Investigation of G-quadruplex formation in promoter region of human CCND1 gene and its interacting with G-quadruplex stabilizers in regulating gene expression

• Main Authors: Chien-Hao Liao, 廖健豪
• Other Authors: Ta-Chau Chang
• Format: Others
• Language: zh-TW
• Published: 2018
• Online Access: http://ndltd.ncl.edu.tw/handle/dt7k6f

碩士 === 國立臺灣大學 === 化學研究所 === 106 === CCND1 (Cell Cyclin D1) is a protein regulating the G1/S transition in the cell cycle. The overexpression of CCND1 is observed in many types of cancer, such as non-small cell lung cancer, head and neck squamous cell carcinomas, and breast carcinoma.Nowadays many pharmaceutical companies focus on developing the CDK (Cyclin-dependent kinase) 4/6 inhibitors for targeting CCND1 pathway for cancer therapies. According to the clinical results, it is suggested that the combination of CDK4/6 inhibitors with other therapies can lead to better efficacy. Therefore, it is of interest in examining whether we can inhibit the expression of CCND1 from transcription level as an additional choice of combination therapies. A number of reports demonstrated that G-quadruplex formed in the promoter region of human genome play important roles in gene regulation. Here we found a potential G-quadruplex-forming sequence at promoter of CCND1 which is 47 base pairs long with 8 continuous G-tracts by using NCBI Genome Data Viewer and QGRS Mapper. The possible G-quadruplex formation of CCND1 was examined by using CD, NMR spectra, and PAGE with UV shadowing. The results revealed that the CCND1 sequence is able to fold into various monomeric G-quadruplex structures. Further studies of G4 ligands were performed to examine whether G4 ligands could stabilize G-quadruplex and inhibit gene expression of CCND1. Three G-quadruplex ligands 3,6-bis(4-methyl-2-vinylpyrazinium iodine)carbazole (BMVC4), meso-tetra(N-methyl-4-pyridyl) porphine tetra tosylate (TMPyP4), and N,N’-(9-(4-(Dimethylamino) phenylamino)acridine-3,6-diyl) bis(3-(pyrrolidin-1-yl)propanamide) (BRACO-19) increase the melting temperature (Tm), implying that they can stabilize the G-quadruplex structures of CCND1. Moreover, PAGE competition assays illustrate that three G-quadruplex stabilizers have better binding affinity to the G-quadruplex of CCND1 than of HT23 (Human telomeres). The studies of qPCR and Western blotting analysis showed that BMVC4 and TMPyP4 treatment for 4 days in H1299 can inhibit the expression of CCND1 in mRNA and protein level with dose dependence. In conclusion, these results revealed that the sequence containing 8 continuous G-tracts at promoter of CCND1 can fold into various monomeric G-quadruplexes. This unique character of the G-quadruplexes of CCND1 provides more binding opportunities for G-quadruplex stabilizers than general type of G-quadruplex, such as HT23 (Human telomeres). Moreover, the bioassay results indicate that G-quadruplex stabilizers can inhibit the expression of CCND1 from transcription level, which may be an additional choice of combination therapies with CDK4/6 inhibitors.