EphA2调控鼻咽癌紫杉醇化疗敏感性及其分子机制的实验研究

发布时间：2018-05-16 16:25

  本文选题：EphA2 + 鼻咽癌　； 参考：《中南大学》2012年博士论文

【摘要】：研究背景鼻咽癌(Nasopharyngeal carcinoma, NPC)是一种鼻咽上皮来源恶性肿瘤,流行于我国南方多省。目前NPC的治疗以放疗为主,化疗是最主要的辅助治疗方法。尽管放、化疗技术不断改进,但是NPC患者的疗效仍改善不明显。其中NPC细胞对化疗抵抗性的产生是影响NPC疗效的重要原因之一。因此寻找与NPC发生、发展及化疗敏感性密切相关的基因,通过对该类有效基因的检测和靶向调控,可以达到辅助诊断NPC、增加NPC的化疗敏感性、改善NPC的疗效、提高NPC患者的生存率等目的,具有潜在的临床应用价值。 促红细胞生成素产生肝细胞受体A2(erythropoietin-producing hepatocellular receptor EphA2)在多种肿瘤中表达上调,且其表达与肿瘤的病理分级、临床分期、转移、预后等密切相关,参与调控肿瘤细胞的生长、增殖、迁移、侵袭、转移等多项恶性生物学行为。目前,国内外有关EphA2的研究多集中在其对肿瘤细胞侵袭转移的影响,而EphA2对肿瘤细胞化疗敏感性的调控国内外报道甚少。紫杉醇为鼻咽癌常用化疗药物,靶向调控其化疗敏感性有助于提高NPC的疗效。本研究将分以下三个阶段探讨EphA2在NPC生长、增殖、侵袭过程中的作用,并重点研究EphA2对NPC紫杉醇化疗敏感性的调控作用及其分子机制。 第一章EphA2在鼻咽癌中的表达及其临床意义 目的：检测EphA2在NPC组织和细胞株中的表达水平,探讨EphA2的表达与NPC临床病理特征的相关性。 方法：利用Western Blot技术检测47例NPC新鲜组织标本和21例鼻咽非肿瘤新鲜组织标本以及NPC细胞株中EphA2蛋白的表达水平,统计分析EphA2的表达与NPC临床病理特征的相关性。 结果：EphA2蛋白在NPC组织中的表达较鼻咽非肿瘤组织中的表达明显升高,在NPC细胞株中也呈现高表达,且EphA2蛋白的表达水平与NPC的T分级(P=0.028)、临床分期(P=0.004)和颈部淋巴结转移(P=0.012)密切相关,组间差异具有统计学意义(P0.05)。 结论：EphA2蛋白在NPC组织和细胞中表达升高,EphA2蛋白的表达与NPC患者的T分级、临床分期以及颈部淋巴结转移密切相关,提示EphA2在NPC的发生、发展过程中可能具有重要作用。 第二章沉默和上调EphA2的表达对鼻咽癌细胞增殖、细胞周期及侵袭能力的影响 目的：观察调控EphA2的表达对NPC细胞的增殖、凋亡、细胞周期及侵袭能力等生物学行为的影响。 方法：利用慢病毒载体介导的EphA2shRNA(EphA2shRNA-LV)和EphA2基因过表达克隆(EphA2cDNA-LV)转染NPC5-8F细胞,利用嘌呤霉素筛选建立稳定沉默和过表达EphA2蛋白的细胞系,Western Blot验证EphA2蛋白沉默和上调效果。CCK-8法分别检测EphA2沉默和过表达前后细胞的生长抑制率,流式细胞术分别检测EphA2沉默和过表达前后细胞的凋亡率及细胞周期,Transwell侵袭实验检测EphA2沉默后细胞侵袭能力的改变。 结果：1.慢病毒介导的RNA干扰和过表达克隆技术成功沉默和上调NPC5-8F细胞中EphA2的表达,并建立了稳定沉默和过表达EphA2的细胞系。 2.沉默EphA2蛋白的表达减弱5-8F细胞的体外增殖能力,72h开始EphA2沉默组细胞与对照组细胞生长抑制率有统计学差异(P0.05)；上调EphA2蛋白的表达增强5-8F细胞的体外增殖能力,72h始EphA2上调组细胞与对照组细胞生长抑制率比较,有统计学差异(P0.05)。 