【作者】 张锐；

【导师】 钱立庭；

【作者基本信息】 山东大学 ， 肿瘤学（专业学位）， 2020， 博士

【摘要】 研究背景:食管癌的发病具有明显的地域差异,欧美等发达国家少见,东亚地区和非洲等地常见,我国是食管癌的高发地区,病理类型以食管鳞癌为主。随着科学技术的发展,肿瘤的治疗方式也出现了多元化,目前食管癌仍然以根治性手术、放射治疗以及化学治疗为主要治疗手段,晚期病人可以考虑靶向以及免疫等新兴治疗方式。而放射治疗在食管癌中的地位仍不可撼动,无论是对于术后患者还是不能手术的患者,放射治疗均有着不可替代的作用。近年来,虽然食管癌的放射治疗技术有了一定的进步,但食管癌放疗后局部复发及纵膈淋巴结转移仍是食管癌复发的常见复发模式,提示常规的放疗剂量可能无法完全杀灭部分食管癌细胞。然而,心脏、肺、脊髓等重要器官的存在,限制了食管癌患者的放疗剂量,且可能会引起放射性肺炎、放射性心包炎甚至放射性脊髓损伤等严重并发症。因此,增加食管癌的放疗敏感性,可能会减少食管癌患者放疗后的野内复发,减轻放射治疗引起的并发症,提高患者的生活质量,并延长患者的总生存。红细胞生成素肝细胞受体(erythropoietin-producing hepatocyte receptor,Eph)是酪氨酸激酶受体(Receptor Tyrosine Kinase,RTK)家族中最大的一个亚群,分为EphA和EphB两类,Eph与其配体ephrin结合后可以介导双向信号传递,调节细胞形态、粘附和运动等。EphA5是Eph家族的成员之一,以往关于EphA5基因的研究多集中在神经系统方面,近来发现EphA5在肿瘤发生发展的调控中也具有重要作用,并且还可能与药物敏感性以及放疗敏感性等相关。但是,EphA5在食管鳞状细胞癌(ESCC)中的表达、作用以及与放疗的关系尚不清楚。本课题,我们探讨了 EphA5在食管鳞癌中的表达情况及其与放疗敏感性的关系。研究目的:一 明确EphA5在食管鳞癌中的表达情况,分析EphA5的表达与食管鳞癌及其预后的关系。二 探讨EphA5在食管鳞癌放射敏感性中的作用及其可能机制。研究方法:第一部分EphA5与食管鳞癌临床病理参数及其预后的相关性分析1.病例收集:一收集2018年09月至2018年10月在江苏省泰兴市人民医院接受手术的4例食管鳞癌患者的肿瘤组织及癌旁正常食管组织,获取新鲜组织立即储存至-80℃冰箱。二纳入2015年01月日至2018年06月在江苏省泰兴市人民医院接受根治性手术的食管鳞癌患者的石蜡包埋标本共77例。收集病例资料包括:年龄、性别、组织学分级、T分期、N分期、淋巴结转移情况、脉管和神经侵犯情况、TNM分期等临床特征。2.Western blot方法检测EphA5在4例新鲜冰冻的食管鳞癌组织以及癌旁组织中的表达情况3.免疫组织化学(Immunohistochemistry,IHC)检测EphA5在77例食管鳞癌及20例相应癌旁组织中的表达情况,并进一步分析EphA5表达与患者性别、年龄、相关病理参数的关系。4.采用Kaplan-Meier法绘制生存曲线,分析EphA5表达与食管鳞癌患者的预后关系。第二部分EphA5影响X线照射后食管鳞癌细胞增殖、周期及侵袭的体外实验1.RT-PCR、Western blot 检测 EphA5 在食管鳞癌细胞株(KYSE30、KYSE70、KYSE140、KYSE150、KYSE180、KYSE410、KYSE450、KYSE510)以及食管正常上皮细胞株(HEEC)中的表达情况。2.选择EphA5高表达的KYSE150和KYSE450细胞株为研究对象,通过转染siRNA(siRNA-1组,siRNA-2组)敲低EphA5的表达。分别采用RT-PCR、Western blot方法对2种食管鳞癌细胞株KYSE150,KYSE450对应的siRNA-1组、siRNA-2组、NC组中EphA5 mRNA和蛋白的表达进行检测,siRNA-1组的EphA5下调显著,选择siRNA-1组做后续试验(后面si-EphA5组即转染siRNA-1组),确定转染后的食管鳞癌细胞中EphA5呈现低表达。3.KYSE150、KYSE450经siRNA转染后,采用6MV X射线进行单次照射,通过CCK8实验、克隆形成实验、划痕实验、Transwell侵袭实验以及流式细胞术,检测6MVX射线单次照射后的食管鳞癌细胞增殖、侵袭能力、凋亡以及周期的变化。第三部分EphA5影响食管鳞癌放射敏感性机制的初步探讨1.通过转染siRNA敲低食管癌细胞中EphA5的表达,给予6MV X线单纯照射,照射后不同时间段,应用免疫荧光技术检测p-ATM以及γH2AX的变化。2.转染siRNA后的食管鳞癌细胞,照射后0h、0.5h、24h提取细胞蛋白,利用 Western blot 检测相关蛋白的表达,如 p-ATM、p53、p-p53(S15)、CHK2、p-CHK2、p21、cdc2、p-cdc2、cyclinB1 等的变化。研究结果:第一部分食管鳞癌组织中EphA5的表达较癌旁组织升高,且与区域淋巴结转移及预后相关1.Western blot结果提示食管鳞癌组织的EphA5表达均较癌旁组织高,两组比较有统计学意义(P<0.05)。2.免疫组化检测共纳入77例食管鳞癌患者,中位年龄为65岁(范围为43-80岁)。58例(75.32%)为男性,19例(24.68%)为女性;按组织学分级,高分化癌和中分化癌共64例(83.12%),低分化癌共13例(16.88%):根据肿瘤侵犯深度,T1、T2患者37例(48.05%),T3、T4患者40例(51.95%);淋巴结转移患者37例(48.05%),淋巴结阴性患者40例(51.95%);脉管/神经阳性者19例(24.68%),阴性者58例(75.32%);参照第8版AJCC TNM分期,所有患者中,Ⅰ期、Ⅱ期 31 例(40.26%),Ⅲ、ⅣA 期 46 例(59.74%)。3.与正常癌旁组织相比,免疫组化提示食管鳞癌组织中EphA5蛋白的表达升高,在食管鳞癌细胞的细胞质和细胞核中均有表达,且在不同食管鳞癌患者中的表达具有差异。EphA5在食管鳞癌组织中的表达明显高于癌旁组织,77例食管癌患者中78%有EphA5表达,而其中相应的20例癌旁组织仅在基底细胞有少许表达,分化好的鳞状上皮不表达,提示食管鳞癌组织和癌旁组织的EphA5表达有明显差异。4.EphA5高表达者局部淋巴结转移较常见,提示EphA5表达水平与食管鳞癌的区域淋巴结转移有关,而与患者年龄、性别、TNM分期、T分期、神经侵犯、脉管癌栓以及肿瘤分化程度等无显著相关性。5.EphA5低表达和高表达患者2年生存率为78.5%和64.3%,2年复发率(包括局部和远处复发)为37.5%和57.14%,但是两组的2年生存率比较没有统计学意义(P=0.16),而2年复发率显示出统计学差异(P<0.05)。提示EphA5高表达患者预后欠佳。第二部分EphA5下调后食管鳞癌细胞的放射敏感性增加1.RT-PCR结果显示,siRNA转染KYSE150和KYSE450细胞后,与NC组相比,siRNA-1和siRNA-2组中EphA5的mRNA的表达均有下调。Western blot结果显示,KYSE150和KYSE450细胞转染siRNA-1和siRNA-2后,与NC组相比,EphA5蛋白条带均减弱。而无论是基因还是蛋白水平,转染siRNA-1的细胞均下调的更加明显。因此,我们选择了 siRNA-1序列(用si-EphA5表示)做后续的实验。