-20a derepress endogenous p21 expression more persistently than corresponding miRNA hairpin inhibitors, which are often used to inhibit miRNA function. Further analysis of the antiseed-mediated derepression of p21 in luciferase reporter constructs - containing the 3′-UTR of p21 and harboring two binding sites for miRNAs of the miR-106b family - provided evidence that the LNA antiseeds inhibit miRNA families while hairpin inhibitors act in a miRNA-specific manner. The derepression caused by LNA antiseeds is specific, as demonstrated via seed mutagenesis of the miR-106b target sites. Importantly, we show functional delivery of LNA (PO) 14-mer antiseeds into cells upon complexation with polyethylenimine (PEI F25-LMW), which leads to the formation of polymeric nanoparticles. In contrast, attempts to deliver a functional seed-directed tiny LNA 8-mer with a phosphorothioate backbone (PS) by formulation with PEI F25-LMW remained unsuccessful. In conclusion, LNA (PO) 14-mer antiseeds are attractive miRNA inhibitors, and their PEI-based delivery may represent a promising new strategy for therapeutic applications.
doi:10.4161/rna.21165
PMCID: PMC3551863  PMID: 22894918
miRNA; miR-17-92; let-7a; antimiR; antiseed; LNA; PEI; p21; cancer
18.  Methylated DNA Binding Domain Protein 2 (MBD2) Coordinately Silences Gene Expression through Activation of the MicroRNA hsa-mir-496 Promoter in Breast Cancer Cell Line 
PLoS ONE  2013;8(10):e74009.
Methylated DNA binding protein 2 (MBD2) binds methylated promoters and suppresses transcription in cis through recruitment of a chromatin modification repressor complex. We show here a new mechanism of action for MBD2: suppression of gene expression indirectly through activation of microRNA hsa-mir-496. Overexpression of MBD2 in breast epithelial cell line MCF-10A results in induced expression and demethylation of hsa-mir-496 while depletion of MBD2 in a human breast cancer cell lines MCF-7 and MDA-MB231 results in suppression of hsa-mir-496. Activation of hsa-mir-496 by MBD2 is associated with silencing of several of its target genes while depletion of MBD2 leads to induction of hsa-mir-496 target genes. Depletion of hsa-mir-496 by locked nucleic acid (LNA) antisense oligonucleotide leads to activation of these target genes in MBD2 overexpressing cells supporting that hsa-mir-496 is mediating in part the effects of MBD2 on gene expression. We demonstrate that MBD2 binds the promoter of hsa-mir-496 in MCF-10A, MCF-7 and MDA-MB-231 cells and that it activates an in vitro methylated hsa-mir-496 promoter driving a CG-less luciferase reporter in a transient transfection assay. The activation of hsa-mir-496 is associated with reduced methylation of the promoter. Taken together these results describe a novel cascade for gene regulation by DNA methylation whereby activation of a methylated microRNA by MBD2 that is associated with loss of methylation triggers repression of downstream targets.
doi:10.1371/journal.pone.0074009
PMCID: PMC3812180  PMID: 24204564
19.  Inhibition of microRNA miR-92a induces apoptosis and necrosis in human acute promyelocytic leukemia 
Background:
MicroRNAs (miRNAs) are endogenous non-coding RNAs, 19-25 nucleotides in length, involved in post-transcriptional regulation of gene expression in a considerable majority of mRNAs. Different aspects of cellular activities like cell growth, proliferation, and differentiation are regulated by miRNAs through their regulatory effects on particular RNA species. In many tumors, up- or down-regulation of different miRNAs has been reported. In acute myeloid leukemia, up-regulation of miR-92a has been reported in human in-vitro studies.
Materials and Methods:
We performed inhibition of miR-92a in an acute promyelocytic leukemia cell line (HL-60), using locked nucleic acid (LNA) Antagomir. At different time points after LNA-anti-miR92a transfection, qRT-Real-Time-polymerase chain reaction (PCR) and Annexin-V/Propidium Iodide staining were performed and the data was analyzed using the Kruskal-Wallis and Mann-Whitney tests.
