【作者】 吴彬；

【导师】 杨兴升；

【作者基本信息】 山东大学 ， 妇产科学（专业学位）， 2019， 博士

【摘要】 卵巢癌(Ovarian cancer,OC)是常见的妇科恶性肿瘤之一,尽管其发病率在妇科生殖系统肿瘤中居第三位,但其死亡率却高居首位。因卵巢癌的发病机制复杂,至今仍缺乏有效的诊断分子标记物及治疗靶点。因此,寻找针对卵巢癌的独特分子标志物仍是卵巢癌基础研究的一项重大课题,一旦有所突破,将能够有效提高患者生存率,为卵巢癌患者带来希望。人滋养层细胞表面抗原-2(TROP2)是一种在多种肿瘤中表达的跨膜糖蛋白,由TACSTD2基因编码,能通过传递细胞内的钙信号、调控ERK/MAPK的信号通路,来参与细胞周期相关蛋白的激活,从而影响肿瘤细胞的增殖、侵袭及凋亡等恶性生物学行为。TROP2被首次发现于胎盘的滋养细胞,它在具有干细胞性质的细胞中表达。研究表明TROP2在子宫内膜样腺癌、宫颈癌、乳腺癌、喉癌和结直肠癌等组织中都有过度表达,该蛋白过表达与多种恶性肿瘤患者的生存率降低以及肿瘤侵袭性和转移性增加有关,且TROP2在转移组织中的过度表达使其成为治疗晚期恶性肿瘤极具吸引力和潜在的治疗靶点。浆液性卵巢癌(Serous ovarian carcinoma,SOC)是卵巢癌中最常见的组织学类型。本研究我们检测出TROP2蛋白在SOC组织中的高表达,评价其与临床病理特征和预后之间的关系,并观察到用siRNA对卵巢癌细胞进行干扰后TROP2的表达明显下调。我们的结果表明TROP2蛋白过表达与SOC侵袭性进展和不良预后密切相关,是评价SOC不良预后的独立因素,能促进卵巢癌细胞增殖、侵袭和转移。TROP2是一个SOC潜在的预后标志物和靶向治疗的新靶点。第一部分TROP2在浆液性卵巢癌中的表达及与Ki-67的相关性研究目的:检测并分析TROP2在卵巢病变组织及正常卵巢、输卵管伞组织中的表达,探讨TROP2的表达在SOC细胞增殖中的作用及与SOC患者临床病理特征的相关性。方法:采用免疫组织化学方法检测126例SOC患者,36例交界性卵巢肿瘤,26例卵巢浆液性囊腺瘤、20例正常卵巢和20例正常输卵管伞石蜡包埋组织标本中TROP2及Ki-67蛋白的表达;比较不同临床病理特征的TROP2表达水平,并分析TROP2和Ki-67表达的相关性。结果:1.TROP2蛋白的表达情况卵巢低级别浆液性腺癌(LGSOC)实验组,采用正常卵巢组织作为正常对照组,正常卵巢组织中TROP2蛋白表达阴性12例(60%),弱阳性7例(35%),中度阳性1例(5%),无强阳性表达。而LGSOC组织中TROP2蛋白阴性6例(10%),弱阳性13例(21.7%),中度阳性20例(5%),强阳性21例(35%),TROP2蛋白表达较正常卵巢、卵巢浆液性囊腺瘤及卵巢交界性肿瘤组织明显增高,具有统计学差异(P<0.05)。卵巢高级别浆液性腺癌(HGSOC)实验组,采用正常输卵管伞组织作为正常对照组,正常输卵管伞组织中TROP2蛋白表达阴性10例(50%),呈弱阳性表达8例(40%),中等阳性2例(10%),无强阳性表达。HGSOC组织中TROP2蛋白表达阴性8例(12.1%),弱阳性16例(24.2%),中等阳性22例(33.3%),强阳性表达20例(30.3%),TROP2蛋白表达较对照组正常输卵管伞组织明显增高(P<0.05)。2.TROP2蛋白表达与SOC的FIGO临床分期、肿瘤病理分级、术前血清CA125水平、腹水和淋巴结转移关系密切(P<0.05),而其表达与卵巢癌患者年龄及铂耐药无相关性(均为P>0.05)。3.Ki-67在卵巢低级别及高级别浆液性腺癌中的表达明显高于对照组及良性病变组织(P<0.05)。Spearman等级相关分析显示,TROP2和Ki-67在SOC组织中的表达具有相关性(P<0.05)。结论:1.TROP2在SOC组织中表达升高,与SOC的不良临床病理特征密切相关,说明TROP2参与SOC的发生发展过程。2.TROP2与Ki-67蛋白表达呈正相关,提示TROP2可能参与SOC细胞的增殖及侵袭过程。第二部分126例浆液性卵巢癌患者的临床资料统计及生存分析目的:进一步探究此项研究第一部分中影响SOC患者预后的相关因素。分析TROP2的表达、FIGO临床分期、肿瘤病理分级、术前血清CA125水平、腹水、淋巴结转移及年龄等不同因素对SOC患者生存时间的影响。方法:采集入组与第一部分中的SOC癌相同的患者病例,共计126例SOC患者。患者在山东大学附属齐鲁医院行手术且及时完成治疗,并保存了完整的病例记录和随访资料,术后随访时间60个月,随访信息包括总生存时间(OS)和无进展生存时间(PFS)。采用Kaplan-Meier法进行生存时间的比较,并绘制生存曲线;采用单因素、多因素Cox比例风险回归分析,筛选影响预后的主要因素。结果:1.单因素分析显示:TROP2蛋白过表达与无进展生存期(PFS)和总生存期(OS)有相关性(均为P<0.05)。在FIGO临床分期、肿瘤病理分级、术前血清CA125水平、腹水和淋巴结转移方面,患者中位生存期具有统计学差异(P<0.05),而年龄<50岁和≥50岁两组患者中位生存期无统计学差异。2.多因素COX回归模型分析结果显示:TROP2的表达、FIGO临床分期、术前血清CA125水平、腹水和淋巴结转移情况是影响SOC患者五年生存率的独立因素。结论:1.TROP2的过表达导致SOC患者的不良预后,是评价SOC预后的独立因素。2.FIGO临床分期、术前血清CA125水平、腹水和淋巴结转移也是影响卵巢癌预后的危险因素。第三部分TROP2的表达对卵巢癌生物学行为的影响目的:观察TROP2表达水平的变化对卵巢癌细胞增殖、凋亡、侵袭及迁移能力等生物学行为的影响,探索TROP2在卵巢癌肿瘤的形成发展中的作用及可能的机制。方法:我们合成了两个独立的siRNA序列siRNA-550和siRNA-1100序列来转染卵巢癌细胞,干扰沉默TROP2的内源性表达。用Western Blot检测转染后TROP2蛋白的表达量;用CCK-8法测定TROP2表达水平的改变对卵巢癌细胞增殖能力的影响;分别用划痕实验和侵袭实验来测定细胞的迁移和侵袭能力;使用Western Blot法测定凋亡相关蛋白Bax和Bcl-2的表达。结果:1.免疫荧光染色分析显示:在A2780、HO8910和SK-OV-3细胞表面均有一层红色荧光染色。Western Blot检测结果表明TROP2蛋白在3种细胞中均有表达,其中A2780细胞系中TROP2的表达比其它两个细胞系更高。2.用转染序列siRNA-550和siRNA-1100对A2780进行干扰后,TROP2蛋白表达明显降低。3.用CCK-8法研究发现:TROP2基因沉默的A2780细胞增殖能力显著降低(P<0.05)。4.通过细胞侵袭实验发现:TROP2基因沉默后的卵巢癌细胞穿过Transwell小室的细胞数量明显降低(P<0.05),说明细胞侵袭能力降低。5.细胞划痕试验显示在进行细胞划痕24和48小时后,TROP2基因沉默后的卵巢癌细胞具有显著较慢的划痕愈合速度(P<0.05),说明细胞迁移能力显著下降。6.Western Blot法测定凋亡相关蛋白显示:TROP2基因沉默后的卵巢癌细胞Bcl-2蛋白的表达显著下降,而Bax蛋白表达显著增强。结论:通过沉默卵巢癌A 2780细胞的TROP2基因,发现TROP2基因参与卵巢癌细胞的增殖、侵袭和转移。TROP2基因通过破坏Bax/Bcl-2家族蛋白之间的平衡调节来诱导细胞凋亡。更多还原

