塞利尼索通过调控miR-205-5p/OTUB1通路对白血病KOCL44细胞增殖、凋亡的影响

Effect of Selinexor on proliferation and apoptosis of leukemia KOCL44 cells by regulating miR-205-5p/OTUB1 pathway

目的 探讨塞利尼索(Selinexor)对急性淋巴细胞白血病细胞KOCL44增殖、凋亡的影响,分析其对微小RNA-205-5p(miR-205-5p)/去泛素化酶卵巢肿瘤蛋白域所含泛素乙酰结合蛋白1(OTUB1)通路的调控作用。方法 体外培养KOCL44细胞,随机分为空白组和低、中、高剂量实验组(125、250、500 nmol·L^(-1)塞利尼索)及高剂量+anti-miR-205-5p组(转染anti-miR-205-5p 48 h后再用500 nmol·L^(-1)塞利尼索处理)、高剂量+pcDNA-OTUB1组(转染pcDNA-OTUB1 48 h后再用500 nmol·L^(-1)塞利尼索处理)。用细胞计数-8(CCK-8)实验检测细胞增殖情况;用流式细胞术检测细胞凋亡情况;用实时荧光定量聚合酶链反应(qRT-PCR)检测miR-205-5p表达水平;用蛋白质印迹法检测OTUB1蛋白表达水平;用双荧光素酶报告实验检测miR-205-5p、OTUB1靶向关系。结果 空白组和低、中、高剂量实验组72 h细胞活力分别为1.21±0.08、1.03±0.08、0.81±0.05和0.67±0.05;细胞凋亡率分别为(5.62±0.49)%、(8.99±0.77)%、(16.81±1.43)%和(25.98±2.22)%;miR-205-5p表达水平分别为1.00±0.00、1.73±0.11、3.08±0.28和5.35±0.36;OTUB1蛋白水平分别为0.72±0.05、0.58±0.05、0.43±0.03和0.24±0.02。空白组与低、中、高剂量实验组比较,各剂量实验组间比较,差异均有统计学意义(均P<0.05)。高剂量实验组、高剂量+anti-miR-205-5p组、高剂量+pcDNA-OTUB1组72 h细胞活力分别为0.65±0.06、1.08±0.08和1.11±0.08;细胞凋亡率分别为(26.27±2.56)%、(9.19±0.76)%和(8.59±0.72)%。高剂量+anti-miR-205-5p组、高剂量+pcDNA-OTUB1组与高剂量实验组比较,差异均有统计学意义(均P<0.05)。结论 塞利尼索可抑制KOCL44细胞增殖,诱导细胞凋亡,其作用机制与调节miR-205-5p/OTUB1表达有关。

Objective To investigate the effects of Selinexor on the proliferation and apoptosis of KOCL44 cell in acute lymphoblastic leukaemia and analyse its regulation on the microRNA-205-5p(miR-205-5p)/de-ubiquitinating enzyme ovarian tumor protein domain-containing ubiquitin acetyl-binding protein 1(OTUB1)pathway.Methods KOCL44 cells were cultured in vitro and randomly divided into blank group,experimental-L,-M,-H groups(125,250,500 nmol·L^(-1) Celinisol),experimental-H+anti-miR-205-5p group(transfected with anti-miR-205-5p for 48 h followed by 500 nmol·L^(-1) Celinisol treatment),experimental-H+pcDNA-OTUB1 group(transfected with pcDNA-OTUB1 for 48 h followed by 500 nmol·L^(-1) Celinisol treatment).Cell count-8(CCK-8)assay was used to detect cell proliferation;flow cytometry was used to detect apoptosis;quantitative real-time fluorescence polymerase chain reaction(qRT-PCR)was used to detect miR-205-5p expression levels;protein immunoblotting was used to detect OTUB1 protein expression levels;dual luciferase reporter assay was used to detect miR-205-5p,OTUB1 targeting relationship.Results Cell viability at 72 h were 1.21±0.08,1.03±0.08,0.81±0.05 and 0.67±0.05 in the blank and experimental-L,-M,-H groups,respectively;apoptosis rates were(5.62±0.49)%,(8.99±0.77)%,(16.81±1.43)%and(25.98±2.22)%,respectively;miR-205-5p expression levels were 1.00±0.00,1.73±0.11,3.08±0.28 and 5.35±0.36,respectively;OTUB1 protein levels were 0.72±0.05,0.58±0.05,0.43±0.03 and 0.24±0.02,respectively.The above indexes in the experimental-L,-M,-H groups and blank group had statistically significant differences(all P<0.05).The cell viability at 72 h was 0.65±0.06,1.08±0.08 and 1.11±0.08 in the experimental-H group,experimental-H+anti-miR-205-5p group and experimental-H+pcDNA-OTUB1 group,respectively;the apoptosis rates were(26.27±2.56)%,(9.19±0.76)%and(8.59±0.72)%,respectively.The above indexes in the experimental-H+anti-miR-205-5p group,experimental-H+pcDNA-OTUB1 group compared with experimental-H group,the differences were statistically significant(all P<0.05).Conclusion Selinexor can inhibit the proliferation of KOCL44 cells and induce apoptosis,and its mechanism of action is related to the regulation of miR-205-5p/OTUB1 expression.