Results:
The assessment of the apoptosis and necrosis indicates that miR-92a inhibition can decrease the viable HL-60 cells and this is at least partially due to induction of apoptosis.
Conclusion:
These findings suggest the inhibition of miR-92a as a novel approach for treatment of Acute Promyelocytic Leukemia (APL).
doi:10.4103/2277-9175.125826
PMCID: PMC3950842  PMID: 24627869
MicroRNA; miR-92a; acute promyelocytic leukemia; locked nucleic acid
20.  MicroRNA-7 inhibition rescues age-associated loss of epidermal growth factor receptor and hyaluronan-dependent differentiation in fibroblasts 
Aging Cell  2013;13(2):235-244.
Age-related defects in fibroblast differentiation were previously shown to be associated with impaired hyaluronan synthase 2 (HAS2) and epidermal growth factor receptor (EGFR) function, with both required for normal fibroblast functionality. In fibroblasts, transforming growth factor-beta 1 (TGF-β1)-dependent phenotypic activation uses two distinct but co-operating pathways that involve TGF-β receptor (TGF-βR)/Smad2 activation and HA-mediated CD44-EGFR co-localization and signalling through extracellular signal-regulated kinase 1/2 (ERK1/2). The HA-mediated CD44-EGFR pathway was found to be compromised with in vitro aging, through loss of EGFR expression and a reduced movement of CD44 throughout the cellular membrane. Here, we also investigate the involvement of microRNAs (miRNAs) in age-related loss of differentiation, through investigation of miRNA-7 (miR-7) regulation of the HA-mediated EGFR-signalling pathway. The transcription of miR-7 was found to be upregulated in aged cells. In young cells, age-related loss of differentiation could be mimicked through transfection of pre-miR-7, and in aged cells, could be reversed through transfection of locked nucleic acids (LNA) targeting miR-7. Additionally, miR-7 was found to be involved in the regulation of CD44 membrane motility, which was downregulated in instances of miR-7 upregulation, and partially restorable through either miR-7 inhibition or HAS2 overexpression. The altered dynamics of CD44 in the cell membrane demonstrated a further action of miR-7 in regulating the HA-dependent CD44/EGFR pathway. We explain this novel mechanism of age-associated functional consequence due to miR-7 upregulation and demonstrate that it is reversible; highlighting miR-7 as a potential target for restoring the healing capabilities in chronic wounds in the elderly.
doi:10.1111/acel.12167
PMCID: PMC4331777  PMID: 24134702
epidermal growth factor receptor; fibroblast; hyaluronan; microRNA-7; transforming growth factor-β1; wound-healing
21.  Inhibition of miR-92a improves re-endothelialization and prevents neointima formation following vascular injury 
Cardiovascular Research  2014;103(4):564-572.
Aims
MicroRNA (miR)-92a is an important regulator of endothelial proliferation and angiogenesis after ischaemia, but the effects of miR-92a on re-endothelialization and neointimal lesion formation after vascular injury remain elusive. We tested the effects of lowering miR-92a levels using specific locked nucleic acid (LNA)-based antimiRs as well as endothelial-specific knock out of miR-92a on re-endothelialization and neointimal formation after wire-induced injury of the femoral artery in mice.
Methods and results
MiR-92a was significantly up-regulated in neointimal lesions following wire-induced injury. Pre-miR-92a overexpression resulted in repression of the direct miR-92a target genes integrin α5 and sirtuin1, and reduced eNOS expression in vitro. MiR-92a impaired proliferation and migration of endothelial cells but not smooth muscle cells. In vivo, systemic inhibition of miR-92a expression with LNA-modified antisense molecules resulted in a significant acceleration of re-endothelialization of the denuded vessel area. Genetic deletion of miR-92a in Tie2-expressing cells, representing mainly endothelial cells, enhanced re-endothelialization, whereas no phenotype was observed in mice lacking miR-92a expression in haematopoietic cells. The enhanced endothelial recovery was associated with reduced accumulation of leucocytes and inhibition of neointimal formation 21 days after injury and led to the de-repression of the miR-92a targets integrin α5 and sirtuin1.
