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

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.