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. 2019 Aug 30;18(1):131.
doi: 10.1186/s12943-019-1056-5.

A novel tumor suppressor protein encoded by circular AKT3 RNA inhibits glioblastoma tumorigenicity by competing with active phosphoinositide-dependent Kinase-1

Xin Xia #  1   2 , Xixi Li #  1   2 , Fanying Li #  1   2 , Xujia Wu  1   2 , Maolei Zhang  1   2 , Huangkai Zhou  1   2 , Nunu Huang  1   2 , Xuesong Yang  1   2 , Feizhe Xiao  3 , Dawei Liu  4 , Lixuan Yang  1   2 , Nu Zhang  5   6
Affiliations

V体育2025版 - A novel tumor suppressor protein encoded by circular AKT3 RNA inhibits glioblastoma tumorigenicity by competing with active phosphoinositide-dependent Kinase-1

Xin Xia et al. Mol Cancer. .

Erratum in (VSports在线直播)

Abstract

Background: The RTK/PI3K/AKT pathway plays key roles in the development and progression of many cancers, including GBM. As a regulatory molecule and a potential drug target, the oncogenic role of AKT has been substantially studied. Three isoforms of AKT have been identified, including AKT1, AKT2 and AKT3, but their individual functions in GBM remain controversial VSports手机版. Moreover, it is not known if there are more AKT alternative splicing variants. .

Methods: High-throughput RNA sequencing and quantitative reverse transcription-PCR were used to identify the differentially expressed circRNAs in GBM samples and in paired normal tissues. High throughput RNA sequencing was used to identify circ-AKT3 regulated signaling pathways. Mass spectrometry, western blotting and immunofluorescence staining analyses were used to validate AKT3-174aa expression. The tumor suppressive role of AKT3-174aa was validated in vitro and in vivo. The competing interaction between AKT3-174aa and p-PDK1 was investigated by mass spectrometry and immunoprecipitation analyses V体育安卓版. .

Results: Circ-AKT3 is a previously uncharacterized AKT transcript variant. Circ-AKT3 is expressed at low levels in GBM tissues compared with the expression in paired adjacent normal brain tissues. Circ-AKT3 encodes a 174 amino acid (aa) novel protein, which we named AKT3-174aa, by utilizing overlapping start-stop codons V体育ios版. AKT3-174aa overexpression decreased the cell proliferation, radiation resistance and in vivo tumorigenicity of GBM cells, while the knockdown of circ-AKT3 enhanced the malignant phenotypes of astrocytoma cells. AKT3-174aa competitively interacts with phosphorylated PDK1, reduces AKT-thr308 phosphorylation, and plays a negative regulatory role in modulating the PI3K/AKT signal intensity. .

Conclusions: Our data indicate that the impaired circRNA expression of the AKT3 gene contributes to GBM tumorigenesis, and our data corroborate the hypothesis that restoring AKT3-174aa while inhibiting activated AKT may provide more benefits for certain GBM patients. VSports最新版本.

Keywords: AKT3; Glioblastoma; PDK1; circRNA. V体育平台登录.

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Conflict of interest statement

The authors declare that they have no competing interests.

