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

doi:10.13481/j.1671-587x.20190616

Abstract

Abstract: 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.

Key words: CRISPR/Cas9, tyrosine kinase inhibitor, non-small cell lung cancer, gefitinib, drug resistance

CLC Number:

R734.2

REN Aihua, WANG Dawei. Effect of CRISPR/Cas9-mediated TKI on sensitivity of non-small cell lung cancer gene targeting gefitinib resistance and its mechanism[J].Journal of Jilin University(Medicine Edition), 2019, 45(06): 1288-1293.

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

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