He C T, Lei Y, Du J R, Jia J J, Hu Q, Niu Q
Department of Occupational Health, School of Public Health, Shanxi Medical University, Taiyuan 030001, China Department of Human Anatomy, College of Basic Medicine, Shanxi Medical University, Taiyuan 030001, China.
Department of Occupational Health, School of Public Health, Shanxi Medical University, Taiyuan 030001, China.
Zhonghua Lao Dong Wei Sheng Zhi Ye Bing Za Zhi. 2023 May 20;41(5):324-332. doi: 10.3760/cma.j.cn121094-20221118-00549.
To investigate the effect and mechanism of miR-96-5p on apoptosis of PC12 cells induced by maltol aluminum. In January 2021, PC12 cells at logarithmic growth phase were divided into blank control group and low, medium and high dose group. Cells in each group were treated with 0, 100, 200 and 400 μmol/L maltol aluminum for 24 hours respectively. Cells were collected and cell apoptosis rates were detected by flow cytometry, miR-96-5p and insulin receptor substrate 1 (IRS1) mRNA expressions were detected by qRT-PCR, and the protein expression levels of cysteine protease 3 (Caspase3) 、activated cysteine protease 3 (Cleaved-caspase3) 、IRS1、phosphorylated protein kinase B (p-AKT) and phosphorylated glucose synthesis kinase 3β (p-GSK3β) were detected by western blotting. The target binding relationship between miR-96-5p and IRS1 was detected by double luciferase reporter gene experiment. The miR-96-5p inhibitor cells and negative control cells were constructed after transfecting PC12 cells with miR-96-5p inhibitor for 24 hours. The cells were divided into blank control group, negative control group, aluminum exposure group, aluminum exposure+negative control group, aluminum exposure+miR-96-5p inhibition group, and miR-96-5p inhibition group. After transfecting PC12 cells with miR-96-5p inhibition and IRS1 siRNA for 24 h, the cells were divided into aluminum exposure+miR-96-5p inhibition+negative control group and aluminum exposure+miR-96-5p inhibition+IRS1 inhibition group. The control group was cultured in complete culture medium, and cells in the aluminum exposure group were treated with 200 μmol/L maltol aluminum for 24 hours. Cells in each group were collected and the apoptosis rate, miR-96-5p and IRS1 mRNA expression levels, as well as protein expression levels of Caspase3, Cleaved-caspase3, IRS1, p-AKT, and p-GSK3β were measured. After 24 hours of exposure, compared with blank control group and low-dose group, the apoptosis rates, relative expressions of Caspase3 and Cleaved-caspase3 proteins, and relative expressions of miR-96-5p in the medium and high-dose groups of PC12 cells were significantly increased, while the relative expression levels of IRS1 mRNA, IRS1, p-AKT and p-GSK3β proteins were significantly decreased (<0.05). Targetscan prediction and double luciferase report experiment both proved that IRS1 was a direct target gene of miR-96-5p. In the transfection experiment, compared with the aluminum exposure group, the apoptosis rate, the relative expressions of Caspase3 and Cleaved-caspase3 proteins, the relative expression of miR-96-5p in the aluminum exposure+miR-96-5p inhibition group were significantly decreased, while the relative expression levels of IRS1 mRNA and IRS1, p-AKT and p-GSK3β proteins were significantly increased (<0.05). In the IRS1 low expression experiment, compared with the aluminum exposure+miR-96-5p inhibition+negative control group, the apoptosis rate, the relative expressions of Caspase3 and Cleaved-caspase3 proteins in the aluminum exposure+miR-96-5p inhibition+IRS1 inhibition group were significantly increased, while the relative expression levels of IRS1 mRNA and IRS1, p-AKT and p-GSK3β proteins were significantly decreased (<0.05) . The increased expression of miR-96-5p and the targeted inhibition of IRS1 may be one of the mechanisms of apoptosis of PC12 cells induced by maltol aluminum exposure.
探讨miR-96-5p对麦芽酚铝诱导的PC12细胞凋亡的影响及机制。2021年1月，将对数生长期的PC12细胞分为空白对照组、低、中、高剂量组。每组细胞分别用0、100、200和400 μmol/L麦芽酚铝处理24小时。收集细胞，采用流式细胞术检测细胞凋亡率，采用qRT-PCR检测miR-96-5p和胰岛素受体底物1（IRS1）mRNA表达，采用蛋白质印迹法检测半胱氨酸蛋白酶3（Caspase3）、活化半胱氨酸蛋白酶3（Cleaved-caspase3）、IRS1、磷酸化蛋白激酶B（p-AKT）和磷酸化葡萄糖合成激酶3β（p-GSK3β）的蛋白表达水平。通过双荧光素酶报告基因实验检测miR-96-5p与IRS1之间的靶标结合关系。用miR-96-5p抑制剂转染PC12细胞24小时后构建miR-96-5p抑制剂细胞和阴性对照细胞。将细胞分为空白对照组、阴性对照组、铝暴露组、铝暴露+阴性对照组、铝暴露+miR-96-5p抑制组和miR-96-5p抑制组。用miR-96-5p抑制剂和IRS1 siRNA转染PC12细胞24小时后，将细胞分为铝暴露+miR-96-5p抑制+阴性对照组和铝暴露+miR-96-5p抑制+IRS1抑制组。对照组在完全培养基中培养，铝暴露组细胞用200 μmol/L麦芽酚铝处理24小时。收集每组细胞，检测细胞凋亡率、miR-96-5p和IRS1 mRNA表达水平，以及Caspase3、Cleaved-caspase3、IRS1、p-AKT和p-GSK3β的蛋白表达水平。暴露24小时后，与空白对照组和低剂量组相比，PC12细胞中、高剂量组的凋亡率、Caspase3和Cleaved-caspase3蛋白的相对表达以及miR-96-5p的相对表达均显著升高，而IRS1 mRNA、IRS1、p-AKT和p-GSK3β蛋白的相对表达水平显著降低（<0.05）。Targetscan预测和双荧光素酶报告实验均证明IRS1是miR-96-5p的直接靶基因。在转染实验中，与铝暴露组相比，铝暴露+miR-96-5p抑制组的凋亡率、Caspase3和Cleaved-caspase3蛋白的相对表达、miR-96-5p的相对表达均显著降低，而IRS1 mRNA和IRS1、p-AKT1、p-AKT和p-GSK3β蛋白的相对表达水平显著升高（<0.05）。在IRS1低表达实验中,与铝暴露+miR-96-5p抑制+阴性对照组相比，铝暴露+miR-96-5p抑制+IRS1抑制组的凋亡率、Caspase3和Cleaved-caspase3蛋白的相对表达均显著升高，而IRS1 mRNA和IRS1、p-AKT和p-GSK3β蛋白的相对表达水平显著降低（<0.05）。miR-96-5p表达增加和对IRS1的靶向抑制可能是麦芽酚铝暴露诱导PC12细胞凋亡的机制之一。
