Identification And Functional Study Of G-quadruplex Structures In The Human AKT1 Promoter And 3'-UTR

Guanine-rich DNA and RNA can fold into special structures,named as G-quadruplexes.These structures are widespread in the human genome and involved in multiple biological processes,such as DNA replication,telomere maintenance,gene expression and regulation and genetic stability.AKT,also known as protein kinase B(PKB),is a serine/threonine kinase involved in a variety of important cellular signal transduction pathways,including cell survival,proliferation,invasion,apoptosis,and angiogenesis.AKT1 plays a key role in cell growth and survival.Activation of AKT1 is mediated by distinct mechanisms,including increased gene expression and post-translational protein phosphorylation.In this study,bioinformatic and biotechnological approaches were employed to investigate the mechanism of AKT1 up-regulation in cancers from the perspective of mRNA levels and DNA/RNA structures,and the potential of G-quadruplex formation by the G-tracts in the human AKT1 promoter and its mRNA 3'-UTR was studied.The results in this study were as follows:1.Bioinformatic analyses indicated that AKT1 was upregulated in various malignancies and physiologically relevant to the clinical outcomes of patients with breast,ovarian,lung,gastric,and liver cancers.In addition,we also explored the relationship between AKT1 expression and immune cell infiltration in different types of malignant tumors.2.Circular dichroism analyses showed that synthetic oligonucleotides based on these G-tract regions of the AKT1 promoter exhibited molar ellipticity peaks at specific wavelengths for G-quadruplex structures.Native polyacrylamide gel electrophoresis,gel-shift assays,and immunostaining by a G4-specific antibody were used to demonstrate the formation of G-quadruplex structures at different annealing conditions.Dimethyl sulfate(DMS)footprinting was used to elucidate the nucleotides involved in forming G-quadruplex structures.Chromatin immunoprecipitation(ChIP)assay coupled with PCR-stop assay revealed the presence of G-quadruplex structures in the AKT1 promoter.In luciferase reporter assay,mutations of the G-tracts in the AKT1 promoter reduced the expression mediated by the promoter,suggesting that G-quadruplex structures positively regulated AKT1 gene expression.Our data also suggested that G-quadruplex formation in the AKT1 promoter could likely alter the binding and regulation of transcription factor SP1.Additionally,the DNA helicase RECQL negatively correlated with AKT1 expression and potentially regulated AKT1 G-quadruplex formation.3.Circular dichroism analysis,gel-shift assay,and DMS footprinting assay were employed to demonstrate the formation of G-quadruplex structures in the AKT1 mRNA 3'-UTR region.These G-quadruplexes could likely alter the binding of miR-193b-3p to the AKT1 mRNA 3'-UTR and impact its effects on AKT1 expression.4.Using molecular docking simulation,the crystal structure of the G-quadruplex in the human MYC gene promoter could be docked to the molecule of Lycorine.The results showed that Lycorine could potentially bind to the G-quadruplex.Native polyacrylamide gel electrophoresis and PCR-stop assay were used to demonstrate that Lycorine induced the formation of the G-quadruplex structure and increased its stability.Therefore,Lycorine represents a new class of G-quadruplex binding ligands and may become a potential anticancer drug.In conclusion,our study proved that the presence of G-quadruplex structures in the promoter and mRNA 3'-UTR region of human AKT1,and these G-quadruplexes positively regulated AKT1 expression,likely through mechanisms of altering the recognition and binding of SP1 to the AKT1 promoter,and interfering with the binding of miR-193b-3p to the 3'-UTR of its mRNA.Meanwhile,RECQL could potentially mediate AKT1 gene expression by unwinding G-quadruplexes in the AKT1 promoter and/or its 3'-UTR.Therefore,the G-quadruplexes in AKT1 may serve as effective targets in cancer therapies.Additionally,Lycorine identified in this study with ability of stabilizing G-quadruplexes can be potentially developed into an anti-cancer agent.