Conclusion
Our data indicate that inhibition of endothelial miR-92a attenuates neointimal lesion formation by accelerating re-endothelialization and thus represents a putative novel mechanism to enhance the functional recovery following vascular injury.
doi:10.1093/cvr/cvu162
PMCID: PMC4145012  PMID: 25020912
Endothelial cells; MicroRNAs; Re-endothelialization; Neointimal formation
22.  EphB6 Receptor Modulates Micro RNA Profile of Breast Carcinoma Cells 
PLoS ONE  2011;6(7):e22484.
Breast carcinoma cells have a specific pattern of expression for Eph receptors and ephrin ligands. EphB6 has previously been characterized as a signature molecule for invasive breast carcinoma cells. The transcription of EphB6 is silenced in breast carcinoma cells and its re-expression leads to decreased invasiveness of MDA-MB-231 cells. Such differences in phenotypes of native and EphB6 expressing MDA-MB-231 cells relate to an altered profile of micro RNAs. Comparative hybridization of total RNA to slides containing all known miRNAs by using locked nucleic acid (LNA) miRCURY platform yielded a significantly altered profile of miRNAs in MDA-MB-231 cells stably transfected with EphB6. After applying a threshold of change and a p-value of <0.001, the list of significantly altered miRNAs included miR-16, miR-23a, miR-24, miR-26a, miR-29a, miR-100, miRPlus-E1172 and miRPlus-E1258. The array-based changes were validated by real-time qPCR of miR-16, miR-23a, miR-24 and miR-100. Except miRPlus-E1172 and miRPlus-E1258, the remaining six miRNAs have been observed in a variety of cancers. The biological relevance of target mRNAs was predicted by using a common-target selection approach that allowed the identification of SMARCA5, SMARCC1, eIF2C2, eIF2C4, eIF4EBP2, FKABP5, FKBP1A, TRIB1, TRIB2, TRIB3, BMPR2, BMPR1A and BMPR1B as important targets of a subset of significantly altered miRNAs. Quantitative PCR revealed that the levels of SMARCC1, eIFC4, eIF4EB2, FKBP1a, FKBP5, TRIB1, TRIB3, BMPR1a and BMPR2 transcripts were significantly decreased in MDA-MB-231 cells transfected with EphB6. These observations confirm targeting of specific mRNAs by miR-100, miR-23a, miR-16 and miR-24, and suggest that the kinase-deficient EphB6 receptor is capable of initiating signal transduction from the cell surface to the nucleus resulting in the altered expression of a variety of genes involved in tumorigenesis and invasion. The alterations in miRNAs and their target mRNAs also suggest indirect involvement of EphB6 in PI3K/Akt/mTOR pathways.
doi:10.1371/journal.pone.0022484
PMCID: PMC3139643  PMID: 21811619
23.  Comparison of different antisense strategies in mammalian cells using locked nucleic acids, 2′-O-methyl RNA, phosphorothioates and small interfering RNA 
Nucleic Acids Research  2003;31(12):3185-3193.
