CRISPR/Cas9介导TKI对非小细胞肺癌基因靶向吉非替尼耐药敏感性的影响及其机制

目的：探讨CRISPR/Cas9介导酪氨酸激酶抑制剂（TKI）对非小细胞肺癌基因靶向吉非替尼耐药敏感性的影响，并阐明其机制。方法：利用CRISPR/Cas9系统建立酪氨酸激酶（TKs）敲除A549细胞株，按照TKI表达情况分为A549、A549TKs^-/+和A549TKs^-/-细胞株。Western blotting法检测细胞株中P53、MDM2、Bcl-2和Bax蛋白表达水平，CCK-8法检测A549、A549TKs^-/+和A549TKs^-/-细胞活性，细胞划痕实验检测A549、A549TKs^-/+和A549TKs^-/-细胞株的细胞迁移情况，流式细胞术检测A549、A549TKs^-/+和A549TKs^-/-细胞凋亡率，克隆形成实验检测细胞集落形成情况。结果：CRISPR/Cas9系统成功建立了TKs敲出A549细胞株，A549、A549TKs^-/+和A549TKs^-/-细胞活性比较差异无统计学意义（P>0.05）；3种细胞凋亡率比较差异无统计学意义（P>0.05），细胞迁移情况变化不大。经6 mol·L^-1吉非替尼干预72 h后，与A549细胞株比较，A549TKs^-/+和A549TKs^-/-细胞株的细胞活性降低（P<0.05），细胞凋亡率升高（P<0.05），细胞划痕愈合率明显降低（P<0.05），细胞迁移能力明显减弱；与A549细胞株比较，A549TKs^-/+和A549TKs^-/-细胞株中P53和Bax蛋白表达水平降低（P<0.05），MDM2和Bcl-2蛋白表达水平明显升高（P<0.05），细胞集落生成率明显降低（P<0.05）。结论：采用吉非替尼对敲除A549细胞株进行干预可以降低细胞活性和迁移能力，升高细胞凋亡率和吉非替尼耐药的敏感性。

Effect of CRISPR/Cas9-mediated TKI on sensitivity of non-small cell lung cancer gene targeting gefitinib resistance and its mechanism

Objective: To investigate the effect of CRISPR/Cas9-mediated tyrosine kinase inhibitor(TKI) on the sensitivity of non-small cell lung cancer gene targeting gefifinib resistance, and to clarify its mechanism. Methods: The A549 cell strains knocked out tyrosine kinases(TKs) by using CRISPR/Cas9 system were established, and then divided into the A549, A549TKs^-/+ and A549 TKs^-/- cell strains according to the expression levels of TKI. The expression levels of P53, MDM2, Bcl-2 and Bax proteins in cell starins were detected by Western blotting method,the activities of A549, A549TKs^-/+, and A549 TKs^-/- cells were detected by CCK-8 method,the cell migration of the A549, A549TKs^-/+, and A549 TKs^-/- cells were detected by cell scratch method, the apoptotic rates of the A549, A549TKs^-/+, and A549 TKs^-/- cells were detected by flow cytometry,and the colony formation status of the A549, A549TKs^-/+, and A549^-/- cells were detected by colony formation assay. Results: The TKs knockout A549 cell strains were successfully established by CRISPR/Cas9 system. There were no statistically significant differences in the activites of the A549, A549TKs^-/+ and A549TKs^-/- cells (P>0.05),there were no significant differences in the apoptostic rates of the A549, A549TKs^-/+ and A549 TKs^-/- cells (P>0.05),and there was no statistically significant difference in the migration of the A549, A549TKs^-/+ and A549TKs^-/- cells. After intervented with gefitinib for 72 h,compared with A549 cell strain, the cell activities of the A549TKs^-/+ and A549TKs^-/- cell strains were significantly decreased(P<0.05),the apoptotic rates were significantly increased (P<0.05), the rates of scratch healing were obviously decreased(P<0.05),and the migration abilities were significantly decreased;compared with A549 cell stain,the expression levels of P53 and Bax proteins in the A549TKs^-/+ and A549TKs^-/- cell strains were significantly decreased (P<0.05),the expression levels of MDM2 and Bcl-2 proteins were significantly increased (P<0.05), and the colony formation rates were significantly decreased (P<0.05). Conclusion: Gefitinib intervention in the knockout A549 cell strain can decrease the cell viability and migration ability and increase the apoptotic rate and sensitivity of gefitinib resistance.