The Effect of KRAS on Proliferation and Apoptosis of T-ALL Cell Lines.
10.19746/j.cnki.issn.1009-2137.2022.04.011
- Author:
Zi-Yang LIU
1
;
Yi SHU
1
;
Guo FU
1
;
Hong-Yu SU
1
;
Dan ZHU
1
;
La-Mei ZENG
1
;
De-Yu MA
1
;
Lin ZOU
2
,
3
Author Information
1. Department of Clinical Molecular Medicine of Children's Hospital in Chongqing Medical University, National Clinical Research Center for Child Health and Disorders, Ministry of Education Key Laboratory of Child Development and Disorders, Chongqing Key Laboratory of Pediatrics, Chongqing 400014, China.
2. Department of Clinical Molecular Medicine of Children's Hospital in Chongqing Medical University, National Clinical Research Center for Child Health and Disorders, Ministry of Education Key Laboratory of Child Development and Disorders, Chongqing Key Laboratory of Pediatrics, Chongqing 400014, China,Clinical Research Unit of Children's Hospital in Shanghai Jiaotong University
3. Institute of Pediatric Infection, Immunity and Critical Care Medicine, Shanghai Jiaotong University School of Medicine, Shanghai 200062, China,E-mail: zoulin74@126.com.
- Publication Type:Journal Article
- Keywords:
RAS;
acute T-lymphocytic leukemia;
cell apoptosis;
cell proliferation
- MeSH:
Apoptosis;
Cell Line;
Cell Proliferation;
Humans;
Mutation;
Precursor T-Cell Lymphoblastic Leukemia-Lymphoma;
Proto-Oncogene Proteins p21(ras)/genetics*
- From:
Journal of Experimental Hematology
2022;30(4):1040-1048
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVE:To investigate the function of RAS protein on the progression of the T-ALL cell lines in vitro.
METHODS:The DNA of the T-ALL cells was purified then amplified the coding regions of three RAS genes (KRAS, NRAS, HRAS) by PCR reaction. After T-A cloning, the coding regions of KRAS, NRAS and HRAS were sequenced by Sanger Sequencing. The siRNA oligonucleotides were cloned into the pSEH-361 vector, which were then packaged into retroviral together with pAMPHO and pVSVG in the HEK-293T cells. The T-ALL cells were infected with the retrovirus. The gene expressions were detected by qRT-PCR and Western blot. The T-ALL cells were stained with Annexin V-PE/7-AAD and the apoptotic cells were detected by flow cytometry. The T-ALL cells were stained with Hoechst 33258, and the cell cycle distribution was determined by flow cytometry. The expression of cleaved-Caspase 3 was stained with antibody and observed with fluorescence microscope.
RESULTS:For RAS genes, beside the Loucy and the P12-ICH cells harbored KRAS c.6187G>A (p.KRASG12D) homozygous mutant, no missense mutation of RAS was found in other T-ALL cells genome. The pan RAS inhibitor compound 3144 showed toxicity to all tested T-ALL cells, except PEER (IC50=47.916 μmol/L). Similarly, Tipifarnib induced apoptosis of multiple T-ALL cell lines except for the PEER cells (IC50=94.2265 μmol/L). After KRAS knock-down, the T-ALL cells showed significant apoptosis and an arrested cell cycle.
CONCLUSION:The KRAS protein is vital for the progression of the T-ALL cells in vitro, it is a potential therapeutic target for T-ALL patients.