2.CCK8结果显示,与NC组细胞相比,转染si-EphA5的KYSE150、KYSE450细胞的细胞生长活力48h内变化不大,72h后生长活力稍有增加。但是经6MVX线单纯照射后,转染si-EphA5的KYSE150,KYSE450细胞的细胞生长活力均被抑制,有统计学差异(P<0.05)。3.克隆形成实验显示,转染si-EphA5的KYSE150,KYSE450细胞的克隆形成率均明显低于NC组,不同剂量(2Gy、4Gy、6Gy、8Gy)的存活分数(Surviving fraction,SF)均明显低于NC组。KYSE150细胞EphA5低表达组的较NC组放射增敏比为1.56,KYSE450细胞EphA5低表达组的较NC组放射增敏比为1.21,表明EphA5下调后的食管鳞癌细胞具有较好的放射治疗敏感性。4.下调EphA5表达后的食管鳞癌细胞,未经6MVX线照射时,si-EphA5组和NC组的细胞凋亡率和细胞周期均无明显差异。经6MV X线照射后,si-EphA5组和NC组的细胞凋亡率未见明显差异,但是细胞周期差异明显,照射24h后,与NC组相比,si-EphA5组细胞S期增多,G2期减少,而G1/S 比值下降,提示下调EphA5影响放疗后细胞的周期可能与G1/S期检查点不能有效激活有关。5.划痕和侵袭实验显示,未经6MV X线照射时,转染si-EphA5后细胞的迁移及侵袭能力未被明显抑制,但是经6MVX线照射后,转染si-EphA5后的食管癌KYSE150,KYSE450细胞的迁移和侵袭能力均明显受抑,有统计学差异(P<0.05)。第三部分EphA5调节食管鳞癌细胞的放射敏感性与ATM激活有关1.免疫荧光实验显示,给予6MVX线照射后0.5小时,NC组细胞出现大量的γH2AX焦点,而si-EphA5组细胞的焦点却比NC组少。同时我们也观察到,NC组细胞在放疗后8小时、24小时,γH2AX焦点均逐渐减少,提示NC组细胞的DNA双链断裂得到了修复。但是,si-EphA5组细胞则表现出了更缓慢的DNA修复速率,在照射后8小时可见大量γH2AX焦点阳性细胞,24小时磷酸化γH2AX焦点阳性细胞数仍较多。以上结果表明,下调EphA5不会导致照射后细胞的DNA双链断裂增加,却显著的延缓了 DNA损伤修复的进程。2.细胞周期结果显示,下调EphA5可以导致放射治疗后的食管癌细胞阻滞在S期,而G2期减少,G1/S比值下降,因此我们检测了相关的细胞周期蛋白。Westen blot结果显示,与NC组相比,给予6MV X线照射后,下调EphA5后p53激活受抑,导致其下游分子p21表达减少;接受X线照射后0.5h,下调EphA5细胞的CHK2磷酸化水平较NC组细胞明显减少,提示CHK2不能被有效激活。而与G2/M检查点相关的cyclinB1、p-cdc2、cdc2在EphA5下调后变化不大。以上结果表明,下调EphA5抑制了 G1/S期检查点的激活,导致的细胞周期的变化。3.DNA损伤引起ATM/ATR的激活,而ATM在DNA双链断裂中起主导作用。Westen blot和免疫荧光结果均显示,给予6MV X线照射后,NC组的AMT在0.5h时明显被激活,24h则p-AMT显著下降;而下调EphA5后,AMT在0.5h时激活不明显,较NC组减少,在24h仍有大量ATM处于激活状态,较NC组增多。上述结果表明,下调EphA5影响了 X线照射后食管鳞癌细胞的ATM激活状态。结论:1.EphA5在食管鳞癌中相对高表达,与区域淋巴结转移等有关,并且预示着较高的局部和远处复发率。2.下调EphA5可降低X线照射后食管鳞癌细胞的增殖及侵袭能力,使得G1/S检查点不能有效激活,细胞不能被有效的阻滞在G1期,更多的细胞进入了 S期。3.下调EphA5影响了 X线照射后食管鳞癌细胞的ATM激活状态,使得DNA损伤不能有效的修复,导致了食管鳞癌细胞的放射敏感性增加。4.EphA5有望成为食管鳞癌患者的预后以及放射敏感性的预测因子。更多还原

【Abstract】 Background:There are obvious regional differences in the incidence of esophageal cancer.Esophageal cancer is rare in developed countries such as Europe and the United States,while is common in East Asia and Africa.Esophageal cancer is common in China,and the main pathological type is esophageal squamous cell carcinoma.With the development of science and technology,the treatment mode of tumors has been diversified.At present,esophageal squamous cell carcinoma is still treated mainly by radical surgery,radiotherapy and chemotherapy.Advanced patients can be treated with some emerging treatments such as targeted therapy and immunity therapy.However,radiation therapy plays an irreplaceable role in both postoperative and inoperable patients.In recent years,although the radiotherapy technology of esophageal cancer has made some progress,the in-field recurrence of esophageal cancer after radiotherapy is still a common recurrence mode of esophageal cancer recurrence.It suggests that the conventional radiotherapy dose for esophageal cancer may not completely kill all esophageal cancer cells.However,the tolerated dose of the heart,lung,spinal cord and other important organs in the thorax limits the dose of radiotherapy for patients with esophageal cancer.Radiotherapy may cause serious complications such as radiation pneumonia,radiation pericarditis and even radiation spinal cord injury.Therefore,increasing the radiosensitivity of esophageal cancer