Department of Pharmaceutical Sciences, School of Pharmacy and Pharmaceutical Sciences, Binghamton University, Binghamton, NY 13902, USA.
Department of Biochemistry and Physiology, School of Pharmacy and Food Sciences, & IN2UB, University of Barcelona, 08028 Barcelona, Spain.
Int J Mol Sci. 2022 Feb 14;23(4):2097. doi: 10.3390/ijms23042097.
KRAS is a GTPase involved in the proliferation signaling of several growth factors. The KRAS gene is GC-rich, containing regions with known and putative G-quadruplex (G4) forming regions. Within the middle of the G-rich proximal promoter, stabilization of the physiologically active G4 structure downregulates transcription of KRAS; the function and formation of other G4s within the gene are unknown. Herein we identify three putative G4-forming sequences (G4FS) within the gene, explore their G4 formation, and develop oligonucleotides targeting these three regions and the G4 forming sequence. We tested Polypurine Reverse Hoogsteen hairpins (PPRHs) for their effects on regulation via enhancing G4 formation or displacing G-rich DNA strands, downregulating transcription and mediating an anti-proliferative effect. Five PPRH were designed, two against the promoter G4 and three others against putative G4FS in the distal promoter, intron 1 and exon 5. PPRH binding was confirmed by gel electrophoresis. The effect on transcription was examined by luciferase, FRET Melt, qRT-PCR. Cytotoxicity was evaluated in pancreatic and ovarian cancer cells. PPRHs decreased activity of a luciferase construct driven by the promoter. PPRH selectively suppressed proliferation in KRAS dependent cancer cells. PPRH demonstrated synergistic activity with a promoter selective G4-stabilizing compound, NSC 317605, in KRAS-dependent pancreatic cells. PPRHs selectively stabilize G4 formation within the KRAS mid promoter region and represent an innovative approach to both G4-stabilization and to modulation with potential for development into novel therapeutics.
KRAS 是一种参与多种生长因子增殖信号的 GTPase。KRAS 基因富含 GC，包含已知和推测的 G-四链体 (G4) 形成区域。在富含 G 的近端启动子中间，生理活性 G4 结构的稳定会下调 KRAS 的转录；基因内其他 G4 的功能和形成尚不清楚。在此，我们在 基因内鉴定了三个假定的 G4 形成序列 (G4FS)，探索了它们的 G4 形成，并开发了针对这三个区域和 G4 形成序列的寡核苷酸。我们测试了聚嘌呤反向 Hoogsteen 发夹 (PPRH) 通过增强 G4 形成或置换富含 G 的 DNA 链来调节 的作用，下调 的转录并介导抗增殖作用。设计了五个 PPRH，两个针对 启动子 G4，另外三个针对远端启动子、内含子 1 和外显子 5 中的假定 G4FS。通过凝胶电泳证实了 PPRH 的结合。通过荧光素酶、FRET 融解、qRT-PCR 检测对 转录的影响。在胰腺和卵巢癌细胞中评估细胞毒性。PPRH 降低了由 启动子驱动的荧光素酶构建体的活性。PPRH 选择性地抑制依赖 KRAS 的癌细胞的增殖。PPRH 与 KRAS 依赖性胰腺细胞中选择性稳定 G4 的 启动子化合物 NSC 317605 表现出协同活性。PPRH 选择性地稳定 KRAS 中启动子区域内的 G4 形成，并代表了一种创新的方法，用于 G4 稳定和 调节，具有开发为新型治疗剂的潜力。
