Effect of MiR-155 Knockout Mediated by Dual sgRNAs on Drug Sensitivity of FLT3-ITD+AML.
10.19746/j.cnki.issn.1009-2137.2022.02.002
- Author:
Ling-Yan WANG
1
;
Pei-Fang JIANG
1
;
Jia-Zheng LI
1
;
Jian-Da HU
2
Author Information
1. Department of Hematology, Fujian Medical University Union Hospital, Fujian Provincial Key Laboratory of Hematology, Fujian Institute of Hematology, Fuzhou 350001, Fujian Province, China.
2. Department of Hematology, Fujian Medical University Union Hospital, Fujian Provincial Key Laboratory of Hematology, Fujian Institute of Hematology, Fuzhou 350001, Fujian Province, China,E-mail: drjiandahu@163.com.
- Publication Type:Journal Article
- Keywords:
CRISPR/Cas9;
FLT3-ITD;
adriamycin;
miR-155;
quizartinib
- MeSH:
CRISPR-Cas Systems;
Doxorubicin/pharmacology*;
Drug Resistance;
Gene Editing;
Humans;
Leukemia, Myeloid, Acute/genetics*;
MicroRNAs/genetics*;
RNA, Guide/genetics*;
fms-Like Tyrosine Kinase 3/genetics*
- From:
Journal of Experimental Hematology
2022;30(2):334-340
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVE:Two sgRNAs transfected FLT3-ITD+AML cell line MV411 with different binding sites were introduced into CRISPR/cas9 to obtain MV411 cells with miR-155 gene knockout. To compare the efficiency of miR-155 gene knockout by single and double sgRNA transfection and their effects on cell phenotypes.
METHODS:The lentiviral vectors were generated containing either single sgRNA or dual sgRNAs and packaged into lentivirus particles. PCR was conducted to measure gene editing efficiency, and miR-155 expression was evaluated by qPCR. CCK-8 assay was used to evaluate the cell proliferation, and calculate drug sensitivity of cells to adriamycin and quizartinib. Annexin V-APC/7-AAD staining was used to label cell apoptosis induced by adriamycin and quizartinib.
RESULTS:In the dual sgRNAs transfected cells, a cleavage band could be observed, meaning the success of gene editing. Compared with the single sgRNA transfected MV411 cells, the expression level of mature miR-155-5p was lower in the dual sgRNA transfected cells. And, dual sgRNA transfected MV411 were more sensitive to adriamycin and quizartinib with lower IC50 and higher apoptosis rate.
CONCLUSION:The inhibition rate of miR-155 gene expression transfected by dual sgRNA is higher than that by single sgRNA. Dual sgRNA transfection can inhibit cell proliferation, reverse drug resistance, and induce apoptosis more significantly. Compared with single sgRNA transfection, dual sgRNA transfection is a highly efficient gene editing scheme.