3.EphA2沉默组与对照组比较,G0/G1期细胞比例(%)(沉默组：74.38±3.71VS对照组：53.82±2.19)明显增加,而S期细胞比例(沉默组：17.89±2.21VS对照组：30.41±1.55)和G2/M期细胞比例(沉默组：7.72±2.24VS对照组：15.80±3.72)则显著降低,组间差异均具有统计学意义(P0.05)。上调EphA2表达,G0/G1期细胞比例较对照组明显降低(上调组47.39±3.75VS53.89±2.22),差异具有统计学意义(P0.05)；而S期细胞比例(38.80±7.11VS38.63±4.15)无明显差异(P0.05),G2/M期细胞比例则较对照组增加(14.15±3.00VS7.81±2.36),差异具有统计学意义(P0.05)。沉默EphA2的表达,细胞的凋亡率(%)较对照组无明显差异(P0.05),而EphA2过表达细胞的凋亡率较对照组减少(4.94±1.45VS8.99±0.84),差异具有统计学意义(P0.05) 4.沉默EphA2的表达,5-8F细胞的体外侵袭能力较对照组明显减弱(穿过小室到下室面的细胞数：沉默组：59±4VS对照组：103±12),差异具有显著统计学意义(P0.01)。 结论：EphA2通过影响NPC细胞的周期进展调控其体外增殖能力,并能影响NPC细胞的体外侵袭能力,提示EphA2在NPC的发展和心恶性进展过程中发挥重要作用。 第三章EphA2对鼻咽癌细胞紫杉醇化疗敏感性的调控作用及其分子机制 目的：探讨EphA2对NPC细胞紫杉醇化疗敏感性的影响及其分子机制。 方法：基因过表达克隆EphA2cDNA和EphA2vector质粒瞬时转染5-8F细胞,72h后Western Blot技术检测EphA2的表达水平。梯度浓度紫杉醇分别作用于稳定沉默EphA2表达的5-8FEphA2-RNAi+细胞及5-8F细胞和瞬时转染上调EphA2表达的5-8FEphA2cDNA+细胞、5-8FEphA2vector细胞及5-8F细胞,48h后CCK-8法检测各孔的吸光度(OD)值,计算各组细胞生存率和紫杉醇IC50浓度。IC30浓度紫杉醇分别作用于5-8FEphA2cDNA+细胞、5-8FEphA2vector组及5-8F细胞,48h后流式细胞术检测各组细胞凋亡率和细胞周期,Western Blot技术检测各组细胞中EphA2、细胞周期调控蛋白(CDK2、Cyclin E、p21、 p27、Rb和p-Rb)和PI3-K/Akt信号通路蛋白(Akt、p-Akt、GSK-3β、p-GSK-3p)的表达水平。以20nM、10nM和0nM浓度的P13-K/Akt抑制剂LY294002分别处理5-8FEphA2.DNA+细胞后,再予以梯度浓度紫杉醇处理,48h后CCK-8法检测各孔吸光度(OD)值,计算各浓度抑制剂处理组细胞生存率,绘制细胞生存曲线,得出紫杉醇的IC30后,再以IC30的紫杉醇作用于以20nM、10nM和0nM浓度的P13-K/Akt抑制剂LY294002处理过的5-8FEphA2.DNA+细胞,Western Blot检测各组EphA2.P13-K/Akt信号通路蛋白和细胞周期调控蛋白的表达水平,流式细胞术检测各组细胞周期和凋亡率的变化。 结果：1.基因过表达克隆EphA2cDNA质粒瞬时转染5-8F细胞,成功上调EphA2的表达,并建立了EphA2过表达的5-8F瞬转细胞系。 2.紫杉醇作用于5-8FEphA2RNAi+细胞和5-8F细胞的IC50值分别为1.6nM和5.6nM,5-8FEphA1RNAi+细胞对紫杉醇作用的敏感性较5-8F细胞明显增加(P0.05)；紫杉醇作用于5-8FEphA2cDNA+、5-8FEphA2vector及5-8F细胞,各组紫杉醇的IC50值分别为5nM、0.7nM、0.6nM,5-8FEphA2cDNA+细胞对紫杉醇的敏感性较5-8FEphA2vector及5-8F细胞明显降低(P0.05)。 