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Fig. 4 | Molecular Cancer

Fig. 4

From: A nuclear function for an oncogenic microRNA as a modulator of snRNA and splicing

Fig. 4

miR-10b regulates U6 snRNA levels, stability, and conformation. a Sequence, putative secondary structure, nucleotide modifications of human U6, and miR-10b binding to U6 (top panel). The structure and modifications of human U6 have not been experimentally determined, and are shown to mimic that of yeast U6 [30]. Positions of U6-targeting ASOs are shown in the lower panel. U6 ASO-1 binds to the 3’end of U6, similarly to miR-10b. b qRT-PCR analysis of U6 levels in glioma cells and GSCs transfected with either U6 ASOs, miR-10b inhibitor, mimic, or corresponding control oligonucletides (mean ± SD, n = 3). P values were calculated using two-tail unpaired t-test. c Northern blot analysis of glioma cells transfected with either U6 ASOs, miR-10b inhibitor, or mimic, with a U6-specific probe, demonstrates that U6 levels are regulated by miR-10b. The lower panel demonstrates that other small RNA species, resolved by denaturing electrophoresis, are not regulated by miR-10b. d qRT-PCR analysis of U6 levels in LN229 cells transfected with miR-10b inhibitor (top) or mimic (bottom), and treated with 5 μg/ml Actinomycin D demonstrates that miR-10b reduces U6 half-life (mean ± SD, n = 3). P values were calculated using two-way ANOVA. e iCLIP with antibodies recognizing pseudouridylation (top) and m6A methylation (bottom) on LN229 and GBM8 cells transfected with either miR-10b inhibitor or mimic, and the corresponding control oligonucleotides, followed by U6 qRT-PCR detection, demonstrate that U6 modification are regulated by miR-10b (mean ± SD, n = 3). P values were calculated using two-tail unpaired t-test. f Representative native Northern blotting with U6-specific probe demonstrates that miR-10b modulation leads to the appearance of an additional U6 structural variant. g Glioma cells treated with Actinomycin D, followed by qRT-PCR for miR-10b and U6 levels, exhibit high stability of miR-10b (mean ± SD, n = 3). P values were calculated using two-way ANOVA. h Expression of miR-10b and U6 in 155 GBM samples representing TCGA/ GDC Pan-Cancer dataset, retrieved from UCSC XENA browser (https://xena.ucsc.edu/). The RNAseq data are FPKM normalized and presented as log2(FPKM-uq + 1). The center line shows the median expression, the box limits indicate the 25th and 75th percentiles, and whiskers extend to the minimum and maximum values. i Expression of miR-10b and U6 in 96 GBM samples retrieved from Clinical Proteomic Tumor Analysis Consortium (CPTAC) (portal.gdc.cancer.gov) [31]. miR-10b and U6 levels were plotted from two separate sequencing libraries generated on the same RNA samples. The data are FPKM normalized using the GDC’s RNA-Seq pipeline and expressed as log2(FPKM-uq + 1). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001

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