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目的通过慢病毒技术构建miR-7-5p稳定过表达的人淋巴母细胞TK6细胞株,探讨miR-7-5p对氢醌诱导TK6细胞损伤的作用机制。方法 (1)构建miR-7-5p过表达慢病毒载体,转染TK6细胞,经嘌呤霉素筛选获得miR-7-5p稳定过表达TK6细胞株(TK6-miR-7-5p细胞)和阴性空载体对照TK6细胞(TK6-NC细胞),采用实时荧光定量聚合酶链式反应鉴定构建效果。(2)以终浓度为0和40μmol/L的氢醌分别处理TK6细胞、TK6-NC细胞和TK6-miR-7-5p细胞48 h后,采用CCK-8法检测细胞存活率,采用流式细胞术检测细胞早期凋亡率;采用免疫印迹法检测以终浓度为40μmol/L的氢醌处理的3种细胞中PARP-1和BRCA1蛋白的相对表达水平。结果 (1)成功筛选出稳定过表达miR-7-5p的TK6细胞株;与正常TK6细胞比较,TK6-miR-7-5p细胞中miR-7-5p的相对表达水平升高约17倍(P<0.01),但细胞形态未出现明显改变。(2)予浓度为40μmol/L氢醌处理3种细胞后,与正常TK6细胞和TK6-NC细胞比较,TK6-miR-7-5p细胞存活率下降(P<0.01),细胞早期凋亡率增加(P<0.01),PARP-1和BRCA1蛋白相对表达水平均下降(P<0.05)。结论 miR-7-5p可能通过抑制PARP-1和BRCA1的DNA损伤修复能力而导致氢醌诱导TK6细胞早期凋亡增加。
Objective To construct miR-7-5p overexpressed human lymphoblastoid cell line TK6 by lentivirus and to explore the mechanism of miR-7-5p on hydroquinone-induced TK6 cell injury. Methods (1) To construct miR-7-5p overexpression lentiviral vector and transfect TK6 cells. After puromycin screening, TK6-miR-7-5p cells (TK6-miR-7-5p cells) Empty vector control TK6 cells (TK6-NC cells), the use of real-time fluorescent quantitative polymerase chain reaction to identify the construction effect. (2) TK6 cells, TK6-NC cells and TK6-miR-7-5p cells were treated with hydroquinone at final concentrations of 0 and 40 μmol / L for 48 h, respectively. Cell viability was determined by CCK-8 assay. The cell apoptosis rate was detected by cytometry. The relative expression levels of PARP-1 and BRCA1 in 3 kinds of cells treated with hydroquinone at the final concentration of 40 μmol / L were detected by Western blotting. Results (1) The TK6 cell line stably overexpressing miR-7-5p was successfully screened. Compared with normal TK6 cells, the relative expression level of miR-7-5p in TK6-miR-7-5p cells increased about 17 times P <0.01), but the cell morphology did not change significantly. (2) Compared with normal TK6 cells and TK6-NC cells, the survival rate of TK6-miR-7-5p cells was decreased (P <0.01) after treated with 40μmol / L hydroquinone for 3 kinds of cells. The early apoptosis rate (P <0.01). The relative expression of PARP-1 and BRCA1 protein decreased (P <0.05). Conclusions miR-7-5p may induce early apoptosis of TK6 cells induced by hydroquinone through inhibition of DNA damage repair ability of PARP-1 and BRCA1.