miR-214介导慢性淋巴细胞白血病对氟达拉滨耐药的作用研究

Effect of miR-214 on Fludarabine Resistance in Chronic Lymphocytic Leukemia

目的:探讨miR-214在慢性淋巴细胞白血病(CLL)细胞对氟达拉滨耐药中的作用及机制。方法:选择2014年08月-2018年07月P本院血液科收治对氟达拉滨(Flu)耐药的CLL患者和健康受试者各10例,采用RTPCR法测定单个核细胞中miR-214表达水平。将CLL患者原代细胞分为空白对照组(control group)、阴性对照组(miR-214-NC group)和miR-214转染组(miR-214-ASO group)。转染病毒24 h后与Flu共同培养48 h,测定细胞增殖和凋亡情况,以及PTEN和PI3K/AKT信号通路下游相关基因和蛋白表达水平。结果:CLL患者单个核细胞miR-214的表达水平较健康受试者显著升高(P<0.05);miR-214转染组细胞miR-214表达水平显著降低(P<0.05);miR-214转染组细胞在Flu浓度为3、10和30μmol/L时的吸光度显著降低(P<0.05);Flu浓度为10 mmol/L时miR-214转染组细胞的凋亡率显著增高(P<0.05),PTEN和BAD m RNA水平显著增高(P<0.05),MDM2和NF-κB m RNA水平显著降低(P<0.05),PTEN和p-BAD蛋白水平显著增高(P<0.05),MDM2和NF-κB蛋白水平显著降低(P<0.05)。结论:抑制miR-214可以提高耐药的CLL细胞对氟达拉滨的敏感性,这种作用可能与其促进细胞凋亡有关,并且与其调节PTEN/AKT信号通路下游分子表达有关。

Objective:To investigate effect and mechanism of miR-214 in fludarabine resistance of chronic lymphocytic leukemia(CLL).Methods:A total of 10 patients with CLL resistante to fludarabine(Flu)and 10 healthy persons admitted to Hematology Department of our hospital in August 2014-July 2018 were selected.Expression level of miR-214 in mononuclear cells in patients with CLL and healthy persons were determined by RT-PCR.Primary CLL cells from patients with CLL were divided into normal control group(control group),negative control group(miR-214-NC group)and viral transinfection group(miR-214-ASO group).After 24 h-transfection,CLL cells were cultured with different con-centration of Flu for 48 h,then the cell proliferation and apoptosis were detected,and the levels of down-stream genes and proteins releted with PTEN and PI3 K/AKT signialing pathway were determined.Results:The expression level of miR-214 in mononuclear cells of CLL patients significantly increased in comparison with healthy persons(P<0.05);the expression level of miR-214 in miR-214-ASO group significantly decreased(P<0.05);Absorbance in control group at Flu concentration of 3,10 and 30μmol/L was significantly decreased(P<0.05).Apoptosis rate in miR-214-ASO group at Flu concentration of 10 mmol/L significantly increased(P<0.05).At Flu concentration of 10 mmol/L,mRNA levels PTEN and BAD in miR-214-ASO group significantly increased(P<0.05),but mRNA levels of MDM2 and NF-κB significantly decreased(P<0.05).At Flu concentration of 10 mmol/L,protein levels of PTEN and p-BAD in miR-214-ASO group significantly increased(P<0.05),but protein levels of MDM2 and NF-κB significantly decreased(P<0.05).Conclusion:Inhibition of miR-214 can enhance the sensitivity of drug-resistant CLL cells to fludarabine,which may be raleted with the promotion of cell apotosis and regulation of down-stream molecules expression of PTEN/AKT signaling pathway.