"VSports注册入口" Figures

Fig. 1
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
Fig. 2
Fig. 2
Circ-AKT3 encodes a 174aa novel protein termed as AKT3-174aa. a Upper panel, the putative ORF in circ-AKT3. Lower panel, the sequences of the putative ORF are shown. Note that the circ-AKT3 used an overlap start-stop codon. b The putative IRES activity in circ-AKT3 was tested. Upper panel, IRES sequences in circ-AKT3 or its different truncations/mutation were cloned between Rluc and Luc reporter genes with independent start and stop codons. Lower panel, the relative luciferase activity of Luc/Rluc in the above vectors was tested. Error bars represent three independent experiments, **, p < 0.01, ***, p < 0.001. c Illustration of AKT3-174aa sequence and AKT3 sequence. The antibody used in the study recognized both proteins. d AKT3 and AKT3-174aa expression were detected in established cell lines and several paired GBM samples. e U251 cells were transfected with empty vector, circ-AKT3 IRES mutated vector, circ-AKT3 vector and linearized AKT3 vector, respectively. Circ-AKT3, AKT3 and AKT3-174aa level were decided. Error bars represent three independent experiments, ***, p < 0.001. f Total proteins from circ-AKT3 or control plasmid-transfected U251 cells were separated via SDS-PAGE. AKT3-174aa overexpression was confirmed by immunoblotting. The differential gel bands between 26 kD and 34 kD was cut and subjected to LC-MS/MS. The identified AKT3-174aa amino acids are shown in red. g NHA cells were transfected with control shRNA or circ-AKT3 shRNAs. Circ-AKT3, AKT3 and AKT3-174aa level were decided. Error bars represent three independent experiments, ***, p < 0.001. h Flag tagged AKT3-174aa was transfected into U251 cells. Immunofluorescence staining using anti-Flag was performed to show the AKT3-174aa cellular localization. Scale bars, 20 μM. i Semi-quantitative analysis of AKT3-174aa expression level and GBM patient overall survival (OS) in the 38 patient cohort. **, p < 0.01. j Left, semi-quantitative analysis of AKT3 expression level and GBM patient overall survival (OS) in the 38 patient cohort. Right, semi-quantitative analysis of AKT3 expression level and GBM patient overall survival (OS) in TCGA data base
Fig. 3
Fig. 3
Tumor suppressive functions of AKT3-174aa in glioma cell lines. a U251, U373 glioma cells were transfected with circ-AKT3, linearized AKT3-174aa and control vectors; SW1783, Hs683 glioma cells were transfected with circ-AKT3 shRNA or control shRNA. AKT1, AKT2, AKT3 and AKT3-174aa level were determined by immunoblot. b Upper, cell proliferation, EdU incorporation of circ-AKT3, linearized AKT3-174aa transfected U251 and U373 cells and their control cells were decided. Lower, cell proliferation, EdU incorporation of circ-AKT3 shRNA transfected SW1783 and Hs683 cells and their control cells were decided. Error bars represent three independent experiments, *, p < 0.05, **, p < 0.01, ***, p < 0.001. c Left, plate colony formation and anchorage independent growth of circ-AKT3, linearized AKT3-174aa transfected U251 and U373 cells and their control cells were decided. Right, plate colony formation and anchorage independent growth of circ-AKT3 shRNA transfected SW1783 and Hs683 cells and their control cells were decided. Error bars represent three independent experiments, *, p < 0.05.d Upper, radiation resistance (cell cytometry or H2AX foci counting followed by 12Gy radiation) of circ-AKT3, linearized AKT3-174aa transfected U251 and U373 cells and their control cells were decided. Lower, radiation resistance (cell cytometry followed by 12Gy radiation) of circ-AKT3 shRNA transfected SW1783 and Hs683 cells and their control cells were decided. Error bars represent three independent experiments, *, p < 0.05
Fig. 4
Fig. 4
AKT3-174aa, but not circ-AKT3, exerts the biological function. a SW1783 and Hs683 cells were transfected with circ-AKT3 shRNA or circ-AKT3 shRNA plus linearized AKT3-174aa overexpression plasmid. AKT3-174aa expression was decided by immunoblot. b Cell proliferation and cell cycle analysis of SW1783 and Hs683 expressing circ-AKT3 shRNA, circ-AKT3 shRNA plus linearized AKT3-174aa and their control cells. Error bars represent three independent experiments, *, p < 0.05, **, p < 0.01, ***, p < 0.001. c Plate colony formation and anchorage independent growth of SW1783 and Hs683 expressing circ-AKT3 shRNA, circ-AKT3 shRNA plus linearized AKT3-174aa and their control cells. Error bars represent three independent experiments, *, p < 0.05. d Radiation resistance (cell cytometry or H2AX foci counting followed by 12Gy radiation) of SW1783 and Hs683 expressing circ-AKT3 shRNA, circ-AKT3 shRNA