【Abstract】 Ovarian cancer(OC)is one of the common gynecological malignancies,although its incidence ranks third among gynecological reproductive tumors,it has the highest death rate.Due to the complexity of the pathogenesis of OC,effective diagnostic molecular markers and therapeutic targets are still lacking.Therefore,finding a unique molecular marker for ovarian cancer is still a major concern in the basic research of OC for the purpose to effectively improve the survival rate of patients and bring new hope for OC patients.Human trophoblast cell surface antigen-2(TROP2)is a transmembrane glycoprotein expressed in a variety of tumors,it’s encoded by the TACSTD2 gene and can participate in the activation of cell cycle related proteins and influence the proliferation,invasion,and apoptosis of tumor cells and malignant biological behavior by transmitting intracellular calcium signals and regulating ERK/MAPK signaling pathways.TROP2 was first discovered in the trophoblasts of the placenta and 1s expressed in stem-like cells.Studies have shown that TROP2 is overexpressed in diseases such as endometrioid adenocarcinoma,cervical cancer,breast cancer,laryngeal cancer and colorectal cancer.Overexpression of this protein has been associated with decreased survival in many patients with malignant tumors and increased tumor invasiveness and metastasis,and the overexpression of TROP2 in metastatic tissues make it an attractive and potential therapeutic target for the treatment of advanced malignancies.Serous ovarian cancer.(SOC)is the most common form of OC.In this study,we detected the high expression of TROP2 in SOC,evaluated its relationship with clinicopathological features and prognosis,and observed that the expression of TROP2 was significantly down-regulated after the interference of siRNA on OC cells.Our results indicated that TROP2 overexpression is closely related to the progression of SOC invasion and poor prognosis,and is an independent factor for evaluating the poor prognosis of SOC,besides,it could promote the proliferation,invasion and metastasis of OC cells.TROP2 might become a potential prognostic marker for SOC and a new target for targeted therapy.PART I Expression of TROP2 in serous ovarian carcinoma and its correlation with Ki-67Objective:To detect and analyze the expression of TROP2 in ovarian lesions and normal ovarian and fallopian tube tissues,and to explore the role of TROP2 expression in SOC cell proliferation and its correlation with clinicopathological features of patients.Methods:Immunohistochemistry was used to determine the expression of TROP2 and Ki-67 proteins in 126 patients with SOC,36 patients with borderline ovarian tumors,26 patients with ovarian serous cystadenoma,20 patients with normal ovaries,and 20 patients with normal fallopian tube parasol parafifin-embedded tissues.TROP2 expression levels were compared among different clinicopathological characteristics,and the correlation between TROP2 and Ki-67 expression was analyzed.Results:1.TROP2 was mainly expressed in the cell membrane of OC tissue.Normal ovarian tissue was used as the control,and the experimental group is ovarian low-grade serous adenocarcinoma.There were 12 negative cases(60%),7 