川北医学院
1.四川省科技厅项目(24NSFSC5546)；2.南充市科学技术局项目(20SXQT0333)；3.大学生创新创业国家级项目(202310634020)；4.大学生创新创业省级项目(202310634040)。
1.North Sichuan Medical College;2.North Sichuan Medical college
目的 基于NOX4/ROS信号通路探讨EFHD2影响乳腺癌发生进展的作用机制。方法 将细胞分为NC-shRNA组和EFHD2-shRNA组，构建沉默EFHD2表达的慢病毒载体及其对照载体后转染乳腺癌细胞MDA-MB-23、MCF-7，qRT-PCR验证转染效率；CCK8检测细胞增殖活性；平板克隆实验检测细胞集落形成能力；划痕实验检测细胞迁移；Transwell实验检测细胞侵袭；流式细胞术检测细胞凋亡及ROS水平；qRT-PCR检测GLUT1、PDK1、PFK1、PKM2、PDH、LDH mRNA的表达；Western blot检测Cleaved caspase-3、MMP-2、NOX4蛋白的表达。 结果 与NC-shRNA组相比，EFHD2-shRNA组细胞中EFHD2的表达明显降低，细胞存活及其集落形成能力减弱；细胞凋亡率及促凋亡蛋白Cleaved caspase-3的表达升高；细胞迁移距离缩短，细胞侵袭数及促迁移及侵袭蛋白MMP-2的表达降低；乳酸及GLUT1、PDK1、PFK1、PKM2、LDH的水平降低，ATP及PDH的水平升高；流式结果表明沉默EFHD2后ROS水平降低，NOX4蛋白下调。结论 EFHD2可通过调节NOX4/ROS信号通路，抑制ROS生成，引起乳酸及葡萄糖堆积从而促进乳腺癌细胞凋亡，抑制细胞增殖、迁移及侵袭。
Objective To investigate the mechanism of EFHD2 affecting the occurrence and progression of breast cancer based on NOX4/ROS signaling pathway. Methods The cells were divided into NC-shRNA group and EFHD2-shRNA group. The lentiviral vector silencing EFHD2 expression and its control vector were constructed and transfected MDA-MB-23 and MCF-7 breast cancer cells. The transfection efficiency was verified by qRT-PCR.CCK8 assay was used to detect cell proliferation activity. Plate cloning assay was used to detect cell colony formation ability. Scratch test was used to detect cell migration. Transwell assay was used to detect cell invasion. Flow cytometry was used to detect apoptosis and ROS levels. qRT-PCR was used to detect the mRNA expression of GLUT1, PDK1, PFK1, PKM2, PDH, and LDH. Western blot was used to detect the expression of Cleaved caspase-3, MMP-2 and NOX4 proteins. Results Compared with NC-shRNA group, EFHD2 expression was significantly decreased in EFHD2-SHRNA group, and cell survival and colony formation ability were weakened. The apoptosis rate and the expression of pro-apoptotic protein Cleaved caspase-3 increased. The cell migration distance was shortened, the number of cell invasion and the expression of MMP-2, which promotes migration and invasion, were decreased. The levels of lactic acid and GLUT1, PDK1, PFK1, PKM2 and LDH decreased, while the levels of ATP and PDH increased. Streaming results showed that ROS levels were reduced and NOX4 protein was down-regulated after silencing EFHD2. Conclusion EFHD2 can inhibit ROS production by regulating NOX4/ROS signaling pathway, cause lactic acid and glucose accumulation, promote apoptosis of breast cancer cells, and inhibit cell proliferation, migration and invasion.
