CRISPR/Cas9-mediated MicroRNA-21 Knockout Increased The TKIs Sensitivity In Chronic Myeloid Leukemia Cells

ObejectiveTyrosine kinase inhibitors（TKIs）targeting BCR-ABL1 kinase are effective in treating chronic myeloid leukemia（CML）,but TKIs resistance occurs in some patients,and the underlying molecular mechanisms of this resistance remain largely unknown.As an onco-miR,micro RNA-21（miR-21）plays an important role in drug resistance,but its relationship with TKI resistance in CML is rarely reported.As a novel and effective gene editing tool,CRISPR/Cas9 has certain advantages in completely knocking out target genes at the gene level.This project aims to:1.Apply CRISPR/Cas9-mediated gene depletion strategy to knockout（KO）miR-21 gene in human chronic myeloid leukemia K562 cell line and its imatinib（IM）resistant K562 cells which was named as KG.To investigate the effectiveness of using CRISPR/Cas9 to establish stable micro RNA knockout cell lines in blood tumor cells.2.Compare the differences in proliferation and drug sensitivity between wild type（WT）and miR-21 KO cells,explored the potential mechanism of increasing drug sensitivity by knockout of miR-21 in cell lines.Methods1.Design and construction of Lentiviruses LV-VMP1-sg RNA（045001）.2.Human chronic myeloid leukemia K562 cells and KG cells were transducted with Lentivirus LV-VMP1-sg RNA（045001）.The induced mutation efficiency was estimated by the surveyor assay.3.Single-cell-derived clones were screened for miR-21 deletion by genomic DNA PCR.4.RQ-PCR assay and Sanger sequence were used to confirm the knockout of miR-21.5.MTT and colony formation assays were applied to assess the cell growth inhibition.They were all used to evaluate the effect on cell proliferation by knockout of miR-21 in K562 and KG cell lines.Imatinib and dasatinib sensitivity was determined by MTT assay and Annexin-V APC/7-AAD double staining flow cytometry.6.Related signaling pathways were examined by western blot.Results1.Lentivirus LV-VMP1-sg RNA（045001）targeting human miR-21 gene was successfully constructed.2.Surveyor mutation detection assay showed that the Lentivirus LV-VMP1-sg RNA（045001）could specifically target to shear human genome DNA,and the mutation rates of K562 and KG cells were about 2.16-21.88%and 7.12-8.11%,respectively.3.Single-cell-derived clones were screened by genomic DNA PCR.We then performed further characterized clones by Sanger sequencing and RQ-PCR to measure the miR-21 level in these clones.Three clones with deletion of miR-21gene fragment were obtained in K562 cell lines and KG cell lines,respectively.The expression levels of miR-21 of#1 K562,#2 K562,#5 K562 cells were13.89±6.32%,26.23±14.36%,0.03±0.04%of that in WT K562 cells,respectively.The expression levels of miR-21 of#1 KG,#2 KG,#6 KG cells were 15.54±3.12%,0.26±0.15%,18.97±6.87%of that in WT KG cells,respectively.4.Both MTT assay and colony formation assay indicated the knockout of miR-21in K562 and KG cells significantly inhibited cell proliferation and colony formation ability.Colony formation rates of WT K562,#1 K562,#2 K562,#5K562 were 73.94%±3.15%,32.39%±3.69%,23.52±1.72%,41.11±2.05%,respectively（P﹤0.05）.Colony formation rates of WT KG,#1 KG,#2 KG,#6 KG were 57.67%±8.25%,26.94%±5.36%,7.17±2.11%,31.5±3.65%,respectively（P﹤0.05）.5.Knockout of miR-21 increased the sensitivity of K562 to dasatinib and KG to imatinib,while the sensitivity of K562 to imatinib was not significantly changed.After miR-21 knockout,IC₅₀ of imatinib in WT K562,#1 K562,#2 K562,#5 K562were 1.26±0.09μm/ml,1.56±0.22μm/ml,1.13±0.20μm/ml,1.18±0.11μm/ml,respectively（P>0.1）.IC₅₀ of dasatinib in WT K562,#1 K562,#2 K562,#5 K562were 206.27±6.95nm/ml,99.36±1.63nm/ml,84.69±3.46nm/ml,101.49±2.47nm/ml,respectively（P<0.05）.IC₅₀ of imatinib in WT KG,#1 KG,#2 KG,#6 KG were21.92±1.36μm/ml,3.98±0.39μm/ml,5.38±1.01μm/ml,9.24±1.36μm/ml,respectively（P<0.05）.IC₅₀ of dasatinib in WT KG,#1 KG,#2 KG,#6 KG were31.41±3.29μm/ml,6.70±0.70μm/ml,5.38±0.33μm/ml,13.54±0.49μm/ml,respectively（P<0.05）.6.Down-regulation of AKT/PI3K signaling pathway and decreased P210^BCR-ABLlevel were found after knockouting of miR-21.ConclusionMiR-21 knockout K562 clones and KG clones were successfully obtained by using CRISPR/Cas9.Knockout of miR-21 could suppress cell proliferation and increase the sensitivity to TKIs.Further mechanism studies suggested that this may be achieved by inhibiting the PI3K/AKT signaling pathway and the expression of BCR-ABL.