miR-155表达与FLT3-ITD^(+)急性髓系白血病细胞系药物敏感性的关系及机制研究

Correlation of miR-155 Expression with Drug Sensitivity of FLT3-ITD^(+)Acute Myeloid Leukemia Cell Line and Its Mechanism

目的:研究miR-155的表达与FLT3-ITD^(+)急性髓系白血病细胞系药物敏感性的关系及潜在的调控机制。方法:通过CRISPR/Cas9基因编辑工具在FLT3-ITD^(+)AML细胞系MV411中实现miR-155编码基因敲除,筛选单克隆细胞,PCR及Sanger测序鉴定单克隆细胞的基因型,实时荧光定量逆转录PCR鉴定成熟miRNA的表达水平。MTT法计算半数抑制率,比较MV411细胞对多柔比星、奎扎替尼以及米哚妥林的药物敏感性。转录组测序分析miR-155敲除后MV411细胞mRNA水平的变化,基因集富集分析获得miR-155敲除后的信号通路的变化水平,Western blot验证信号通路关键分子的表达。结果:通过PCR初筛和Sanger测序获得了4个基因敲除的杂合子和1个基因插入的杂合子。实时荧光定量逆转录PCR结果显示,这些单克隆细胞中成熟miR-155的表达显著低于野生型克隆。MTT结果显示,与野生型相比,miR-155基因敲除后MV411细胞对多种抗FLT3-ITD^(+)AML的药物敏感性有了显著增加。RNA测序显示miR-155敲除后mTOR信号通路和Wnt信号通路受到抑制。Western blot结果显示,信号通路关键分子p-mTOR、Wnt5α、β-catenin的表达下调。结论:miR-155敲除后能显著提升MV411细胞对多柔比星、奎扎替尼和米哚妥林的药物敏感性,这与包括mTOR及Wnt信号通路在内的多条信号通路的抑制有关。

Objective:To investigate the correlation of miR-155 expression with drug sensitivity of FLT3-ITD^(+)acute myeloid leukemia(AML)cell line and its potential regulatory mechanism.Methods:By knocking out miR-155 gene in FLT3-ITD^(+)AML cell line MV411 through CRISPR/Cas9 gene-editing technology,monoclonal cells were screened.The genotype of these monoclonal cells was validated by PCR and Sanger sequencing.The expression of mature miRNA was measured by RT-qPCR.The treatment response of doxorubicin,quizartinib and midostaurin were measured by MTT assay and IC50 of these drugs were calculated to identify the sensitivity.Transcriptome sequencing was used to analyze change of mRNA level in MV411 cells after miR-155 knockout,gene set enrichment analysis to analyze change of signaling pathway,and Western blot to verify expressions of key molecules in signaling pathway.Results:Four heterozygotes with gene knockout and one heterozygote with gene insertion were obtained through PCR screening and Sanger sequencing.RT-qPCR results showed that the expression of mature miR-155 in the monoclonal cells was significantly lower than wild-type clones.MTT results showed that the sensitivity of MV411 cells to various anti FLT3-ITD^(+)AML drugs increased significantly after miR-155 knockout compared with wild-type clones.RNA sequencing showed that the mTOR signaling pathway and Wnt signaling pathway were inhibited after miR-155 knockout.Western blot showed that the expressions of key molecules p-mTOR,Wnt5αandβ-catenin in signaling pathway were down-regulated.Conclusion:Drug sensitivity of MV411 cells to doxorubicin,quizartinib and midostaurin can be enhanced significantly after miR-155 knockout,which is related to the inhibition of multiple signaling pathways including mTOR and Wnt signaling pathways.