CRISPR/Cas9-mediated microRNA-21 Cleavage Sensitizes the Treatment Response to Dasatinib in Chronic Myeloid Leukemia Cell Line K562

Abstract

Background: Tyrosine kinase inhibitors (TKIs) targeting BCR-ABL1 kinase are effective in treating chronic myeloid leukemia (CML), but TKI resistance occurs in a significant number of patients, and the underlying molecular mechanisms of this resistance remain largely unknown. As an oncomiR, microRNA-21(miR-21) functions directly in drug resistance, but its relationship with TKI resistance in CML is rarely reported. As a novel and effective gene editing tool, clustered regularly interspaced short palindromic repeats–CRISPR-associated protein 9(CRISPR/Cas9) has certain advantages in completely knocking out target genes at the gene level. Methods: We successfully constructed lentiviruses LV-VMP1-sgRNA (045001) and human chronic myeloid leukemia K562 cells were transducted with them. Single-cell-derived clones were screened for miR-21 deletion by genomic DNA PCR and Sanger sequence. RQ-PCR assays was used to confirm the knockout of miR-21. 3-(4,5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide(MTT) and colony formation assays were applied to assess the cell growth inhibition. Imatinib and dasatinib sensitivity was determined by MTT assay and Annexin-V APC/7-AAD double staining flow cytometry. Western blot assay was performed to measure the levels of Phosphatase and tensin homolog(PTEN), Phosphatidylinositol 3-kinase(PI3K), Serine/threonine Kinase(AKT), p-AKT, BCR-ABL(P210), p-BCR-ABL (p-P210) . Result: miR-21 knockout inhibited proliferation of K562 cells, promoted their apoptosis and increased their sensitivity to dasatinib. Further mechanism studies suggest that this is achieved by inhibiting the PI3K/AKT signaling pathway and destroying BCR-ABL. Conclusions: Our study reveals the efficacy of CRISPR/Cas9 gene editing to miR-21 in K562 and indicates miR-21 as a potential target in sensitizing dasatinib treatment for CML patients with pool response to the TKI targeting therapy.

Transfecção celular de anti-miR-21 aplicada em linhagem de carcinoma renal de células claras metastáticas

“…Consideramos nossos resultados in vitro promissores, pois o miR-21 pode apresentar um papel importante para a progressão do câncer de rim metastático.Além disso, nossos achados corroboram com dados da literatura que mostram que o miR-21 foi aumentado em amostras de CRCC, sugerindo que o miR-21 é um regulador crítico no desenvolvimento do CRCC humano(53). Além disso, muitos autores relatam que o miR-21 diminuído está associado à diminuição da progressão de outros tipos de câncer, como o câncer de ovário, leucemia mieloide crônica e carcinoma de nasofaringe(54)(55)(56).…”