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. 2021 Mar 14;42(3):243-249.
doi: 10.3760/cma.j.issn.0253-2727.2021.03.011.

[CRISPR/Cas9-mediated microRNA-21 knockout increased imatinib sensitivity in chronic myeloid leukemia cells]

[Article in Chinese]
Y Zhang  1 , L Y Wang  1 , J Z Li  1 , P F Jiang  1 , J D Hu  1 , B Y Chen  1
Affiliations

[CRISPR/Cas9-mediated microRNA-21 knockout increased imatinib sensitivity in chronic myeloid leukemia cells]

[Article in Chinese]
Y Zhang et al. Zhonghua Xue Ye Xue Za Zhi. .

Abstract
in English, Chinese

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(B)CR-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.

目的： 观察microRNA-21（miR-21）敲除对耐伊马替尼的人慢性髓性白血病细胞株K562/G01细胞在增殖、药物敏感性等方面的影响，初步探讨miR-21影响K562/G01细胞伊马替尼敏感性的可能机制。 方法： 运用CRISPR/Cas9技术敲除K562/G01细胞的miR-21，经PCR筛选、Sanger测序鉴定和实时定量PCR检测获得miR-21敲除的单细胞克隆。扩增培养后，采用MTT法、细胞克隆形成实验检测miR-21敲除对K562/G01细胞增殖的影响。使用伊马替尼处理细胞后，用MTT法和Annexin Ⅴ-APC/7-AAD双染流式细胞检测法观察敲除miR-21后K562/G01细胞对伊马替尼的敏感性的变化。Western blot法检测miR-21敲除前后K562/G01细胞PTEN、AKT、p-AKT、PI3K、p-PI3K、P210(BCR-ABL)、p-P210(BCR-ABL)蛋白表达量的变化。 结果： 成功构建了3个miR-21敲除的K562/G01单细胞克隆，CRISPR/Cas9介导的突变效率为7.12%~8.11%。miR-21敲除使K562/G01细胞的增殖受抑，野生型和1#、2#、6#单细胞克隆的克隆形成率依次为（57.67±8.25）%、（26.94±5.36）%、（7.17±2.11）%、（31.50±3.65）%，差异有统计学意义（P<0.05）。miR-21敲除使K562/G01细胞对伊马替尼的敏感性增加，野生型和1#、2#、6#单细胞克隆对伊马替尼的IC(50)值分别为（21.92±1.36）µmol/ml、（3.98±0.39）µmol/ml、（5.38±1.01）µmol/ml、（9.24±1.36）µmol/ml，差异有统计学意义（P<0.05）。miR-21敲除后，其靶基因PTEN的蛋白表达水平未见明显变化，但PI3K、AKT信号分子的活化受到抑制，并且P210(BCR-ABL)、p-P210(BCR-ABL)蛋白表达也下调。 结论： miR-21敲除抑制K562/G01细胞增殖，提高其对伊马替尼的敏感性，这可能是通过抑制PI3K/AKT信号通路和BCR-ABL表达实现的。.

Keywords: CRISPR/Cas9; Chronic myeloid leukemia; Knockout; imatinib; miR-21.

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Figures

图1
图1. CRISPR/Cas9诱导的突变效率
M：Marker；1：野生型K562/G01细胞；2、3：MOI分别为100、200的CRISPR/Cas9慢病毒转导的K562/G01细胞；4：阴性对照
图2
图2. 筛选单细胞克隆的PCR结果
以亲本K562/G01细胞作为野生型（WT）。M：Marker；1、2、6、7泳道显示仅具有较短片段的单细胞克隆；3、4、5、8泳道显示除了有和WT相等的PCR扩增片段外，还有一个长度较短的产物片段
图3
图3. Sanger测序检测单细胞克隆miR-21的碱基缺失情况
miR-21 KO：miR-21敲除的K562/G01单细胞克隆。红色部分为miR-21的5p和3p序列，5p端带红色下划线的碱基对为种子序列，删除线部分为删除序列
图4
图4. MTT法分析miR-21敲除克隆的细胞活力（实验重复3次）
miR-21 KO：miR-21敲除的K562/G01单细胞克隆
图5
图5. 甲基纤维素克隆形成实验显微镜下观察平均克隆大小（×400）
miR-21 KO：miR-21敲除的K562/G01单细胞克隆
图6
图6. 以不同浓度的伊马替尼分别处理细胞48 h后用Annexin V-APC和7-AAD双染色流式细胞术分析细胞凋亡率
miR-21 KO：miR-21敲除的K562/G01单细胞克隆。与野生型比较，aP<0.05，bP<0.01
图7
图7. Western blot法比较野生型和miR-21敲除的K562/G01细胞中PTEN/PI3K/AKT信号通路和BCR-ABL融合蛋白表达量
1：野生型；2～4分别为miR-21敲除的1#、2#、6#单细胞克隆
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