may reduce the in-field recurrence of esophageal cancer patients after radiotherapy,alleviate the complications caused by radiotherapy,improve the quality of life of patients and prolong the survival of patients.Erythropoietin-producing hepatocyte receptor is the largest subgroup of the tyrosine kinase receptor family,divided into two categories:EphA and EphB.Eph receptor binding to its ligand can mediate bidirectional signaling and regulate cell morphology,adhesion and movement.EphA5 is a member of the Eph family.Most of the previous studies on EphA5 gene have focused on the nervous system.It has recently been found that EphA5 plays an important role in the regulation of tumorigenesis and development,and may also be related to drug sensitivity and radiotherapy sensitivity.However,the roles of EphA5 in esophageal cancer are unclear.In this study,we explored the expression of EphA5 in esophageal squamous cell carcinoma and its relationship with radiotherapy sensitivity.Purpose:1.To clarify the expression of EphA5 in esophageal squamous cell carcinoma,to analyze the relationship between EphA5 expression and clinicopathological parameters and prognosis in esophageal squamous cell carcinoma patients.2.To explore the role of EphA5 in the radiosensitivity of esophageal squamous cell carcinoma and its possible mechanisms.Methods:Part 1.Correlation between EphA5 and clinicopathological parameters and prognosis of esophageal cancer.1.Cases:We got tumor tissues and adjacent normal esophageal tissues of 4 patients with esophageal squamous cell carcinoma undergoing surgery in Taixing People’s Hospital of Jiangsu Province from September 2018 to October 2018.Fresh tissues were immediately stored in-80℃ refrigerator.A total of 77 patients with esophageal squamous cell carcinoma undergoing radical surgery were included from Taixing City People’s Hospital,Jiangsu Province from January 2015 to June 201 8.We collected the relevant information:age,sex,histological grade,T stage,N stage,lymph node metastasis,vascular and nerve invasion,pTNM stage and other clinical characteristics.2.Western blot was used to detect the expression of EphA5 in 4 cases of fresh frozen esophageal carcinoma and adjacent tissues.3.Immunohistochemistry was used to detect the expression of Epha5 in 77 esophageal cancer cases and 20 corresponding paracancerous tissues.Statistical Product and Service Solutions(SPSS)was used to analyze the relationship between Epha5 expression and related pathological parameters.Part 2.The effects of EphA5 on proliferation,cell cycle and invasion of esophageal squamous carcinoma after X-ray irradiation in vitro.1.RT-PCR and western blot was used to detect the expression of EphA5 in esophageal squamous carcinoma cell lines(KYSE30,KYSE70,KYSE140,KYSE150,KYSE180,KYSE410,KYSE450,KYSE510)and the normal esophageal epithelial cell line(HEEC).2.EphA5 highly expressed KYSE150,KYSE450 cell lines were selected to knock down EphA5 expression by transfected siRNAs(siRNA-1,siRNA-2).Western blot and RT-PCR was used to detect the expression of EphA5 protein and gene in esophageal squamous cell carcinoma cell lines KYSE150,KYSE450 