3.IC30紫杉醇作用于5.8FEphA2cDNA+细胞.5-8FEphA2vector细胞及5-8F细胞,流式细胞检测各组凋亡率(%)分别为：9.84±2.08、8.66±1.59、7.74±1.34,两两比较,差异均无明显统计学意义(PO.05).5-8FEphA2cDNA+组细胞较5-8Fvector组和5-8F组G0/G1期细胞比例明显减少(分别为45.25±3.89VS65.85±2.28、64.52±3.31),差异具有统计学意义(P0.05),而S期细胞比例(分别为31.56±1.59VS25.76±1.89、24.55±3.64)和G2/M期细胞比例(分别为23.10±4.55VS8.39±0.8K10.94±3.27)则明显增多,差异具有统计学意义(P0.05);5-8Fvector细胞与5-8F细胞比较,各周期细胞比例无明显统计学差异(P0.05)。 4.EphA2的过表达,可以抑制紫杉醇作用后的5-8F细胞中周期进展阻滞蛋白p21和p27的表达；引起细胞周期抑制蛋白Rb的表达活化、p-Rb表达水平增高；同时可以引起5-8F细胞中p-Akt表达上调,而Akt的表达总量不变,但是对细胞周期启动蛋白CDK2和CyclinE蛋白的表达无明显影响。 5.PI3-K/Akt抑制剂LY294002可以抑制Akt和GSK-3β的磷酸化导致p-Akt和p-GSK-3β的表达下调,但是Akt和GSK-3β的表达总量不变；同时细胞周期进展阻滞蛋白p21和p27的表达升高,而促细胞周期进展蛋白p-Rb的表达水平下调,细胞周期启动蛋白CDK2和Cyclin E的表达无明显改变。20nM、10nM和0nM浓度的PI3-K/Akt抑制剂LY294002处理IC30浓度紫杉醇干预的5-8FEphA2cDNA+细胞,20nM和10nM抑制剂处理组细胞凋亡率分别为：9.55±0.84和9.92±2.13,较不加抑制剂(0nM)组细胞凋亡率(11.67±2.30)低,但差异无统计学意义(P0.05)；20nM和10nM抑制剂处理组G0/G1期细胞比例分别为：65.69±2.78、58.61±2.13,较不加抑制剂(0nM)组G0/G1期细胞比例(46.62±1.77)明显增加,差异有统计学意义(P0.05),而S期细胞(20nM组：24.37±3.70,10nM组：27.08±2.19,0nM组：32.14±1.98)和G2/M期细胞(20nM组：9.94±3.97,10nM组：14.31±1.61,0nM组：21.24±1.47)则明显增加,差异有统计学意义(P0.05)。PI3-K/Akt抑制剂LY294002使5-8FEphA2cDNA+细胞对紫杉醇作用的敏感性增加。 结论：EphA2通过PI3-K/Akt/GSK-3β介导的信号通路影响细胞周期调控蛋白的表达,进而调控NPC细胞对紫杉醇化疗的敏感性。EphA2可能作为有效的分子靶点应用于调控NPC对紫杉醇化疗的敏感性,对提高NPC的疗效具有重要意义。
[Abstract]:Background of the Invention Nasopharyngeal carcinoma ( NPC ) is a kind of malignant tumor of nasopharyngeal epithelial origin , which is popular in many provinces in southern China . The treatment of NPC is the most important auxiliary treatment method . Although the radiotherapy and chemotherapy technique has been improved continuously , it is important to find a gene which is closely related to the occurrence , development and chemosensitivity of NPC .

