miRNA-106a在人淋巴瘤Jurkat细胞中的表达及作用机制

Expression and mechanism of miRNA-106a in human lymphoma Jurkat cells

目的探讨miRNA-106a在人淋巴瘤Jurkat细胞中的表达及作用机制。方法取对数生长期人淋巴瘤Jurkat细胞,分别向培养基中加入5 ml生理盐水配制的浓度为0、0.5、1.0、1.5μg/ml的多柔比星,取健康体检者(对照组)的单个核细胞。采用定量逆转录聚合酶链反应(qRT-PCR)检测miRNA-106a以及视网膜母细胞瘤1(RB1)、E2F转录因子1(E2F1)、胱天蛋白酶3(caspase 3)mRNA的表达水平,采用噻唑蓝(MTT)法检测细胞增殖能力,流式细胞术检测细胞凋亡能力,采用蛋白质印迹法(Western blot)检测RB1、E2F1、caspase 3蛋白的表达水平。结果0μg/ml多柔比星干预人淋巴瘤Jurkat细胞miRNA-106a的表达水平明显高于对照组单个核细胞(P﹤0.01)。随多柔比星浓度升高、作用时间延长,miRNA-106a表达水平逐渐降低,光密度(OD)值逐渐降低,细胞增殖抑制率(IR)和凋亡率均逐渐升高,RB1、caspase 3 mRNA及其蛋白的表达水平均逐渐升高,E2F1 mRNA及其蛋白的表达水平均逐渐降低,差异均有统计学意义(P﹤0.05)。相关性分析结果显示,miRNA-106a与RB1、caspase 3的表达均呈负相关(P﹤0.01),与E2F1的表达呈正相关(P﹤0.01)。结论miRNA-106a在人淋巴瘤Jurkat细胞中高表达,其可能通过调控RB/E2F1通路相关蛋白的表达来调节淋巴瘤进展。

Objective To study the expression and mechanism of miRNA-106a in human lymphoma Jurkat cells.Method Take human lymphoma Jurkat cells in logarithmic growth phase,and prepare 5 ml of physiological saline in the culture medium with concentrations of 0,0.5,1.0,and 1.5μg/ml of doxorubicin.Take blood mononuclear cells from healthy individuals(control group).Quantitative reverse transcription-polymerase chain reaction(qRT-PCR)was used to detect the expression levels of miRNA-106a and retinoblastoma 1(RB1),E2F transcription factor 1(E2F1),and caspase 3 mRNA.Methyl thiazolyl terazolium(MTT)assay was used to detect cell proliferation ability,flow cytometry was used to detect cell apoptosis ability,and Western blot was used to detect the expression levels of RB1,E2F1,and caspase 3 protein.Result The expression levels of miRNA-106a in Jurkat cells of lymphoma treated with 0μg/ml doxorubicin were higher than that in blood mononuclear cells from control group(P<0.01).As the concentration of doxorubicin increased and the duration of action prolonged,the expression levels of miRNA-106a gradually decreased,the optical density(OD)gradually decreased,and the inhibition rate(IR)of cell proliferation and apoptosis gradually increased,the expression levels of RB1,caspase 3 mRNA and their proteins also gradually increased,while the expression levels of E2F1 mRNA and its protein gradually decreased,and the differences were statistically significant(P<0.05).The correlation analysis results showed that miRNA-106a were negatively correlated with the expression of RB1 and caspase 3(P<0.01),and positively correlated with the expression of E2F1(P<0.01).Conclusion miRNA-106a is highly expressed in human lymphoma Jurkat cells,and may regulate lymphoma progression by regulating the expression of RB/E2F1 pathway related proteins.