transfected with siRNA-1,siRNA-2 and negative control sequence(NC),respectively.The EphA5 of siRNA-1 group was down-regulated significantly and siRNA-1 sequence was selected for the follow experiment(the latter si-EphA5 group is transfected with siRNA-1 sequence).3.KYSE150,KYSE450 cells after siRNA transfection,were treated with ionizing radiation with 6MV X-ray.The proliferation,invasion,apoptosis and cycle changes of esophageal squamous cell carcinoma cells were detected by CCK8 experiment,clone formation experiment,scratch test,transwell invasion experiment and flow cytometry after ionizng radiation.Part 3.Discussion on the potential mechanism of EphA5 affecting radiosensitivity in esophageal squamous cell carcinoma1.KYSE150 and KYSE450 cell lines with EphA5 expression knocked down were selected to detect the p-ATM and yH2AX changes by immunofluorescence in different time periods after exposure to ionizing radiation.2.The esophageal squamous cell carcinoma cells were transfected with siRNA,and the total cell protein was extracted after irradiation for 0 h,0.5h and 24h.The expression of related proteins such as p-ATM,p53,p-p53,chk2,p-chk2,p21,cdc2,p-cdc2 and cyclinB1 was detected by Western blot.Results:Part 1.Expression of EphA5 in esophageal squamous cell carcinoma is higher than that adjacent tissues and it is associated with regional lymph node metastasis and prognosis.1.The results of Western blot indicated that the expression of EphA5 in esophageal squamous cell carcinoma was higher than that adjacent tissues(P<0.05).2.77 patients with esophageal squamous cell carcinoma were collected for immunohistochemical assays.The median age was 65 years(range 43 to 80 years),75.32%of the patients(58 cases)were men and 24.68%of the patients(19 cases)were women.According to histological grade,there were 64 cases(83.12%)with highly differentiated and moderately differentiated cancers,and 13 cases(16.88%)with poorly differentiated and undifferentiated cancers;According to tumor invasion depth,there were 37 patients(48.05%)with T1/T2 stage and 40 patients(51.95%)with T3/T4 stage.37 patients(48.05%)had lymph node metastasis while 40 patients(51.95%)with lymph node negative.19 patients(24.68%)were vascular/neural positive while 58 patients(75.32%)negative.Accoding to the 8th version of AJCC TNM staging,there were 31 patients(40.26%)in stage Ⅰ,Ⅱ and 46 patients(59.74%)in stage Ⅲ、ⅣA.3.The expression of EphA5 protein in esophageal squamous cell carcinoma tissue was increased,mainly in the cytoplasm and nuclei of esophageal squamous cell carcinoma cells,and the expression was different in different patients.The expression of EphA5 in esophageal squamous cell carcinoma tissue was significantly higher than that in paracancerous tissues.78%of the patients had EphA5 expression,while the corresponding paracancerous tissue was only slightly expressed in basal cells,and the differentiated squamous epithelium was not expressed.4.Local lymph node metastasis was more common in patients with high EphA5 expression,suggesting that EphA5 expression level was related to regional lymph node metastasis of esophageal squamous cell carcinoma.But there was no significant correlation between EphA5 expression and age,sex,TNM stage,T stage,nerve invasion,vascular tumor thrombus and tumor differentiation degree.5.The 2-year survival rates of patients with low and high expression of EphA5 were 78.5%and 64.3%,and the 2-year recurrence rates(including local and distant recurrences)were 37.5%and 57.14%.The 2-year relapse rate showed statistical difference(P=0.08)while the 2-year survival rates of the two groups were not statistically significant(P=0.16).The results indicates that the prognosis of patients with high expression of EphA5 is poor.Part 2.EphA5 downregulation increased radiosensitivity of esophageal cancer cells.1.RT-PCR results showed that the expression of EphA5 mRNA in the siRNA-1 group and siRNA-2 group was decreased compared with the NC group after transfection with siRNA.Western blot results showed that after transfection,the EphA5 protein bands were weakened compared with the NC group.Regardless of gene or protein level,the cells transfected with siRNA-1 were more down-regulated.Therefore,we choose the siRNA-1 sequence(denoted by si-EphA5)for subsequent experiments.2.CCK8 results showed that compared with NC groups,the cell growth activity of the transfected si-EphA5 cells did not change much within 48 h,and the growth activity increased slightly after 72h.Nevertheless,after exposure to ionizing radiation(IR),the cell growth activity of KYSE150 and KYSE450 cells transfected with si-EphA5 was inhibited with statistical difference(P<0.05).3.Clone formation experiments showed that the clone formation rate of KYSE150 and KYSE450 cells transfected with si-EphA5 was significantly lower than that of NC group,and the surviving fraction of different doses was significantly lower than that of NC group.The sensitivity enhancement ratio(SER)of KYSE150 cells of EphA5 lower expression group was 1.56,and that of KYSE450 cells was 1.21,which indicated that the esophageal squamous cells with EphA5 knockdown had better radiotherapy sensitivity.4.Scratch and invasion experiments showed that the migration and invasion ability of cells after transfection of si-EphA5 was not significantly inhibited without X-rays irradiation.However,after ionizing radiation,the migration and invasion ability of esophageal cancer cells with EphA5 knockdown were significantly inhibited,with statistical differences(P<0.05).5.There was no significant difference in apoptosis rate and cell cycle between si-EphA5 group and NC group without irradiation.When irradiated by X-rays,there was no significant difference in apoptosis rate between si-EphA5 group and NC group,but the difference of cell cycle was obvious.After 24 h of irradiation,compared with the NC group,the cell S phase increased,the G2 phase decreased and the G1/S ratio decreased,suggesting that EphA5 down-regulation affects the cell cycle after radiotherapy may be related to the ineffective activation of the checkpoint in the G1/S phase.Part 3.EphA5 regulating radiosensitivity of esophageal cancer cells is associated with ATM activation1.The immunofluorescence assay showed that NC cells had a large number of yH2AX foci after X-ray irradiation for 0.5 hours,while the si-EphA5 group had less foci than the NC group.We also observed that the yH2AX foci of NC groups decreased after 8 hours and 24 hours after radiotherapy,suggesting that the DNA double strand breaks of cells were repaired.But it showed a slower rate of DNA repair in si-EphA5 group cells.A large number γH2AX foci positive cells after irradiation for 8 hours and numbers of phosphorylated yH2AX foci positive cells for 24 hours were observed.The results showed that the down-regulation of EphA5 did not lead to an increasement of DNA double strand breaks after irradiation,but significantly delayed the repair of DNA damage.2.The cell cycle results showed that downregulation of EphA5 could lead to esophageal cancer cell arrest after radiotherapy in the S phase,while the G2 phase decreased,so we detected the associated cyclins.Westen blot results showed that after 6 MV X-ray irradiation,the activation of p53 in the down-regulated EphA5 cells was inhibited,which resulted in the decrease p21 expression of downstream molecules.Similar results were found for both 0.5 h and 24 h after 6 MV X-ray irradiation.The phosphorylation of CHK2 after down-regulation of EphA5 decreased compared with NC group,suggesting that CHK2 could not be effectively activated.The cyclinB1 and p-CDC2 associated with the G2/M checkpoint did not change significantly compared with the NC group after EphA5 downregulation.The above results indicate that the EphA5 down-regulation inhibits the activation of G1/S checkpoint,resulting in the changes of cell cycle.3.DNA damage causes ATM/ATR activation,while ATM plays a dominant role in the DNA double-strand break.Westen blot and immunofluorescence results showed that the AMT of NC group was significantly activated after IR for 0.5 h and decreased significantly for 24 h.But when EphA5 was down-regulated,the activation of AMT was not obvious at 0.5 h,which was less than that of the NC group.While there were still a large amount of ATM in the activation state at 24h,which was more than that of the NC groups.The above results indicated that the down-regulation of EphA5 affected the ATM activation status of esophageal cancer cells after X-ray irradiation.Conclusion1.EphA5 is relatively highly expressed in esophageal squamous cell carcinoma.High EphA5 expression is associated with regional lymph node metastasis and indicates higher local and distant recurrence rates.2.The down-regulation of EphA5 reduced the proliferation and invasion ability of esophageal squamous cell carcinoma cells after IR,which leading to G1/S checkpoint activated ineffectively.The cells can not be effectively blocked in the G1 stage,and more cells enter the S stage.3.The down-regulation of EphA5 affected the ATM activation state of esophageal cancer cells after X-ray irradiation,which caused the result that the DNA injury could not be effectively repaired,resulting in an increase of radiosensitivity in esophageal squamous cell carcinoma.4.EphA5 is expected to be a predictor of prognosis and radiosensitivity in the patients with esophageal squamous cell carcinoma.更多还原