活性氧影响SKOV3细胞间充质转化及其机制研究

发布时间：2019-07-09 16:51

【摘要】：目的:探讨活性氧(ROS)对卵巢上皮性癌细胞株SKOV3细胞的间充质转化的影响及其中可能存在的分子机制。方法:1通过采用ROS生成剂大黄素(Emodin)、ROS清除剂二硫苏糖醇(DTT)调节卵巢上皮性癌细胞株SKOV3细胞内ROS水平,采用PCR技术及蛋白印迹法测定3组(Emodin组、DTT组、对照组)细胞中间充质转化标志分子E钙黏蛋白(E-cadherin)mRNA及蛋白的表达,比较细胞株侵袭转移能力。2结合改变活性氧水平,构建缺氧诱导因子-1α(HIF-1α)的化学合成,小干扰RNA(SiRNA)沉默HIF-1α表达(HIF-1αSiRNA组、DTT组、对照组),比较HIF-1αmRNA的表达。3使用赖氨酰氧化酶(LOX)抑制剂β-氨基丙腈(β-APN)抑制LOX表达,观察各组(Emodin组、DTT组、β-APN组、Emodin+β-APN组、对照组)细胞HIF-1α、LOX、E-cadherin mRNA及蛋白表达。结果:1SKOV3细胞Emodin组ROS水平较对照组明显升高[(164.85±8.93)%vs(97.42±6.47)%,P0.01],E-cadherin mRNA及蛋白的表达均较对照组显著降低(P0.05),Emodin组的细胞侵袭率高于对照组[(19.48±0.49)%vs(10.74±2.38)%,P0.01],而DTT组结果与之相反,与对照组比较差异有统计学意义(P0.05)。2HIF-1αSiRNA作用下,HIF-1αSiRNA组的HIF-1αmRNA表达较对照组降低(0.20±0.09 vs 0.87±0.10,P0.05),LOX mRNA表达较对照组降低(0.40±0.08 vs 3.76±0.66,P0.05),而E-cadherin mRNA表达较对照组增高(1.01±0.15 vs 0.30±0.09,P0.05)。DTT组的效果较HIF-1αSiRNA组更为显著,两组比较,差异均有统计学意义(P0.05)。3β-APN作用下,β-APN组的HIF-1α蛋白表达较对照组无明显变化(0.92±0.19 vs 0.94±0.09,P0.05),LOX蛋白表达较对照组降低(0.35±0.11 vs 0.81±0.13,P0.05),而E-cadherin蛋白表达较对照组增高(4.65±0.90 vs 2.23±0.61,P0.05)。结论:使ROS升高,上调HIF-1α,使LOX过表达,进而下调E-cadherin,ROS可能介导卵巢癌细胞间充质转化,继而发生侵袭转移。
[Abstract]:Objective: To study the effect of reactive oxygen (ROS) on the mesenchymal transition of ovarian epithelial cell line SKOV3 cells and the possible molecular mechanism of ROS. Methods:1. The ROS level in the SKOV3 cells of the epithelial ovarian cancer cell line SKOV3 was adjusted by using the ROS-generating agent Emodin and the ROS-scavenger dithiothreitol (DTT), and the three groups (Emodin group and DTT group) were determined by PCR and Western blot. In the control group, the expression of E-cadherin (E-cadherin) mRNA and protein was compared with the expression of E-cadherin (E-cadherin) mRNA and protein, and the invasion and metastasis ability of the cell line was compared. The expression of HIF-1 mRNA was compared with the expression of HIF-1. The expression of HIF-1, LOX, E-cadherin and the expression of protein in each group (Emodin group, DTT group, P-APN group, Emodin + 1-APN group and control group) were observed. Results: The level of ROS in the Emodin group of 1SKOV3 cells was significantly higher than that in the control group[(164.85-8.93)% vs (97.42-6.47)%, and the expression of E-cadherin mRNA and protein in the Emodin group was significantly lower than that in the control group (P0.05), and the cell invasion rate of the Emodin group was higher than that of the control group[(19.48-0.49)% vs (10.74-2.38)%, P0.01], Compared with the control group, the expression of HIF-1 mRNA in the HIF-1-SiRNA group was lower than that in the control group (0.20-0.09 vs 0.87-0.10, P0.05), while the expression of LOX mRNA was lower in the control group (0.40% 0.08 vs 3.76-0.66, P0.05). The expression of E-cadherin mRNA was higher in the control group (1.01-0.15 vs 0.30-0.09, P0.05). The effect of the DTT group was more significant than that of the HIF-1/ SiRNA group. The difference between the two groups was statistically significant (P <0.05). The expression of HIF-1 in the P-APN group was not significantly changed in the control group (0.92, 0.19 vs. 0.94, 0.09, P0.05), and the expression of LOX protein was lower in the control group (0.35-0.11 vs. 0.81-0.13, The expression of E-cadherin was higher in the control group (4.65, 0.90 vs 2.23, 0.61, P0.05). Conclusion: The increase of ROS, the up-regulation of HIF-1, the overexpression of LOX, and the further down-regulation of E-cadherin, ROS may mediate the transformation of human ovarian cancer cells, and then the invasion and metastasis.
【作者单位】：上海交通大学医学院附属仁济医院;上海交通大学医学院基础医学院细胞生物学教研室;
【基金】：上海市卫生局科研项目(编号:2010272)
【分类号】：R737.31

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