Zhongguo shi yan xue ye xue za zhi · 2022
[The Effect of KRAS on Proliferation and Apoptosis of T-ALL Cell Lines].
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Liu Zi-Yang, Shu Yi, Fu Guo, Su Hong-Yu, Zhu Dan, Zeng La-Mei, Ma De-Yu, Zou Lin
Аннотация
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.
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.