Dataset Information

Polysome shift assay for direct measurement of miRNA inhibition by anti-miRNA drugs.

ABSTRACT: Anti-miRNA (anti-miR) oligonucleotide drugs are being developed to inhibit overactive miRNAs linked to disease. To help facilitate the transition from concept to clinic, new research tools are required. Here we report a novel method--miRNA Polysome Shift Assay (miPSA)--for direct measurement of miRNA engagement by anti-miR, which is more robust than conventional pharmacodynamics using downstream target gene derepression. The method takes advantage of size differences between active and inhibited miRNA complexes. Active miRNAs bind target mRNAs in high molecular weight polysome complexes, while inhibited miRNAs are sterically blocked by anti-miRs from forming this interaction. These two states can be assessed by fractionating tissue or cell lysates using differential ultracentrifugation through sucrose gradients. Accordingly, anti-miR treatment causes a specific shift of cognate miRNA from heavy to light density fractions. The magnitude of this shift is dose-responsive and maintains a linear relationship with downstream target gene derepression while providing a substantially higher dynamic window for aiding drug discovery. In contrast, we found that the commonly used 'RT-interference' approach, which assumes that inhibited miRNA is undetectable by RT-qPCR, can yield unreliable results that poorly reflect the binding stoichiometry of anti-miR to miRNA. We also demonstrate that the miPSA has additional utility in assessing anti-miR cross-reactivity with miRNAs sharing similar seed sequences.

SUBMITTER: Androsavich JR

PROVIDER: S-EPMC4737174 | biostudies-literature | 2016 Jan

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Polysome shift assay for direct measurement of miRNA inhibition by anti-miRNA drugs.

Androsavich John R JR Sobczynski Daniel J DJ Liu Xueqing X Pandya Shweta S Kaimal Vivek V Owen Tate T Liu Kai K MacKenna Deidre A DA Chau B Nelson BN

Nucleic acids research 20150917 2

Anti-miRNA (anti-miR) oligonucleotide drugs are being developed to inhibit overactive miRNAs linked to disease. To help facilitate the transition from concept to clinic, new research tools are required. Here we report a novel method--miRNA Polysome Shift Assay (miPSA)--for direct measurement of miRNA engagement by anti-miR, which is more robust than conventional pharmacodynamics using downstream target gene derepression. The method takes advantage of size differences between active and inhibited ...[more]

PMID: 26384419

Similar Datasets

Project description:Dysregulation of the anti-apoptotic protein, cellular FLICE-like inhibitory protein (c-FLIP), has been associated with tumorigenesis and chemoresistance in various human cancers. Therefore, c-FLIP is an excellent target for therapeutic intervention. MicroRNAs (miRNAs) are small non-coding RNAs that are involved in tumorigenesis, tumor suppression, and resistance or sensitivity to anti-cancer drugs. However, whether miRNAs can suppress c-FLIPL expression in cancer cells is unclear. The aim of this study was to identify miRNAs that could inhibit the growth of renal cancer cells and induce cell death by inhibiting c-FLIPL expression. We found that MiRNA-708 and c-FLIPL expression were inversely correlated. While c-FLIPL expression was upregulated, miRNA-708 was rarely expressed in renal cancer cells. Luciferase reporter assays demonstrated that miRNA-708 negatively regulated c-FLIPL expression by binding to the miRNA-708 binding site in the 3' untranslated region (3'UTR) of c-FLIPL. Ectopic expression of miRNA-708 increased the accumulation of sub-G1 populations and cleavage of procaspase-3 and PARP, which could be prevented by pretreatment with the pan-caspase inhibitor, Z-VAD. Ectopic expression of miRNA-708 also increased the sensitivity to various apoptotic stimuli such as tumor necrosis factor-related apoptosis-inducing ligand, doxorubicin (Dox), and thapsigargin in Caki cells. Interestingly, miRNA-708 specifically repressed c-FLIPL without any change in c-FLIPs expression. In contrast, inhibition of endogenous miRNA-708 using antago-miRNAs resulted in an increase in c-FLIPL protein expression. The expression of c-FLIPL was upregulated in renal cell carcinoma (RCC) tissues compared to normal tissues. In contrast, miRNA-708 expression was reduced in RCC tissues. Finally, miRNA-708 enhanced the tumor-suppressive effect of Dox in a xenograft model of human RCC. In conclusion, miRNA-708 acts as a tumor suppressor because it negatively regulates the anti-apoptotic protein c-FLIPL and regulates the sensitivity of renal cancer cells to various apoptotic stimuli.

Dataset Information

Polysome shift assay for direct measurement of miRNA inhibition by anti-miRNA drugs.

Publications

Polysome shift assay for direct measurement of miRNA inhibition by anti-miRNA drugs.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets