EphA2经P-gp及自噬作用调控鼻咽癌紫杉醇耐药性

发布时间：2018-02-11 03:36

本文关键词： EphA2 鼻咽癌化疗耐药性 P-gp PI3-K/Akt 自噬作用紫杉醇　出处：《中南大学》2014年博士论文　论文类型：学位论文

【摘要】：目的：鼻咽癌紫杉醇耐药性的形成是一复杂调控机制,鉴于经典耐药理论中P-gp蛋白发挥的重要作用以及近年来自噬作用在肿瘤化疗耐药领域的新兴热点地位,本研究在发现EphA2可对鼻咽癌细胞紫杉醇耐药性起调控作用的前期研究基础上,旨在进一步探讨EphA2调控鼻咽癌细胞紫杉醇耐药性的P-gp相关及自噬相关分子机制。方法：1.利用Western Blot分别检测鼻咽癌细胞中慢病毒介导的EphA2沉默及过表达组与阴性对照组(转染无义shRNA或无义cDNA)和空白对照组(转染空载体)内P-gp蛋白的表达情况,探讨EphA2是否具有调控鼻咽癌细胞P-gp表达的能力； 2.利用siRNA干扰技术在鼻咽癌细胞中下调P-gp的表达,同时给予空白对照组(原始细胞)、阴性对照组(转染无义序列)和P-gp表达下调组0.1nM/L的紫杉醇(该浓度为前期研究所得的原始细胞的IC30浓度,结合文献,考虑到P-gp表达下调后的紫杉醇增敏作用,故选择该浓度)刺激,48小时后CCK-8法检测吸光度,得到各组的存活率,以说明P-gp蛋白的表达改变是否影响鼻咽癌细胞的紫杉醇耐药行为； 3.利用siRNA干扰技术在EphA2过表达鼻咽癌细胞中下调P-gp的表达,同时给予空白对照组(EphA2过表达细胞)、阴性对照组(EphA2过表达并转染无义序列细胞)和P-gp表达下调组(EphA2过表达并P-gp表达下调细胞)5nM/L的紫杉醇(前期研究基础中该浓度下实验组与对照组存活率差异最大)刺激,48小时后CCK-8法检测吸光度,得到各组的存活率,以探讨在EphA2过表达细胞中,调控P-gp表达对鼻咽癌细胞紫杉醇耐药性的影响； 4.鉴于文献报道PI3-K/Akt通路参与P-gp蛋白表达调控以及前期研究发现EphA2通过PI3-K/Akt通路影响鼻咽癌紫杉醇耐药性,本研究在EphA2过表达鼻咽癌细胞中,分别加入0nM/L和20nM/L的PI3-K/Akt抑制剂LY294002,再给予5nM/L的紫杉醇刺激,48小时后利用Western Blot检测各组PI3-K/Akt信号通路蛋白Akt和p-Akt,以及P-gp的表达水平,以探讨在EphA2过表达细胞中,PI3-K/Akt通路是否参与P-gp蛋白的表达调控； 5.给予实验组(EphA2过表达鼻咽癌细胞)及对照组(转染无义cDNA)5nM/L紫杉醇刺激,48小时后利用Western Blot检测自噬标志性LC3B-Ⅱ蛋白在实验组及对照组中的表达差异,初步探讨EphA2调控鼻咽癌紫杉醇耐药性的可能机制(自噬作用)。结果：1.EphA2沉默组中P-gp蛋白较两对照组表达下调,EphA2过表达组中P-gp蛋白较两对照组表达上调； 2.紫杉醇作用于空白对照组、阴性对照组和P-gp表达下调组的存活率分别是68.9±4.0%,72.4±6.2%v.s.52.1±3.7%,P-gp表达下调组对紫杉醇的耐药性较两对照组降低(P0.05)； 3.在EphA2过表达鼻咽癌细胞中,紫杉醇作用于空白对照组、阴性对照组和P-gp表达下调组的存活率分别是57.9±6.2%,58.2±5.8%v.s.36.1±5.1%,P-gp表达下调组对紫杉醇的耐药性较两对照组降低(P0.05)； 4.在EphA2过表达鼻咽癌细胞中,给予通路抑制剂组较对照组Akt表达基本不变,而磷酸化的Akt表达下降,P-gp表达下降； 5. EphA2过表达鼻咽癌细胞株中自噬标志性LC3B-Ⅱ蛋白的表达较对照组显著上调。结论：1.EphA2具有调控鼻咽癌细胞P-gp表达的能力； 2.下调P-gp蛋白表达可降低鼻咽癌细胞紫杉醇耐药性； 3.下调P-gp蛋白表达可逆转EphA2介导的鼻咽癌紫杉醇耐药性； 4.在EphA2过表达细胞中,PI3-K/Akt通路参与P-gp蛋白的表达调控； 5.在经紫杉醇作用的鼻咽癌细胞中,EphA2表达的改变能影响自噬相关蛋白的表达水平,提示EphA2也可能经白噬途径调控鼻咽癌细胞的紫杉醇耐药性。综上所述,EphA2可通过介导P-gp蛋白表达,进而调控鼻咽癌细胞紫杉醇耐药性,PI3-K/Akt通路可能参与这一过程；此外,EphA2还可能经自噬途径调控鼻咽癌细胞紫杉醇耐药性。EphA2有望成为鼻咽癌紫杉醇增敏靶点。
[Abstract]:Objective: the formation of taxol resistance is a complex regulatory mechanism, in view of the important role of P-gp protein in the theory of classical resistance and apoptosis in play in tumor chemotherapy resistance in the field of emerging hot status, this study found that EphA2 in paclitaxel resistance in nasopharyngeal carcinoma cells based on the regulation of early research, aimed at further to explore the P-gp and autophagy related molecular mechanism of EphA2 regulation of paclitaxel resistance in nasopharyngeal carcinoma cells.
Methods: 1. Blot were detected by Western in nasopharyngeal carcinoma cells with lentivirus mediated EphA2 silencing and overexpression group and negative control group (transfected with shRNA or nonsense nonsense cDNA) and control group (transfected empty vector) expression of P-gp protein, and investigate whether EphA2 has the ability to control the expression of P-gp in nasopharyngeal carcinoma cells;
2. expression by siRNA interference down-regulation of P-gp in nasopharyngeal carcinoma cells, while giving the blank control group (original cells), negative control group (transfected antisense sequence) downregulation of P-gp and 0.1nM/L group (the concentration of paclitaxel for preliminary research the original cell concentration of IC30, combined with the literature, considering the expression of P-gp after the decline of paclitaxel sensitization, the choice of the concentration) stimulation, CCK-8 method was used to detect the absorbance after 48 hours, get the survival rate of each group, to illustrate the expression of P-gp protein changes effect of paclitaxel resistance in nasopharyngeal carcinoma cells;
