Identification, amplification and characterization of miR-17-92 from canine tissue

Boggs, R. Michelle; Moody, Jessica A.; Long, Charles R.; Tsai, Kate L.; Murphy, Keith E.

doi:10.1016/j.gene.2007.08.015

Cited by 30 publications

(19 citation statements)

References 25 publications

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“…However, clustered miRNAs may accumulate differentially in vivo as a result of posttranscriptional processing or stability, 29 and endogenous miR-18 appears to be expressed at lower levels than the other miRNAs in the liver. 30 Thus, it might not be possible to engineer this miRNA scaffold to achieve high-level expression of mature exogenous miRNAs in the liver, and the use of the last miRNA in the cluster (i.e., miR-92), as an exogenous miRNA scaffold, may be a better choice.…”

Section: Discussionmentioning

confidence: 99%

Inhibition of hepatitis C virus replication using adeno-associated virus vector delivery of an exogenous anti-hepatitis C virus microrna cluster

et al. 2010

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RNA interference (RNAi) is being evaluated as an alternative therapeutic strategy for hepatitis C virus (HCV) infection. The use of viral vectors encoding short hairpin RNAs (shRNAs) has been the most common strategy employed to provide sustained expression of RNAi effectors. However, overexpression and incomplete processing of shRNAs has led to saturation of the endogenous miRNA pathway, resulting in toxicity. The use of endogenous microRNAs (miRNAs) as scaffolds for short interfering (siRNAs) may avoid these problems, and miRNA clusters can be engineered to express multiple RNAi effectors, a feature that may prevent RNAi-resistant HCV mutant generation. We exploited the endogenous miRNA-17-92 cluster to generate a polycistronic primary miRNA that is processed into five mature miRNAs that target different regions of the HCV genome. All five anti-HCV miRNAs were active, achieving up to 97% inhibition of Renilla luciferase (RLuc) HCV reporter plasmids. Self-complementary recombinant adeno-associated virus (scAAV) vectors were chosen for therapeutic delivery of the miRNA cluster. Expression of the miRNAs from scAAV inhibited the replication of cell culture-propagated HCV (HCVcc) by 98%, and resulted in up to 93% gene silencing of RLuc-HCV reporter plasmids in mouse liver. No hepatocellular toxicity was observed at scAAV doses as high as 5 3 10 11 vector genomes per mouse, a dose that is approximately five-fold higher than doses of scAAV-shRNA vectors that others have shown previously to be toxic in mouse liver. Conclusion: We have demonstrated that exogenous anti-HCV miRNAs induce gene silencing, and when expressed from scAAV vectors inhibit the replication of HCVcc without inducing toxicity. The combination of an AAV vector delivery system and exploitation of the endogenous RNAi pathway is a potentially viable alternative to current HCV treatment regimens. (HEPATOLOGY 2010;52:1877-1887 H epatitis C virus (HCV) infection remains a major worldwide health care problem, because approximately 3% of the world population is chronically infected with this virus, which causes viral hepatitis and can lead to cirrhosis and hepatocellular carcinoma.1 HCV replicates in the cytoplasm by a virally encoded RNA-dependent RNA polymerase (nonstructural protein 5B [NS5B]), and like most RNA polymerases, NS5B has low fidelity and incorporates mutations into its genome at a rate of $10 À4 base substitutions/nucleotide, 2 generating $one mutation per round of replication. Thus, HCV shows extraordinary genetic diversity with six major genotypes, at least 50 subtypes, and millions of quasispecies. This feature of HCV has made vaccine and drug development extremely challenging. Although HCV infections are currently managed with a combination of pegylated interferon-a and ribavirin, this regimen is successful in achieving a sustained virological response in only approximately 50% of patients infected with

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Section: Discussionmentioning

confidence: 99%

Inhibition of hepatitis C virus replication using adeno-associated virus vector delivery of an exogenous anti-hepatitis C virus microrna cluster

et al. 2010

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“…To date, microarray (4,(7)(8)(9)18,19,(21)(22)(23)25,35,36), northern blot analysis (6,18,24,37), and RT-PCR (7,11,16,27,38,39) have been reported for screening endogenous miRNAs. However, none of these methods were used to characterize exogenous, synthetic miRNAs.…”

Section: Discussionmentioning

confidence: 99%

A Novel Ultrasensitive Hybridization-Based ELISA Method for 2-Methoxyphosphorothiolate MicroRNAs and Its In vitro and In vivo Application