weakly positive cases(35%),1 moderately positive case(5%),and no strong positive cases.And among the ovarian low-grade SOC group,6 cases were negative(10%),1 3 cases were weakly positive(2 1.7%),20 cases were moderately positive(5%),and 21 cases were strongly positive(35%).The expression of TROP2 protein was significantly higher than that of normal ovarian,ovarian serous cystadenoma and ovarian borderline tumor tissues(P<0.001).Normal ovarian tissue was used as the control,and the experimental group is ovarian high-grade serous adenocarcinoma.There were 10 negative cases(50%),8 weakly positive cases(40%),2 moderately positive case(10%),and no strong positive cases.And among the ovarian low-grade SOC group,8 cases were negative(12.1%),16 cases were weakly positive(24.2%).22 cases were moderately positive(33.3%),and 20 cases were strongly positive(30.3%).The expression of TROP2 protein was significantly higher than that of normal ovarian,ovarian serous cystadenoma and ovarian borderline tumor tissues(P<0.001).The expression of TROP2 protein was significantly higher than that of normal oviduct(P<0.001).2.TROP2 expression was closely correlated with SOC clinical stage,pathological grade,preoperative serum CA125 level,ascites and lymph node metastasis(P<0.05),but was not significantly correlated with age(P=0.280)or platinum resistance(P=0.944).3.Ki-67 expression in low grade and high grade SOC was significantly higher than that in the control group and benign lesion tissues(P<0.001).Spearman correlation analysis showed that the expression of TROP2 is associated with Ki-67 expression in SOC(rs=0.453,P<0.001).Conclusions:1.The expression of TROP2 in SOC was significantly higher than that in the control group and benign lesions,and the high expression of TROP2 was significantly associated with the adverse clinicopathological features of OC,indicating that TROP21s involved in the development of ovarian cancer.2.TROP2 was positively correlated with Ki-67 expression in the same SOC tissues,indicating that TROP2 might be involved in the growth and proliferation of OC cells.PART Ⅱ Clinical data statistics and survival analysis of 126 patients with SOCObjective:To further explore the factors affecting survival in patients with SOC in part Ⅰ of this study.To analyze the effects of TROP2 expression,FIGO clinical stage,tumor pathological grade,preoperative serum CA125 level,ascites,lymph node metastasis and age on the survival time of SOC patients.Methods:A total of 126 patients with SOC were enrolled with the same criteria as in part Ⅰ.The patients underwent surgery in Qilu Hospital of Shandong University and completed the treatment in time,the complete case records and follow-up data were saved.The follow-up time was 60 months.The follow-up information included total survival time(OS)and progression-free survival time(PFS).The survival time was compared by Kaplan-Meier method,and the survival curve was drawn.Univariate and multivariate Cox proportional risk regression analysis was used to screen the main factors affecting prognosis.Results:1.Univariate analysis showed that TROP2 protein overexpression was associated with PFS(P<0.001)and OS(P=0.017).The median