3. by siRNA interference expression in nasopharyngeal carcinoma cells down-regulation of P-gp in EphA2, while giving the control group (EphA2 cells), negative control group (transfected with EphA2 overexpression and antisense sequence of cells) and P-gp expression group (EphA2 overexpression and P-gp expression down regulated cells of paclitaxel (5nM/L) the basis of the preliminary study in the concentration of the experimental group and the control group survival rate difference) stimulation, CCK-8 method was used to detect the absorbance after 48 hours, get the survival rate of each group, in order to explore the expression of EphA2 in cells, regulate the expression of P-gp on paclitaxel resistance in nasopharyngeal carcinoma cells alcohol;
In view of the 4. reported that PI3-K/Akt pathway is involved in regulation of P-gp protein expression and the previous study found that EphA2 through PI3-K/Akt signaling pathway of taxol resistance in this study, overexpression of EphA2 in nasopharyngeal carcinoma cells, the PI3-K/Akt inhibitor LY294002 were added to 0nM/L and 20nM/L, then given 5nM/L paclitaxel stimulation, 48 hours after the use of Western Blot to detect the PI3-K/Akt signal pathway protein Akt and p-Akt, and the expression level of P-gp, to investigate the expression of EphA2 in cells, regulating the expression of PI3-K/Akt pathway P-gp protein;
5. given the experimental group (EphA2 overexpression in nasopharyngeal carcinoma cells) and control group (transfection nonsense cDNA 5nM/L paclitaxel) stimulation, 48 hours after the use of Western Blot to detect marker expression difference of LC3B- protein in the experimental group and control group, to discuss the possible mechanism of EphA2 regulation of nasopharyngeal carcinoma paclitaxel resistance (autophagy).
Results: the expression of P-gp protein in the 1.EphA2 silencing group was lower than that in the two control group, and the expression of P-gp protein in the EphA2 overexpression group was up up compared with the two control group.
2. paclitaxel played a role in the blank control group. The survival rate of the negative control group and the P-gp expression downregulation group was 68.9 + 4%, 72.4 + 6.2%v.s.52.1 + 3.7%, and the P-gp expression down-regulation group had lower resistance to paclitaxel than the two control group (P0.05).
3., in EphA2 overexpressing nasopharyngeal carcinoma cells, paclitaxel acted on the blank control group. The survival rates of the negative control group and the P-gp expression downregulation group were 57.9 + 6.2%, 58.2 + 5.8%v.s.36.1 + 5.1%, and the P-gp expression downregulation group was lower than that of the two control group (P0.05).
4. in EphA2 over expression of nasopharyngeal carcinoma cells, the expression of Akt in the given pathway inhibitor group was less than that of the control group, but the expression of phosphorylated Akt decreased and the expression of P-gp decreased.
The expression of autophagic marker LC3B- II protein in 5. EphA2 overexpressed nasopharyngeal carcinoma cell lines was significantly higher than that in the control group.
Conclusion: 1.EphA2 has the ability to regulate the expression of P-gp in nasopharyngeal carcinoma cells.
2. down regulation of P-gp protein could reduce the drug resistance of taxol in nasopharyngeal carcinoma cells.
3. the expression of P-gp protein could reverse the drug resistance of taxol in nasopharyngeal carcinoma (NPC) mediated by EphA2.
4. in EphA2 overexpressed cells, PI3-K/Akt pathway participates in the regulation of the expression of P-gp protein.
5., in the paclitaxel treated nasopharyngeal carcinoma cells, the change of EphA2 expression can affect the expression level of autophagy related protein, suggesting that EphA2 may also regulate the paclitaxel resistance of nasopharyngeal carcinoma cells through the white channel.
In conclusion, EphA2 can guide the protein expression of P-gp mediated by, and regulation of paclitaxel resistant nasopharyngeal carcinoma cells, PI3-K/Akt pathway may be involved in this process; in addition, EphA2 may also through the autophagy pathway regulation of paclitaxel resistance in nasopharyngeal carcinoma cells.EphA2 is expected to become the target of taxol sensitization.

【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R739.63

【参考文献】