The expression of EphA2 is closely related to the pathological grade , clinical stage , metastasis , prognosis and so on . At present , the study of EphA2 has little effect on tumor cell invasion and metastasis , and EphA2 plays a role in regulating the chemosensitivity of tumor cells .

The Expression of EphA2 in Nasopharyngeal Carcinoma and Its Clinical Significance

Objective : To investigate the expression level of EphA2 in NPC tissues and cell lines , and to investigate the correlation between EphA2 expression and clinical pathological characteristics of NPC .

Methods : The expression level of EphA2 protein in 47 NPC fresh tissue samples and 21 cases of nasopharyngeal non - tumor fresh tissue samples and NPC cell lines were detected by Western Blot . The correlation between EphA2 expression and clinical pathological characteristics of NPC were analyzed .

Results : The expression of EphA2 protein in NPC tissue was significantly higher than that in nasopharyngeal non - tumor tissues , and the expression level of EphA2 protein was closely related to NPC ' s T grade ( P = 0.028 ) , clinical stage ( P = 0.004 ) and cervical lymph node metastasis ( P = 0.012 ) .

Conclusion : EphA2 protein expression increases in NPC tissues and cells . EphA2 protein expression is closely related to T grade , clinical stage and cervical lymph node metastasis in NPC patients , suggesting that EphA2 may play an important role in NPC ' s development and development .

Chapter 2 silencing and up - regulation of EphA2 expression on the proliferation , cell cycle and invasion ability of nasopharyngeal carcinoma cells

Objective : To investigate the effects of EphA2 expression on the proliferation , apoptosis , cell cycle and invasion ability of NPC cells .

Methods : Using EphA2 shRNA ( EphA2shRNA - LV ) and EphA2 ( EphA2 cDNA - LV ) mediated by lentivirus vector , NPC5 - 8F cells were transfected into NPC5 - 8F cells . The inhibitory rates of EphA2 silencing and overexpression of EphA2 protein were determined by Western Blot .

Results : 1 . lentivirus - mediated RNA interference and overexpression cloning techniques successfully silenced and up - regulate EphA2 expression in NPC5 - 8F cells , and established a cell line that stabilizes silent and overexpressed EphA2 .

2 . The expression of the silent EphA2 protein attenuated the in vitro proliferation ability of 5 - 8F cells , and the growth inhibition rate of EphA2 silent group cells and control group was significantly different at 72 h ( P0.05 ) .
The increase of EphA2 protein expression enhanced the in vitro proliferation ability of 5 - 8F cells , and EphA2 up - regulated the growth inhibitory rate of EphA2 at 72h . There was statistical difference ( P0.05 ) .

3 . The percentage of cells in G0 / G1 phase ( 74.38 卤 3.71VS control group : 53.82 卤 2.19 ) increased significantly compared with control group , while the percentage of S phase cells ( silence group : 17.89 卤 2.21VS control group : 30.41 卤 1.55 ) and G2 / M phase cell ratio ( 15.80 卤 3.72 ) decreased significantly ( P0.05 ) .
There was no significant difference in S phase cell proportion ( 38.80 卤 7.11VS38.63 卤 4.15 ) ( P0.05 ) . The percentage of apoptotic cells in G2 / M phase increased ( 14.15 卤 3.00VS7.81 卤 2.36 ) , and the difference was statistically significant ( P0.05 ) . The apoptosis rate of EphA2 overexpression cells was lower than that in control group ( 4.94 卤 1.45V8.99 卤 0.84 ) , and the difference was statistically significant ( P0.05 ) .

4 . The expression of EphA2 and the in vitro invasion ability of 5 - 8F cells were significantly decreased compared with those in the control group ( the number of cells passing through the chamber to the lower chamber : the silent group : 59 卤 4VS control group : 103 卤 12 ) , the difference was statistically significant ( P0.01 ) .

