CRISPR/Cas9-mediated microRNA-21 knockout increased imatinib sensitivity in chronic myeloid leukemia cells
10.3760/cma.j.issn.0253-2727.2021.03.011
- VernacularTitle:CRISPR/Cas9介导的microRNA-21敲除对慢性髓性白血病细胞伊马替尼敏感性的影响
- Author:
Yun ZHANG
1
;
Lingyan WANG
;
Jiazheng LI
;
Peifang JIANG
;
Jianda HU
;
Buyuan CHEN
Author Information
1. 福建医科大学附属协和医院、福建省血液病研究所、福建省血液病学重点实验室，福州 350001
- Keywords:
CRISPR/Cas9;
miR-21;
Knockout;
imatinib;
Chronic myeloid leukemia
- From:
Chinese Journal of Hematology
2021;42(3):243-249
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To observe the effects of miR-21 knockout on proliferation and drug resistance in K562/G01 cells, and to preliminarily explore the mechanism of imatinib sensitivity by knocking out miR-21 in K562/G01 cells.Methods:Using CRISPR/Cas9 to knock out the miR-21 gene in K562/G01 cells, and single-cell-derived clones of miR-21 knockout were obtained by genomic DNA PCR screening, Sanger sequencing, and real-time PCR. We used MTT and cell colony formation assays to assess the cell proliferation, and determined imatinib sensitivity by MTT assay and Annexin-Ⅴ-APC/7-AAD double staining flow cytometry. Using western blot, we examined the potential mechanisms affecting imatinib sensitivity by knocking out miR-21 in K562/G01 cells.Results:Three miR-21 knockout K562/G01 single-cell-derived clones were successfully constructed. The mutation efficiency mediated by CRISPR/Cas9 was 7.12%-8.11%. MiR-21 knockout inhibited the proliferation of K562/G01 cells; the clone formation rates of WT and 1#, 2#, 6# K562/G01 single-cell clones were (57.67±8.25) %, (26.94± 5.36) %, (7.17±2.11) %, (31.50±3.65) %, respectively. MiR-21 knockout increased the sensitivity of K562/G01 cells to imatinib, IC 50 of imatinib in WT, and 1#, 2#, 6# K562/G01 single-cell clones were (21.92±1.36) μmol/ml, (3.98±0.39) μmol/ml, (5.38±1.01) μmol/ml, (9.24±1.36) μmol/ml. After the knockout of miR-21, the activation of PI3K/Akt signaling molecules was inhibited, while the expression of P210 BCR-ABL and p-P210 BCR-ABL was downregulated; however, the expression of PTEN was not affected. Conclusion:The knockout of miR-21 can suppress cell proliferation and improve sensitivity to imatinib in K562/G01 cells, which may be achieved by inhibiting the PI3K/AKT signaling pathway and BCR-ABL expression.