plus linearized AKT3-174aa and their control cells. Error bars represent three independent experiments, *, p < 0.05. e U251 and U373 cells were transfected with linearized AKT3-174aa plasmid or ATG mutated circ-AKT3 plasmid, and AKT3-174aa was determined by immunoblot. f Cell proliferation and cell cycle analysis of U251 and U373 cells transfected with linearized AKT3-174aa plasmid or ATG mutated circ-AKT3 plasmid. Error bars represent three independent experiments, *, p < 0.05, **, p < 0.01. g Plate colony formation and anchorage independent growth of U251 and U373 cells transfected with linearized AKT3-174aa plasmid or ATG mutated circ-AKT3 plasmid. Error bars represent three independent experiments, *, p < 0.05. h Radiation resistance (cell cytometry or H2AX foci counting followed by 12Gy radiation) of U251 and U373 cells transfected with linearized AKT3-174aa plasmid or ATG mutated circ-AKT3 plasmid. Error bars represent three independent experiments, *, p < 0.05, **, p < 0.01
Fig. 5
Fig. 5
AKT3-174aa interacts with p-PDK1 and prevents AKT thr308 phosphorylation. a RNA sequencing was conducted in U251-circ-AKT3 IRES mut, U251-circ-AKT3, U373-circ-AKT3 IRES mut, U373-circ-AKT3 cells. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was performed to find the most differentially changed signaling pathways. b Upper, U251, U373, SW1783 and Hs683 cells were overexpressed or knocked down circ-AKT3, according to their endogenous AKT3-174aa expression level. The AKT3-174aa, AKT1, AKT2, AKT3, AKT-pan, AKT-thr308, AKT-ser473, p-EGFR, PTEN level were determined. Lower, AKT1, AKT2 and AKT3 mRNA level were determined in above cells, respectively. c IP assay using AKT1, AKT2 and AKT3 antibodies were performed in indicated cells followed by immunoblot using AKT1, AKT2, AKT3, AKT-thr308, AKT-ser473 antibodies. d Total proteins from Flag-AKT3-174aa plasmid-transfected HEK293T cells were separated via SDS-PAGE. P-PDK1 was identified by LC/LC-MS in AKT3-174aa protein complex. e Mutual interaction of p-PDK1 and Flag-AKT3-174aa were determined by IP. f Left, prokaryotic purified His-tagged-PDK1, GST-tagged-AKT3-174aa and GST was subjected to GST pull down or His-IP. Right, eukaryotic purified His-tagged-PDK1, prokaryotic purified GST-tagged-AKT3-174aa and GST was subjected to GST pull down or His-IP. g Flag-tagged AKT3-174aa was transfected into U251 cells and immunofluorescence was performed using anti-Flag and anti-p-PDK1 antibody. Scale bar, 20 μm
Fig. 6
Fig. 6
AKT3-174aa competitively interacts with p-PDK1 form ATK2/3. a PDK1 kinase activity was determined in U251 and U373 cells with circ-AKT3 overexpression and their control cells at the indicated time point. Error bars represent three independent experiments, **, p < 0.01. b Upper, AKT kinase activity was determined in U251 and U373 cells with circ-AKT3 overexpression and their control cells (48 h); or in SW1783 and Hs683 cells with circ-AKT3 knocking down and their control cells (48 h). Error bars represent three independent experiments, *, p < 0.05, **, p < 0.01. Lower, p-SGK level was determined in indicated cells. c U251 cells were transfected with increasingly dose of circ-AKT3. IP was performed by using AKT2, AKT3 and p-PDK1 antibodies and followed by immunoblot using indicated antibodies. d SW1783 cells were transfected with increasingly dose of circ-AKT3 shRNA. IP was performed by using AKT2, AKT3 and p-PDK1 antibodies and followed by immunoblot using indicated antibodies
Fig. 7
Fig. 7
AKT3-174aa negatively correlates the tumorigenicity of GBM cells in vivo. a U251, U373 cells with circ-AKT3 overexpression and their control cells were intracranially injected into nude mice (1X105 per mice, five mice per group). In vivo tumorigenicity was monitored, and mice were sacrificed when showing clinical symptoms. Mice brain were subjected to HE is staining or IHC staining as indicated. Survival analysis was calculated by Kaplan-Merier curve. *, p < 0.05. b SW1783, Hs683 cells with circ-AKT3 koncking down and their control cells were intracranially injected into nude mice (1X105 per mice, five mice per group). In vivo tumorigenicity was monitored, and mice were sacrificed when showing clinical symptoms. Mice brain were subjected to HE is staining or IHC staining as indicated. Survival analysis was calculated by Kaplan-Merier curve. *, p < 0.05. c Illustration of AKT3-174aa function. Normally, AKT3-174aa interacts p-PDK1 and limits AKT3 thr308 phosphorylation as a molecular decoy. In GBM, loss of AKT3-174aa exposed AKT thr308 to p-PDK1 more easily, promotes AKT activation and sequential signaling cascades
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