miR-7-5p对氢醌诱导TK6细胞损伤作用

发布时间：2018-08-18 20:57

【摘要】：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(P0.01),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(P0.01),细胞早期凋亡率增加(P0.01),PARP-1和BRCA1蛋白相对表达水平均下降(P0.05)。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。
[Abstract]:Objective to construct a stable human lymphoblastocyte TK6 cell line with miR-7-5p overexpression by lentivirus technique and to explore the mechanism of miR-7-5p on the damage of TK6 cells induced by hydroquinone. Methods (1) miR-7-5p overexpression lentivirus vector was constructed and transfected into TK6 cells. MiR-7-5p overexpression TK6 cell lines (TK6-miR-7-5p cells) and negative empty vector control TK6 cells (TK6-NC cells) were obtained by purine mycin screening. The construction effect was evaluated by real-time fluorescence quantitative polymerase chain reaction. (2) the final concentration of hydrogen was 0 and 40 渭 mol/L. TK6-NC cells and TK6-miR-7-5p cells were treated with quinone for 48 h, respectively. CCK-8 assay was used to detect cell survival rate, flow cytometry was used to detect early apoptosis rate, and Western blot was used to detect the relative expression of PARP-1 and BRCA1 protein in three kinds of cells treated with hydroquinone at the final concentration of 40 渭 mol/L. Results (1) TK6 cell lines with stable expression of miR-7-5p were successfully screened, and the relative expression level of miR-7-5p in TK6-miR-7-5p cells was increased by about 17 times (P0.01) compared with that in normal TK6 cells. (2) after treated with 40 渭 mol/L hydroquinone at 40 渭 mol/L, the relative expression level of miR-7-5p in TK6-miR-7-5p cells was increased by 17 times (P0.01). (2) three kinds of cells were treated with 40 渭 mol/L hydroquinone. Compared with normal TK6 cells and TK6-NC cells, the survival rate of TK6-miR-7-5p cells decreased (P0.01), and the early apoptosis rate increased (P0.01). The relative expression of PARP-1 and BRCA1 protein decreased (P0.05). Conclusion miR-7-5p may induce early apoptosis of TK6 cells induced by hydroquinone by inhibiting DNA damage and repair ability of PARP-1 and BRCA1.
【作者单位】：广东医科大学公共卫生学院广东医科大学环境与健康研究所东莞环境医学重点实验室;
【基金】：广东省普通高校重点平台特色创新类项目(自然科学类)(2015KTSCX052) 广东省自然科学基金(S2013010015153) 东莞市社会科技发展(一般)项目(2017507152413) 广东医科大学科研基金(M2016012)
【分类号】：R114

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