| Author: | Chan, Ka Hin |
| Title: | Understanding the biofunction of RNA G-quadruplexes in human cells with the novel fluorescent binding ligands |
| Advisors: | Wong, W. L. (ABCT) Wong, K. Y. (ABCT) |
| Degree: | M.Phil. |
| Year: | 2023 |
| Subject: | Quadruplex nucleic acids Genetic regulation Protein binding RNA-protein interactions Hong Kong Polytechnic University -- Dissertations |
| Department: | Department of Applied Biology and Chemical Technology |
| Pages: | vi, 130 pages : color illustrations |
| Language: | English |
| Abstract: | Cancer is a leading cause of death worldwide. Despite the mechanism of causing cancer is not very clear at present, the overexpression of some oncogenes and their corresponding proteins such as RAS GTPase, RAF kinase, receptor tyrosine kinase KIT and transcription factor MYC in cancer cells usually leads to the suppression of antitumor mechanism and flavor the abnormal cancer cell proliferation. Currently, some drugs such as AMG 510, Sorafenib and Regorafenib have been developed and found able to target several cancers clinically; however, the in vivo cytotoxicity selectivity and off-target side effects of these clinical drug remain a great challenge to be overcome. In addition, there are still number of critically important and undruggable targets of cancer which result in a low survival rate and limited treatment options. One of the typically undruggable target of cancer hallmarks is encoded from the RAS family genes. The mutated genes express RAS proteins, the mutated GTPases, for abnormal cancer cell proliferation and only one drug is available for treating KRAS-driven cancers currently. Over the past decades, many studies have revealed that nucleic acids consisting of guanine(G)-rich sequences can adopt a four-stranded structure in vitro, which is termed G-quadruplex (G4), and play important roles in both gene transcription and translation in cancer cells. It is reported that the mRNA of oncogenes contains G-rich regions, particularly in the 5’-untranslated region, that may form G4 structures. These mRNA G4s may provide alternative drug targets for their undruggable RAS proteins including KRAS, NRAS and HRAS. It is because the protein expression can be inhibited at translational level through targeting RAS mRNA G4 structures with small-molecules. In this study, we designed and synthesized a series of novel fluorescent ligands that is able to interact and stabilize RNA G4s. Among eighteen ligands synthesized and examined in various in vitro and cellular bioassays, one of the ligands, B3C, was identified to show the best affinity and selectivity interacting with NRAS mRNA G4s. We found that the ligand markedly downregulated NRAS proteins, arrested the progression of cell cycle and induced apoptosis in HeLa cells in a dose-dependent manner. Further experiments suggested that the downregulation of NRAS might restore the activation of DNA damage response, initiate the DNA repair mechanism, cause S phase prolongation for DNA repair, and ultimately induce apoptosis. To the best of our knowledge, this is the first study targeting NRAS mRNA G4 with RNA G4-ligand and provides evidence on restoring the DNA damage response that abrogated by RAS proteins in cancer cells. Cell-based MTT results also showed that B3C exhibit a low IC50 value about 6.5 against cancer cell lines HeLa, and a relatively high IC50 value in non-cancer human cell line HFF1. The results obtained may illustrate that the use of small ligands selectively targeting mRNA G4s of oncogenes such as NRAS is a promising anticancer strategy. Our study may provide novel insights into the development of effective therapy against RAS-related cancers. |
| Rights: | All rights reserved |
| Access: | open access |
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