Chan

Liu

Xie

et al. 2010

AAPS J

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Abstract. MicroRNAs (miRNAs) are endogenous, small non-coding RNAs that bind to target mRNAs and regulate their expression. Recent evidence has indicated the involvement of miRNAs in human malignancies. It has been suggested that aberrantly down-regulated or up-regulated miRNAs may be replaced with synthetic miRNAs or antagomiRNAs, respectively, and restore normal cell functions. As therapeutic development requires analytical support, we developed and validated an ultrasensitive and selective assay for quantification of synthetic 2′-methoxyphosphorothiolate-miRNA in mouse plasma and cell lysate for the first time. The method is based on a hybridization-ligation fluorescence enzyme-linked immunosorbent assay and has provided a linear dynamic range of 10-1,000,000 pM for three synthetic miRNAs both singly and in a mixture. The intra-and inter-day coefficients of variation were <20% and the accuracy values nearly 100%. Using this assay, we performed pharmacokinetic studies of three synthetic miRNAs in mice treated with a single i.v. bolus dose of 7.5 mg kg −1. The 2-methoxyphosphorothiolate-miRNAs reached peak concentrations in the μM and nM ranges in plasma and bone marrow, respectively, and remained measurable at 24 h. These concentrations are in a range that shows biological activities. We conclude that this method provides a general and valuable tool for the pharmacologic study and clinical development of synthetic miRNAs.

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“…In addition to their role in cancer, miR-17-92 members play important roles in the normal development and differentiation of various cells in animals. In mouse, miR-17-5p was detected in the heart, brain, kidney, and lung, whereas miR-20a was detected in the heart, brain, liver, and lung [34][35][36]. The miRNAs miR-106, miR-17-5p, and miR-20a were differentially expressed during mouse embryonic development and controlled the differentiation of embryonic stem cells [24].…”

Section: Regulation Of Glucose Phosphate Isomerase By Mirnamentioning

confidence: 99%

Regulation of Glucose Phosphate Isomerase by the 3′UTR-Specific miRNAs miR-302b and miR-17-5p in Chicken Primordial Germ Cells1

Rengaraj

Park

Lee

et al. 2013

Biology of Reproduction

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Glucose phosphate isomerase (GPI) involves in the reversible isomerization of glucose-6-phosphate to fructose-6-phosphate in glucose pathways. Because glucose metabolism is crucial for the proliferation and differentiation of embryonic stem and germ cells, reducing GPI expression may affect the characteristic features of these cells. MicroRNAs (miRNAs) have been shown to regulate genes. In the present study, we investigated the regulation of chicken GPI by its predicted miRNAs. We determined the expression patterns of seven GPI 3'-untranslated region (3'UTR)-targeting miRNAs, including the gga-miR-302 cluster, gga-miR-106, gga-miR-17-5p, and gga-miR-20 cluster in chicken primordial germ cells (PGCs), compared with GPI mRNA. Among the miRNAs, gga-miR-302b, gga-miR-302d, and gga-miR-17-5p were expressed at lower levels than GPI mRNA. The remaining four miRNAs-gga-miR-302c, gga-miR-106, gga-miR-20a, and gga-miR-20b-were expressed at higher levels than the expression of GPI mRNA. Next, we cotransfected four candidate miRNAs-gga-miR-302b, gga-miR-106, gga-miR-17-5p, and gga-miR-20a-with GPI 3'UTR into 293FT cells by dual fluorescence reporter assay. Overexpression of gga-miR-302b and gga-miR-17-5p miRNAs in 293FT cells significantly downregulated GPI expression, whereas the other two miRNAs had no effect. Then, knockdown and overexpression of these four candidate miRNAs were performed by RNA interference assay to regulate GPI in PGCs. In the RNA interference assay, the expression of GPI was greatly regulated by gga-miR-302b and gga-miR-17-5p. Finally, we examined the effects of GPI regulation on PGC proliferation and migration. Our results suggested that the regulation of GPI by gga-miR-302b and gga-miR-17-5p affected PGCs proliferation. However, regulation of GPI using these two miRNAs did not affect the migration of PGCs into embryonic gonads.

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Identification, amplification and characterization of miR-17-92 from canine tissue

Cited by 30 publications

References 25 publications

Inhibition of hepatitis C virus replication using adeno-associated virus vector delivery of an exogenous anti-hepatitis C virus microrna cluster

Inhibition of hepatitis C virus replication using adeno-associated virus vector delivery of an exogenous anti-hepatitis C virus microrna cluster

A Novel Ultrasensitive Hybridization-Based ELISA Method for 2-Methoxyphosphorothiolate MicroRNAs and Its In vitro and In vivo Application

Regulation of Glucose Phosphate Isomerase by the 3′UTR-Specific miRNAs miR-302b and miR-17-5p in Chicken Primordial Germ Cells1

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