HO-1诱导表达在调控乳腺癌细胞对阿霉素敏感性中的作用及分子机制研究

发布时间：2018-01-26 05:16

  本文关键词： 乳腺癌 HO-1 阿霉素 化疗敏感性　出处：《广西医科大学》2015年硕士论文　论文类型：学位论文

【摘要】：目的 研究血红素加氧酶-1(heme oxygenase, HO-1)在调控乳腺癌细胞对阿霉素敏感性中的作用及其相关分子机制。方法 (1)PI染色结合细胞流式术测定乳腺癌MDA-MB-453和MDA-MB-231细胞对阿霉素的凋亡反应；(2)利用Western blot检测乳腺癌MDA-MB-453和MDA-MB-231细胞在阿霉素刺激前后HO-1蛋白的表达水平；(3)采用siRNA介导的RNA干扰技术阻断阿霉素对HO-1的诱导表达,用细胞流式术检测其对细胞凋亡的影响；(4)采用细胞自噬检测试剂盒(ENZO)染色,并分别应用共激聚焦显微镜、细胞流式术检测细胞自噬水平；(5)应用双荧光素酶基因检测、Western blot等方法检测阿霉素刺激STAT3等转录因子及Src等蛋白激酶的活性变化；(6)应用siRNA干扰、Western blot等技术探讨阿霉素诱导HO-1表达及其影响乳腺癌细胞对阿霉素敏感性的分子机制。结果 阿霉素(0.2μM)刺激48小时后,MDA-MB-453细胞凋亡率明显上升(从1.5%到40%),而MDA-MB-231细胞的凋亡率较低(从3.29%到13.7%)。Western blot结果显示MDA-MB-231细胞经阿霉素刺激后,HO-1蛋白出现了显著的诱导表达上调,而在MDA-MB-453细胞中并未观察到此现象。沉默HO-1基因表达后,MDA-MB-231细胞凋亡率显著上升。进一步研究发现,阿霉素可诱导MDA-MB-231细胞的自噬水平明显升高,抑制自噬后MDA-MB-231细胞凋亡率明显上升。抑制HO-1基因表达后,MDA-MB-231细胞的自噬水平显著下降。阿霉素的刺激作用下,MDA-MB-231细胞出现了STAT3和磷酸化STAT3蛋白表达上调,双荧光素酶报告基因表达分析显示STAT3的转录活性增强,抑制STAT3的表达后H0-1蛋白的诱导表达及细胞自噬水平均显著被抑制。在MDA-MB-231细胞中,阿霉素还显著诱导Src活化水平上调,抑制Src的表达后STAT3转录活性、H0-1蛋白及自唆表达水平均明显下调。结论 阿霉素特异性诱导乳腺癌MDA-MB-231细胞HO-1高表达,阻断HO-1的诱导表达能显著增加其对阿霉素的敏感性;HO-1维持乳腺癌细胞对阿霉素的抗性作用可能是通过诱导细胞自嚼来介导的;阿霉素诱导HO-1表达的可能机制是通过激活Src/STAT3信号通路,进而上调HO-1的表达水平;抑制Src/STAT3/HO-l/autophagy信号途径的激活能显著增加乳腺癌细胞对阿霉素的敏感性。
[Abstract]:Objective to study heme oxygenase of heme oxygenase-1. The role of HO-1 in regulating the sensitivity of breast cancer cells to adriamycin and its related molecular mechanisms. Pi staining and flow cytometry were used to detect the apoptosis of MDA-MB-453 and MDA-MB-231 cells to doxorubicin. (2) Western blot was used to detect the expression of HO-1 protein in MDA-MB-453 and MDA-MB-231 cells before and after adriamycin stimulation. (3) RNA interference mediated by siRNA was used to block the expression of HO-1 induced by adriamycin, and its effect on apoptosis was detected by flow cytometry. The level of autophagy was detected by confocal microscope and flow cytometry. (5) double luciferase gene assay was used to detect the activity of STAT3 and protein kinase such as Src stimulated by doxorubicin. Application of siRNA interference. Western blot and other techniques to investigate the expression of HO-1 induced by adriamycin and its molecular mechanism affecting the sensitivity of breast cancer cells to adriamycin. After 48 hours of stimulation. The apoptosis rate of MDA-MB-453 cells increased significantly (from 1.5% to 40). However, the apoptosis rate of MDA-MB-231 cells was lower (from 3.29% to 13.70.Western blot results showed that MDA-MB-231 cells were stimulated by adriamycin). The expression of HO-1 protein was significantly up-regulated, but not observed in MDA-MB-453 cells. The expression of HO-1 gene was silenced. The apoptosis rate of MDA-MB-231 cells was significantly increased. Further studies showed that doxorubicin could induce a marked increase in autophagy level of MDA-MB-231 cells. After inhibiting autophagy, the apoptosis rate of MDA-MB-231 cells increased significantly. After inhibiting the expression of HO-1 gene, the autophagy level of MDA-MB-231 cells decreased significantly. The expression of STAT3 and phosphorylated STAT3 protein was up-regulated in MDA-MB-231 cells, and the expression of double luciferase reporter gene showed that the transcriptional activity of STAT3 was enhanced. The expression of H0-1 protein and the level of autophagy were significantly inhibited after inhibiting the expression of STAT3. In MDA-MB-231 cells, doxorubicin also significantly upregulated the level of Src activation. The transcriptional activity of STAT3 was inhibited after the expression of Src. Conclusion doxorubicin specifically induces the overexpression of HO-1 in breast cancer MDA-MB-231 cells. Blocking the induced expression of HO-1 could significantly increase its sensitivity to adriamycin. The maintenance of HO-1 resistance to adriamycin in breast cancer cells may be mediated by inducing self-chewing. The possible mechanism of HO-1 expression induced by doxorubicin is by activating the Src/STAT3 signaling pathway and then upregulating the level of HO-1 expression. Inhibition of activation of Src/STAT3/HO-l/autophagy signaling pathway significantly increased the sensitivity of breast cancer cells to adriamycin.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R737.9
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