miR-17-5p Regulates Endocytic Trafficking through Targeting TBC1D2/Armus

abstract: ...miRNA cluster miR-17-92 is known as oncomir-1 due to its potent oncogenic function. miR-17-92 is a polycistronic cluster that encodes 6 miRNAs, and can both facilitate and inhibit cell proliferation. Known targets of miRNAs encoded by this cluster are largely regulators of cell cycle progression and apoptosis. Here, we show that miRNAs encoded by this cluster and sharing the seed sequence of miR-17 exert their influence on one of the most essential cellular processes endocytic trafficking. By mRNA expression analysis we identified that regulation of endocytic trafficking by miR-17 can potentially be achieved by targeting of a number of trafficking regulators. We have thoroughly validated TBC1D2/Armus, a GAP of Rab7 GTPase, as a novel target of miR-17. Our study reveals regulation of endocytic trafficking as a novel function of miR-17, which might act cooperatively with other functions of miR-17 and related miRNAs in health and disease.

...PLoS One PLoS ONE plos plosone PLoS ONE 1932-6203 Public Library of Science San Francisco, USA 23285084 3527550 PONE-D-12-23156 10.1371/journal.pone.0052555 Research Article Biology Genetics Molecular Genetics Gene Regulation Gene Expression Molecular Cell Biology Nucleic Acids RNA RNA interference Cell Growth Gene Expression Membranes and Sorting Medicine Oncology Basic Cancer Research miR-17-5p Regulates Endocytic Trafficking through Targeting TBC1D2/Armus miR-17 Regulates Endocytic

... regulation of endocytic trafficking by miR-17 can potentially be achieved by targeting of a number of trafficking regulators. We have thoroughly validated TBC1D2/Armus, a GAP of Rab7 GTPase, as a novel target of miR-17. Our study reveals regulation of endocytic trafficking as a novel function of miR-17, which might act cooperatively with other functions of miR-17 and related miRNAs in health and disease. This work was supported by BMBF FORSYS ViroQuant (#0313923), BMBF SysTec (0315523A) and Baden

... we could identify several trafficking-related genes (TBC1D2/Armus, LDLR, M6PR and ASAP2), that were down-regulated when miR-17 was over-expressed. Furthermore, by combining different approaches we validated TBC1D2/Armus, a GAP of Rab7 GTPase, as a novel target of miR-17. Our work expands the repertoire of miR-17 cellular activities beyond regulation of cell cycle and transcription. By regulating endocytic trafficking, miR-17 seed family potentially influences signalling and cell adhesion that, in turn, might cooperate with other functions of miR-17 in health and disease. Materials and Methods Materials Polyclonal rabbit anti-TBC1D2 antibody were described in [30] . Polyclonal rabbit anti-LDLR antibody was from Cayman Chemicals (Ann Arbor, MI, USA), monoclonal mouse anti-tubulin- (clone DM1A) was from Thermo Fisher Scientific (Waltham MA, USA), polyclonal anti-FLAG antibodies were from Sigma-Aldrich (St. Louis, USA) Secondary anti-mouse and anti-rabbit IgG HRP-conjugated antibodies were from R&D Systems (Minneapolis, MN, USA). Secondary goat Alexa647-conjugated anti-mouse IgG antibody, Dil-LDL, EGF-Alexa555 and transferrin-Alexa568 were from Invitrogen (Carlsbad, CA, USA). siRNAs targeting human TBC1D2 (SI02807518 and SI04239494), LDLR (SI00011186 and SI03024525), EGFR (SI02663983 and SI02660140), TfR (SI02780715 and SI02781121) and non-silencing siRNA All Stars (SI03650318) were from Qiagen (Hilden, Germany). Cy3-labeled siRNA targeting INCENP (28431) and Pre-miRs were from Ambion

... for miR-320a, and ID 001093 for small nuclear RNU6B) and TaqMan gene expression assays (ID Hs00917985_m1 for TBC1D2, ID Hs00181192_m1 for LDLR, and ID Hs99999905_m1 for endogenous control GAPDH gene) were from Applied Biosystems (Foster City, CA, USA). Cloning of Luciferase Reporters and Plasmid Encoding LDLR Double stranded DNA fragments encoding miRNA-binding sites and full-length 3UTRs of TBC1D2 and LDLR genes were inserted into Xho I- and Not I-digested psiCheck-2 vector (Promega, Madison, WI, USA) immediately downstream of the stop codon of the Renilla luciferase gene. The plasmids with mutated or deleted miR-17-5p binding site in the TBC1D2 3UTR were generated by using the Phusion site-directed mutagenesis kit (Finnzymes, Vantaa, Finland). HeLa cells were co-transfected with the respective luciferase reporter construct, Pre-miRs and controls at a final concentration of 50 nM. Renilla and firefly luciferase activities were measured consecutively using the Dual-Luciferase Assay System

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Gene: TBC1D2 - TBC1 domain family, member 2 - (NCBI)

Protein : 2dhk

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