Fig. 1

Identification of circ-AKT3 as a novel AKT gene alternative splicing transcript. a Upper, Volcano plot of circRNA expression. X-axis: log2 ratio of circRNA expression levels between normal and tumor tissues. Y-axis: the FDR value (−log10 transformed) of circRNAs. The green dot indicates hsa_circ_0017250 (circ-AKT3). Lower, identified three circRNAs from AKT gene in our RNA-seq. b Illustration of the complete ORF in circ-AKT3. Circ-AKT3 used an overlap start-stop codon UAAUGA. c Illustration of the annotated genomic region of AKT3, the putative different mRNA splicing forms (linear splicing and ‘head-to-tail’ splicing) and the validation strategy for the circular exon 3–7 (circ-AKT3). Sanger sequencing following PCR conducted using the indicated divergent flanking primers showed the ‘head-to-tail’ splicing of circ-AKT3 in HEK293T cells. d Left, relative RNA level of circ-AKT3 and linear AKT3 in different time point. Right, relative RNA level of circ-AKT3 and linear AKT3 treated with RNase R. Error bars represent three independent experiments, **, p < 0.01, ***, p < 0.001. e Left, circ-AKT3 overexpression plasmid vector (not shown) and two circ-AKT3 junction shRNAs and a control shRNA were designed, transfected into HEK293T cells. Right, relative circ-AKT3 and linear AKT3 RNA level were decided by q-PCR. f Left, Fluorescence in situ hybridization (FISH) with junction-specific probes were used to decide the localization of circ-AKT3. Circ-AKT3 overexpression or shRNA were used independently or in combination to show the specificity of these probes. Scale bars, 20 μM. Middle, cytoplasmic and nuclear fractions were isolated, circ-AKT3 and linear AKT3 expression were decided. β-actin and U2 RNA served as cytoplasmic and nuclear RNA markers. Right, total RNA from HEK293T cells were reversely transcript with Oligo dT primers or random primers, and circ-AKT3 or linear AKT3 mRNA were decided by q-PCR. Error bars represent three independent experiments, *, p < 0.05, ***, p < 0.001. g Northern blots using the junction-specific circular probe were used to detect circ-AKT3 in circ-AKT3 overexpressed or plus circ-AKT3 shRNA transfected NSC and NHA cells. h Relative circ-AKT3 and linear AKT3 RNA level of GBM clinical samples and paired adjacent normal tissues in a cohort of 38 GBM patients, or of NSC, NHA and GBM cell lines. Error bars represent three independent experiments, ***, p < 0.001. i Circ-AKT3 RNA level and its correlation with IDH1 status or molecular subtypes. Error bars represent three independent experiments