Rapamycin, a natural product inhibitor of the Raptormammalian target of rapamycin complex (mTORC1), is known to induce Protein kinase B (Akt/PKB) Ser-473 phosphorylation in a subset of human cancer cell lines through inactivation of S6K1, stabilization of insulin receptor substrate (IRS)-1, and increased signaling through the insulin/insulin-like growth factor-I/phosphatidylinositol 3-kinase (PI3K) axis. We report that A-443654, a potent small-molecule inhibitor of Akt serine/threonine kinases, induces Akt Ser-473 phosphorylation in all human cancer cell lines tested, including PTEN-and TSC2-deficient lines. This phenomenon is dose-dependent, manifests coincident with Akt inhibition and likely represents an alternative, rapid-feedback pathway that can be functionally dissociated from mTORC1 inhibition. Experiments performed in TSC2 À/À cells indicate that TSC2 and IRS-1 cooperate with, but are dispensable for, A-443654-mediated Akt phosphorylation. This feedback event does require PI3K activity, however, as it can be inhibited by LY294002 or wortmannin. Small interfering RNA-mediated knockdown of mTOR or Rictor, components of the rapamycin-insensitive mTORC2 complex, but not the mTORC1 component Raptor, also inhibited Akt Ser-473 phosphorylation induced by A-443654. Our data thus indicate that Akt phosphorylation and activity are coupled in a manner not previously appreciated and provide a novel mode of Akt regulation that is distinct from the previously described rapamycin-induced IRS-1 stabilization mechanism.
Increasing evidence supports the essential roles of circular RNAs (circRNAs) and microRNAs (miRNAs/miRs) in different types of human cancer. For example, hsa_ circ_0137008 functions as a sponge for mi-338-5p and inhibits the malignant phenotype in colorectal cancer. Furthermore, hsa_circ_RNA_0011780 downregulates FBXW7 by targeting miR-554a and suppressing the progression of non-small cell lung cancer. Thus far, only a single report has identified that the miRNA miR-331-3p exerts a pivotal effect on human colorectal cancer (CRC) evolution. However, both the upand downstream regulatory mechanisms of miR-331-3p are unclear. In the present study, it was predicted via bioinformatics analysis that the circRNA, hsa_circ_0038646, and the glutamate receptor ionotropic kainate 3 (GRIK3) gene contain binding sites that can interact with miR-331-3p. Thus, hsa_ circ_0038646/miR-331-3p/GRIK3 may be a novel therapeutic pathway for CRC. Reverse transcription-quantitative PCR and western blotting analyses were performed, as well as cell proliferation, luciferase reporter and Transwell migration assays. Hsa_circ_0038646 was overexpressed in both CRC cells and tissues, and this aberrant expression was positively related with increasing tumor grade. Knockdown of hsa_circ_0038646 significantly weakened human CRC cell proliferation and migration. It was shown that hsa_circ_0038646 can sponge miR-331-3p to suppress its expression, and that suppression of miR-331-3p can reverse the effects of hsa_circ_0038646 inhibition in CRC cells. It was determined that GRIK3 is a downstream target of miR-331-3p, and that hsa_circ_0038646 could increase the levels of GRIK3 by suppressing miR-331-3p in CRC cells. Restoring GRIK3 expression rescued the weakened CRC cell proliferation and migration following hsa_circ_0038646 knockdown. The present study indicated that hsa_circ_0038646 functions as a tumor promoter in CRC by increasing GRIK3 expression via sponging of miR-331-3p. The hsa_circ_0038646/miR-331-3p/GRIK3 axis may be a novel therapeutic and diagnostic target of CRC.
Background: Circular RNAs (CircRNAs) have been recently implicated in the progression of pancreatic cancer (PC). Aims: To investigate the involvement of CircCCT3 in PC and studying its interactions and functioning during the progression of PC in vitro and in vivo, using methods of molecular biology and bioinformatics. Study Desig n: Experimental study. Methods: The expressions of CircCCT3 and miR-613 in pancreatic carcinoma tissues and cell lines were evaluated by quantitative real-time polymerase chain reaction (PCR). The relationship between clinical pathologic features as well as the survival rate and CircCCT3 expression was analyzed with chi-square test and the Kaplan–Meier method. CCK-8, wound healing, transwell assays, and the fluorescein isothiocyanate-AnnexinV/propidium iodide (FITC-AnnexinV/PI) assay were used to assess cell proliferation, migration, invasion, and apoptosis after CircCCT3 overexpression or downregulation. The Dual-Luciferase reporter assay, RNA immunoprecipitation (RIP), RNA pull-down and fluorescence in situ hybridization (FISH) assays were performed to validate the potential interaction of CircCCT3, miR-613, and vascular endothelial growth factor (VEGFA). The nude mouse xenograft tumor assay was used to detect CircCCT3 effects on pancreatic tumorigenesis in vivo. Western blotting analysis was performed to examine the VEGFA and the vascular endothelial growth factor receptor 2 (VEGFR2) protein expressions following. Results: CircCCT3 expression was significantly increased in PC tissues (3.41 ± 0.57 vs. 1.00 ± 0.10, P < .01) and cell lines (Patu8988 2.57 ± 0.20; SW1990 2.88 ± 0.10; BxPC-3 2.45 ± 0.20; Panc02 2.99 ± 0.10 vs. H6c7 1.00 ± 0.10; all P < .001). CircCCT3 expression was negatively correlated with miR-613 expression. PC patients with high CircCCT3 expression exhibited significantly poorer overall survival rate than those patients with low CircCCT3 expression ( P = .013). Moreover, it was found that CircCCT3 promoted cell proliferation, migration, and invasion, and inhibited cell apoptosis in PC cells. The CircCCT3 acted as a sponge for the miR-613 to facilitate VEGFA and VEGFR2 expression. si-CirCCT3 also inhibited tumor growth of PC in nude mice. si-CircCCT3 reduced VEGFA and VEGFR2 expression, whereas overexpression of CircCCT3 increased VEGFA and VEGFR2 expression. Conclusion: Increased CircCCT3 suggests a poor prognosis for PC patients and promotes the migration and invasion through targeting VEGFA/VEGFR2 signaling. CircCCT3 may serve as a potential and promising therapeutic target for PC treatment.
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