survival of patients with FIGO clinical stage,tumor pathological grade,preoperative serum CA125 level,ascites and lymph node metastasis was statistically different(P<0.05),while there was no correlation between median survival and age group.2.Multivariate COX regression model analysis showed that TROP2 expression,FIGO clinical stage,preoperative serum CA125 level,ascites and lymph node metastasis were independent factors affeecting the five-year survival rate of SOC patients.Conclusions:1.High expression of TROP2 is positively correlated with poor prognosis of SOC,and TROP2 is an independent prognostic indicator for SOC.2.FIGO clinical stage,preoperative serum CA125 level,ascites and lymph node metastasis are also independent factors affecting the prognosis of SOC.PART Ⅲ Effects of TROP2 expression on biological behavior of OC cellsObjective:To observe the effects of changes in TROP2 expression levels on biological behaviors such as proliferation,apoptosis,invasion and migration of OC cells,and explore the role and possible mechanism of TROP2 in the development of OC.Methods:We synthesized two independent siRNA sequences siRNA-550 and siRNA-1100 to transfect OC cells and interfere with the endogenous expression of TROP2.The expression of TROP2 protein after transfection was determined by Western blot.The effect of TROP2 expression on the proliferation of ovarian cancer cells was determined by CCK-8 method.The migration and invasion abilities of cells were measured by scratch test and invasion test respectively.The expressions of apoptosis-related proteins Bax and Bcl-2 were determined by Western blot.Results:1.Immunofluorescence staining showed a red fluorescent stain on the surface of A2780,HO8910 and SK-OV-3 cells.Western blot analysis showed that TROP2 protein was expressed in all three kinds of cells,and the expression of TROP2 in A2780 cell line was higher than that of the other two.2.TROP2 protein expression was significantly decreased after transfecting siRNA-550 and siRNA-1100 in A2780 cell.3.CCK-8 showed that TROP2 gene silencing significantly decreased the proliferation ability of A2780 cells(P<0.05).4.Cell invasion experiments found that the number of OC cells passing through Transwell compartment after TROP2 gene silencing was significantly decreased(P<0.05),indicating that the ability of cell invasion was decreased.5.The cell scratch test indicated that after 24 and 48 hours of the cell scratch,the OC cells with TROP2 gene silencing showed a significantly slow scratch healing speed(P<0.05),indicating that the migration ability of the cells was significantly decreased.6.Western blot analysis showed that the expression of Bcl-2 protein in OC cells was significantly decreased after TROP2 gene silencing,while the expression of Bax protein was significantly enhanced.Conclusions:TROP2 expression is related to the proliferation,apoptosis,migration and invasion of OC cells,and it might induce apoptosis by disrupting the balance regulation between Bax/Bcl-2 family proteins.TROP2 plays an important role in the development and progression of OC,and it might become a new target for OC therapy and provide a theoretical basis for targeted therapy.更多还原