Development of Pan-RAF inhibitors targeting KRAS mutations
- 발행기관 강릉원주대 일반대학원
- 발행년도 2022
- 학위수여년월 2023. 2
- 학위명 석사
- 학과 및 전공 도움말 일반대학원 웰니스바이오산업학과
- 세부분야 해당없음
- 실제URI http://www.dcollection.net/handler/kangnung/000000011376
- UCI I804:42001-000000011376
- 본문언어 영어
초록/요약 도움말
RAF is a serine/threonine kinase that plays important roles in the RAS/RAF/MEK/ERK protein kinase signaling pathway, which is involved in cell proliferation, differentiation, survival, and angiogenesis. Existing firstgeneration BRAF inhibitors selectively act on BRAF V600E mutant cancer cells but rapidly lead to resistance and paradoxical activation. To overcome these limitations, a second-generation Pan-RAF inhibitor of CRAF and BRAF V600E mutations should be developed. Through WST and crystal violet assays, we found that cell growth and the rates of cell death were increased in the Q12b-treated group in a concentration-dependent manner in mutant cells compared to those in the KRAS wild-type cells. Western blot analysis showed that in the Q12b-treated group, the expression levels of BRAF and CRAF were decreased from 10 μM in wild-type and 1 μM in mutant cells. RNA sequencing analysis confirmed that RAF transcript expression was decreased compared to that in the control group. Further investigation of the inhibitory mechanism of AKT and MYC signaling in cell proliferation is needed. Our results demonstrate the potential of developing a second-generation RAF inhibitor of both BRAF and CRAF, suggesting a mechanism by which the MAPK pathway is inhibited to reduce cancer cell survival.
more목차 도움말
Ⅰ. Introduction ...........................................................................................9
Ⅱ. Materials and Methods .......................................................................9
1. Materials and Antibodies .................................................................................. 9
2. Cell culture ..................................................................................................... 10
3. Drug screening................................................................................................. 10
4. Cell proliferation assay.................................................................................... 11
5. Cell viability assay........................................................................................... 11
6. Flow cytometry (FACS) assay......................................................................... 12
7. Western blot assay ........................................................................................... 12
8. RNA sequencing.............................................................................................. 13
9. In VIVO........................................................................................................... 14
10. Statistical analysis.......................................................................................... 14
Ⅲ. Results................................................................................................15
1-1. Drug screening in KRAS WT (Wild type) and mutant cells...............15
1-2. Inhibition of cell growth and reduction of cell viability in respective
KRASWT (Wild type) and mutant cells..............................................19
2. Analysis of each KRAS WT (Wild type) and mutant apoptosis cells...23
3. Analysis of MAPK pathway protein expression in KRAS WT(Wild type)
and mutant cells.....................................................................................29
4. Analysis of gene expression through RNA sequencing ........................33
5. Evaluation of mouse drug efficacy using WT (Wild type) and KRAS
mutant cells ·······································································38
Ⅳ. Conclusion..........................................................................................41
Ⅴ. References............................................................................................43