Conclusion : EphA2 regulates its in vitro proliferation ability by influencing the cycle progression of NPC cells , and can affect the in vitro invasion ability of NPC cells , suggesting that EphA2 plays an important role in the development of NPC and the progression of cardiac malignant progression .

The Effect of EphA2 on the Sensitivity of Paclitaxel Chemotherapy in Nasopharyngeal Carcinoma and Its Molecular Mechanism

Objective : To investigate the effect of EphA2 on the chemosensitivity of paclitaxel in NPC cells and its molecular mechanism .

Methods : The expression levels of EphA2 , CDK2 , Cyclin E , p21 , p27 , Rb and p - Rb , and PI3 - K / A were detected by Western Blot . The expression levels of EphA2 , CDK2 , Cyclin E , p21 , p27 , Rb and p - Rb in each group were determined by Western Blot . The expression levels of EphA2 , cyclin E , p21 , p27 , Rb and p - Rb in each group were determined by Western Blot .

Results : 1 . The gene overexpression of EphA2 cDNA plasmid transiently transfected 5 - 8F cells , successfully raised EphA2 expression , and established a 5 - 8F transient cell line of EphA2 overexpression .

2 . IC50 values of paclitaxel acting on 5 - 8FEphA2RNAi + cells and 5 - 8F cells were 1.6 nM and 5.6 nM , respectively , and the sensitivity of 5 - 8FEphA1RNAi + cells to paclitaxel increased significantly ( P0.05 ) .
The IC50 values of 5 - 8FEphA2vector and 5 - 8F cells were 5 nM , 0.7 nM , 0.6 nM , 5 - 8FEphA2cDNA + cells were significantly lower than those of 5 - 8FEphA2vector and 5 - 8F cells ( P0.05 ) .

3 . The percentage of apoptosis rate ( % ) was 9.84 卤 2.08 , 8.66 卤 1 . 59 , 7.74 卤 1 . 34 in 5 - 8FEphA2vector cells and 5 - 8F cells . The percentage of cells in S phase ( 31.56 卤 1.59VS25 . 76 卤 1 . 89 , 24.55 卤 3.64 ) and G _ 2 / M phase were significantly increased ( P < 0 . 05 ) .

4 . The overexpression of EphA2 can inhibit the expression of cyclin p21 and p27 in 5 - 8F cells after paclitaxel .
the expression of the cyclin Rb is activated , and the expression level of the p - Rb is increased ;
At the same time , the expression of p - in 5 - 8F cells was up - regulated , while the total amount of the expression was unchanged , but there was no significant effect on the expression of CDK2 and Cyclin E protein in the cell cycle .

5 . PI3 - K - 3.beta . phosphorylation led to downregulation of p - and p - gsk - 3.beta . , but the total amount of p3 - 3 beta was unchanged .
At the same time , the expression level of cyclin E was down regulated , and the expression level of cyclin E was down regulated . The expression of cyclin E was 9.55 卤 0.84 and 9.92 卤 2.13 respectively . The apoptosis rate was 11.67 卤 2.30 , but there was no statistical significance ( P0.05 ) .
The percentage of G0 / G1 phase cells was 65.69 卤 2.78 , 58.61 卤 2.13 , and the percentage of G0 / G1 phase cells ( 46.62 卤 1.77 ) increased significantly in the 20 nM and 10 nM groups ( P 0.05 ) , while the S phase cells ( 20 nM group : 24.37 卤 3.70 , 10 nM group : 27.08 卤 2.19 , 0nM group : 14.31 卤 1.61 , 0nM group : 21.24 卤 1.47 ) increased significantly ( P0.05 ) .

Conclusion : EphA2 can regulate the expression of cell cycle regulatory protein by PI3 - K - / - / - 3 - 尾 - mediated signal pathway , and then regulate the sensitivity of NPC cells to paclitaxel chemotherapy . EphA2 may be used as an effective molecular target to regulate NPC ' s sensitivity to paclitaxel chemotherapy , and it is of great significance to improve NPC ' s efficacy .

【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R739.63

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