Locked nucleic acids (LNAs) and double-stranded small interfering RNAs (siRNAs) are rather new promising antisense molecules for cell culture and in vivo applications. Here, we compare LNA–DNA–LNA gapmer oligonucleotides and siRNAs with a phosphorothioate and a chimeric 2′-O-methyl RNA–DNA gapmer with respect to their capacities to knock down the expression of the vanilloid receptor subtype 1 (VR1). LNA–DNA–LNA gapmers with four or five LNAs on either side and a central stretch of 10 or 8 DNA monomers in the center were found to be active gapmers that inhibit gene expression. A comparative co-transfection study showed that siRNA is the most potent inhibitor of VR1–green fluorescent protein (GFP) expression. A specific inhibition was observed with an estimated IC50 of 0.06 nM. An LNA gapmer was found to be the most efficient single-stranded antisense oligonucleotide, with an IC50 of 0.4 nM being 175-fold lower than that of commonly used phosphorothioates (IC50 ∼70 nM). In contrast, the efficiency of a 2′-O-methyl-modified oligonucleotide (IC50 ∼220 nM) was 3-fold lower compared with the phosphorothioate. The high potency of siRNAs and chimeric LNA–DNA oligonucleotides make them valuable candidates for cell culture and in vivo applications targeting the VR1 mRNA.
PMCID: PMC162243  PMID: 12799446
24.  Temporal and Spatial Expression Patterns of miR-302 and miR-367 During Early Embryonic Chick Development 
The microRNAs (miRNAs) are small, non-coding RNAs that modulate protein expression by interfering with target mRNA translation or stability. miRNAs play crucial roles in various functions such as cellular, developmental, and physiological processes. The spatial expression patterns of miRNAs are very essential for identifying their functions. The expressions of miR-302 and miR-367 are critical in maintaining stemness of pluripotent stem cells, including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) but their functions in early development are not fully elucidated. So, we used Locked Nucleic Acid (LNA) probes to perform in situ hybridization and confirmed the temporal and spatial distribution patterns during early chick development. As a result, we found that miR-302 and miR-367 were expressed in various tissues such as primitive steak, neural ectoderm, neural plate, neural fold, neural tube, notochord, and oral cavity. Specially, we confirmed that miR-302 and miR-367 were strongly expressed in neural folds in HH8 to HH10. miR-302 was expressed on dorsal part of the neural tube but miR-367 was expressed on lateral and ventral parts of the neural tube. And also we performed quantitative stem-loop real-time PCR to analyze global expression level of miR-302 and miR-367. miR-302 and miR-367 expression was sustained before Hamburger and Hamilton stage (HH) 14. Thus, the temporal and spatial expression patterns of miR-302 and miR-367 may provide us information of the role of these miRNAs on tissue formation during early chick development.
doi:10.15283/ijsc.2014.7.2.162
PMCID: PMC4249900  PMID: 25473455
Early chick development; miRNAs; LNA; In situ hybridization
25.  Intracellular inhibition of hepatitis C virus (HCV) internal ribosomal entry site (IRES)-dependent translation by peptide nucleic acids (PNAs) and locked nucleic acids (LNAs) 
Nucleic Acids Research  2004;32(13):3792-3798.
Hepatitis C virus (HCV) is the major etiological agent of non-A, non-B hepatitis. Current therapies are not effective in all patients and can result in the generation of resistant mutants, leading to a need for new therapeutic options. HCV has an RNA genome that contains a well-defined and highly conserved secondary structure within the 5′-untranslated region. This structure is known as the internal ribosomal entry site (IRES) and is necessary for translation and viral replication. Here, we test the hypothesis that antisense peptide nucleic acid (PNA) and locked nucleic acid (LNA) oligomers can bind key IRES sequences and block translation. We used lipid-mediated transfections to introduce PNAs and LNAs into cells. Our data suggest that PNAs and LNAs can invade critical sequences within the HCV IRES and inhibit translation. Seventeen base PNA or LNA oligomers targeting different regions of the HCV IRES demonstrated a sequence-specific dose–response inhibition of translation with EC50 values of 50–150 nM. Inhibition was also achieved by PNAs ranging in length from 15 to 21 bases. IRES-directed inhibition of gene expression widens the range of mechanisms for antisense inhibition by PNAs and LNAs and may provide further therapeutic lead compounds for the treatment of HCV.
doi:10.1093/nar/gkh706
PMCID: PMC506796  PMID: 15263060

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