miR-124靶向RAD51对Jurkat细胞增殖和凋亡及依托泊苷化疗敏感性的影响

Effects of miR-124 targeting RAD51 on the proliferation and apoptosis of Jurkat cells and the chemosensitivity of etoposide

目的研究微小RNA-124(miR-124)对人白血病T淋巴细胞(Jurkat细胞)增殖、凋亡及依托泊苷化疗敏感性的影响,并探讨其作用机制。方法体外培养Jurkat细胞,随机分为对照组、miR-124 NC组和miR-124 mimics组;实时荧光定量PCR(RT-qPCR)法检测转染后各组细胞miR-124及DNA双链断裂修复蛋白51(RAD51)mRNA表达情况;细胞计数试剂盒8(CCK-8)法检测转染后各组细胞存活率变化情况及依托泊苷化疗敏感性;膜联蛋白V-异硫氰酸荧光素/碘化丙啶(Annexin V-FITC/PI)法检测各组细胞凋亡情况;蛋白印迹分析法检测转染后各组细胞RAD51蛋白的表达情况;双荧光素酶报告实验验证miR-124与RAD51的靶向关系。结果与对照组和miR-124NC组相比,miR-124 mimics组Jurkat细胞miR-124表达水平、凋亡率显著升高,差异有统计学意义(P<0.05),细胞存活率、IC50、RAD51 mRNA及蛋白表达水平显著降低,差异有统计学意义(P<0.05)。经mirtarbase数据库预测,miR-124与RAD51 3′UTR有结合位点。与RAD51-3′UTR-WT+miR-124 NC组比较,RAD51-3′UTR-WT+miR-124 mimics组荧光素酶活性降低,差异有统计学意义(P<0.05)。结论 miR-124过表达能够抑制人白血病T淋巴细胞增殖,诱导凋亡,可能通过靶向抑制RAD51表达,提高依托泊苷化疗敏感性。

Objective To study the effects of microRNA-124(miR-124) on proliferation,apoptosis and etoposide sensitivity of human leukemia T lymphocytes(Jurkat cells) and explore its mechanism.Methods Jurkat cells were cultured in vitro and randomly divided into control group,miR-124 NC group and miR-124 mimics group;real-time PCR was used to detect the expression of DNA double-strand break repair protein 51(RAD51)mRNA.Cell counting kit-8(CCK-8)assay was used to detect the cell survival rate and etoposide sensitivity.Annexin V-fluorescein isothiocyanate/propidium iodide(Annexin V-FITC/PI)assay was used to detect apoptosis;the expression of RAD51 protein was detected by Western blot.In addition,double luciferase reporter assay was used to verify the targeting relationship between miR-124 and RAD51.Results Compared with those in control group and miR-124 NC group,the miR-124 expression level and apoptosis rate of Jurkat cells in miR-124 mimics group were significantly higher(P<0.05),and cell survival rate,IC50,RAD51 mRNA and protein expression levels were significantly lower(P<0.05).Mirtarbase database prediction showed that miR-124 had binding sites with RAD51 3’UTR.Compared with that of RAD51-3’UTR-WT + miR-124 NC group,the luciferase activity of RAD51-3’UTR-WT + miR-124 mimics group was lower(P<0.05).Conclusion Overexpression of miR-124 could inhibit the proliferation and induce apoptosis of human leukemic T lymphocytes,which might increase the chemosensitivity of etoposide by targetingly inhibiting the expression of RAD51.