microRNA家族成员let-7e对人单核细胞系THP-1细胞活性的影响及机制研究

Effecst of let-7e on the cell activities of monocytic cell line THP-1 and the possible mechanism

目的：研究let-7e对单核细胞系THP-1细胞凋亡和细胞因子分泌的影响及其机制。方法免疫荧光法检测cy3标记的mimic 对照转染THP-1细胞转染率，qRT-PCR法检测let-7e mimic及对照、let-7e inhibitor及对照转染THP1-细胞后let-7e的表达；MTT法检测转染后THP-1细胞活性，流式细胞术检测细胞凋亡，Western blot法检测let-7e靶基因IFI6（interferon alpha-inducible protein 6）、EZH2（enhancer of zeste homolog 2）、caspase-3的表达；转染48h 后THP-1细胞用LPS刺激2h收集细胞培养上清，细胞因子芯片法检测上清中炎性因子的表达。结果 miRNA模拟物可以转染THP-1细胞，转染率约为75％，转染 let-7e mimic 12h 、24 h、48 h 后THP-1细胞的 let-7e 表达倍数分别为8．551±0．365、83．893±15．941、38．858±2．743，转染 let-7e inhibitor 12 h、24 h、48 h后THP-1细胞的let-7e表达倍数分别为0．594±0．174、2．427±1．229、3．053±0．207；let-7e mimic转染组THP-1细胞活性升高、凋亡率降低；miranda数据库分析证实let-7e与EZH2、IFI6、caspase-3结合的mirSVR score 分别为－0．1608、－0．5693、－09．423，证实IFI6、EZH2、caspase-3可能是let-7e的靶基因；let7-e mimic转染组IFI6、EZH2、caspase-3表达都有所下降；let-7e mimic 转染组 CD154、粒细胞集落刺激因子（ G-CSF）、CD54、IL-13、IL-1RA和IL-23表达有所下降。结论 let-7e有抗THP-1细胞凋亡的作用，可影响LPS诱导的细胞因子的表达。 let-7e可能通过调节其靶基因caspase-3、IFI6、EZH2对THP-1细胞的生物学功能产生影响。

Objective To investigate the effects of a microRNA family member , let-7e, on mono-cytic cell line THP-1 with regard to cell apoptosis and cytokine secretion and to analyze the possible mecha -nism.Methods THP-1 cells were transfected with mimic negative control （cy3） and observed with immu-nofluorescence microscopy for the evaluation of transfection rate .The expression of let-7e in THP-1 cells re-spectively transfected with let-7e mimic, mimic negative control, let-7e inhibitor and inhibitor negative con-trol were detected by qRT-PCR.MTT assay and flow cytometry analysis were used to detect the activities and apoptosis of transfected THP-1 cells.Western blot assay was performed to measure the expression of the genes encoding interferon alpha-inducible protein 6（ IFI6 ） , enhancer of zeste homolog 2 （EZH 2 ） and caspase -3 that were target genes of let-7e predicted by bioinformatics analysis .THP-1 cells were transfected with let-7e mimic and mimic negative control for 48 h and then stimulated with LPS for 2 h for further detec-tion.The supernatants of cell culture were collected for the detection of secreted cytokines by Human Cyto -kine Array.Results The monocytic THP-1 cells were transfected with mimic negative control with a trans-fection efficiency of about 75%.There were 8.551±0.365, 83.893±15.941, 38.858±2.743 and 0.594± 0.174, 2.427±1.229, 3.053±0.207 fold increases in let-7e expression after the transient transfection of THP-1 cells with let-7e mimic and let-7e inhibitor for 12 h, 24 h and 48 h, respectively.The transfection of＆amp;nbsp;let-7e mimic into THP-1 cells enhanced the cell activities and inhibited the apoptosis of the transfected cells . Bioinformatics analysis showed that let-7e bound to the genes encoding EZH 2, IFI6 and caspase-3 with the mirSVR scores of -0.1608,-0.5693 and-0.9423, suggesting them as the predicted target genes of let-7e. The expressions of IFI6, EZH2 and caspase-3 in let-7e mimic transfected THP-1 cells were decreased as in-dicated by Western blot assay .The results of Human Cytokine Array showed that the expression of LPS-in-duced cytokines including CD154, G-CSF, CD54, IL-13, IL-1RA and IL-23 were inhibited in let-7e mimic transfected THP-1 cells. Con clusion Let-7e had an anti-apoptosis effect on monocytic THP-1 cells and in-fluenced the secretion of LPS-induced cytokines in THP-1 cells.Let-7e might regulate the biological function of THP-1 cells through inhibiting the expression of target genes encoding caspase -3, IFI6 and EZH2.