【作者】 李辉；

【导师】 陈奎生；

【作者基本信息】 郑州大学 ， 病理学与病理生理学， 2022， 硕士

【摘要】 背景与目的食管癌是全世界恶性肿瘤最高发的一种,每年全世界约30万死于该病,2020年全球癌症报告指出,食管癌发病率位列所有肿瘤发病率的第七位,死亡率位于第六位。食管癌的发病具有显著的地域差异,我国是食管癌的高发区,其发病率居我国大陆各类肿瘤第五位,病死率居我国大陆恶性肿瘤第四位。我国食管癌的主要类型为食管鳞状细胞癌(esophageal squamous cell carcinoma,ESCC),约占食管癌患者的95%,严重威胁人类健康。ESCC作为一种高侵袭性恶性肿瘤,起病隐匿且进展迅速,大多数患者初次确诊已为中晚期,根治性切除手术是食管癌治疗的首选方案,但患者局部复发率较高导致预后较差,五年生存率较低。迄今为止,已有大量的研究试图阐明ESCC的基本分子机制和生物学行为,但具体发生发展的分子机制尚不清楚。寻找新的可能在ESCC发生发展过程中具有重要作用的基因、蛋白以及阐明其相关的分子机制将会在ESCC的诊断和治疗中具有非常重要的意义。早期有丝分裂抑制剂1(early mitoticinhibitor 1,Emi1)属于F-box家族蛋白,可参与组成Skp1-Cul1-F-box-protein(SCF)泛素连接酶,介导蛋白的泛素化降解。Emi1在许多恶性肿瘤中表达上调,其过表达导致有丝分裂缺陷,可能促使肿瘤的发生。大量研究证实Emi1与多种肿瘤的发生密切相关,但Emi1在ESCC的表达水平及功能研究鲜有报道,有待研究。泛素结合酶10(ubiquitin-conjugating enzyme 10,UBCH10)又称泛素偶联酶E2C(ubiquitin conjugating enzyme E2C,UBE2C),属于泛素结合酶E2家族的成员,是细胞周期调控(细胞有丝分裂、纺锤体形成等)的重要调节器。UBCH10在非小细胞肺癌、脑膜瘤、结肠癌、乳腺癌、骨肉瘤等肿瘤中高表达并且与肿瘤分期正相关,说明UBCH10可能为潜在的癌基因。但关于UBCH10在ESCC中的表达情况研究甚少,尚无明确的研究结果。Cyclin B1是调控G2期的关键蛋白,对整个细胞周期的进程至关重要。Cyclin B1在多种肿瘤中异常表达,例如乳腺癌、肝癌、鼻咽癌、胃癌等。深入研究ESCC中Cyclin B1介导的细胞周期调控异常与肿瘤组织中的无序增殖特征之间的联系,能够为ESCC的治疗提供一个新的靶点。Emi1能够调控后期促进复合物/环体(anaphase-promoting complex/cyclosome,APC/C)对靶蛋白的泛素化降解作用,Cyclin B1是APC/C重要的靶蛋白,而在APC/C介导Cyclin B1蛋白降解过程中,需要UBCH10的参与。有研究指出,Emi1能够拮抗APC中的D-box结合域,阻碍UBCH10与APC/C的底物结合,最终抑制APC/C的靶蛋白Cyclin B降解。然而,有关Emi1、UBCH10和Cyclin B1联合检测在ESCC中的表达及其与肿瘤增殖和凋亡的关系尚未见文献报道。本研究应用免疫组织化学和原位杂交方法检测ESCC组织和正常食管黏膜组织中Emi1、UBCH10和Cyclin B1蛋白及m RNA的表达,探讨其相关性;进一步采用TUNEL法和免疫组织化学检测Ki67指标分析ESCC组织中细胞的凋亡和增殖,探讨Emi1、UBCH10和Cyclin B1表达与ESCC凋亡和增殖的关系。希望为提高ESCC治疗效果,改善预后提供新的理论依据。方法1运用免疫组织化学方法检测50例正常食管黏膜组织和50例ESCC组织中Emi1、UBCH10及Cyclin B1蛋白表达,探讨蛋白表达与ESCC发生发展的关系。2采用原位杂交技术检测50例正常食管黏膜组织和50例ESCC组织中Emi1、UBCH10和Cyclin B1 m RNA表达,探讨m RNA表达与ESCC发生发展的关系。3采用免疫组织化学方法检测50例正常食管黏膜组织和50例ESCC组织中Ki-67蛋白表达,探讨Emi1、UBCH10及Cyclin B1表达与ESCC增殖的关系。4采用TUNEL方法检测50例正常食管黏膜组织和50例ESCC组织中凋亡情况,探讨Emi1、UBCH10及Cyclin B1表达与ESCC凋亡的关系。结果1 Emi1、UBCH10和Cyclin B1与ESCC的关系1.1 ESCC组织中Emi1蛋白及m RNA的阳性表达率均为86.00%,显著高于癌旁组织18.00%和12.00%,两组间Emi1蛋白及m RNA阳性表达率相比较,差异具有统计学意义(p(27)0.05)。1.2 ESCC组织中UBCH10蛋白及m RNA的阳性表达率均为88.00%,显著高于癌旁组织14.00%和16.00%,两组间UBCH10蛋白及m RNA阳性表达率相比较,差异具有统计学意义(p(27)0.05)。1.3 ESCC组织中Cyclin B1蛋白及m RNA的阳性表达率分别为86.00%和82.00%,显著高于癌旁组织12.00%和14.00%,两组间Cyclin B1蛋白及m RNA阳性表达率相比较,差异具有统计学意义(p(27)0.05)。1.4 ESCC组织中Emi1、UBCH10和Cyclin B1蛋白及m RNA的表达,与肿瘤组织分级、病理分期和淋巴结转移有关(p(27)0.05),而与性别、年龄、肿瘤直径、浸润深度没有相关性(p>0.05)。1.5通过相关性分析显示ESCC组织中Emi1、UBCH10和Cyclin B1蛋白与其对应m RNA表达具有正相关性(r=0.6077、r=0.4595、r=0.5484;p(27)0.0001、p(27)0.0008、p(27)0.0001)。2 ESCC和癌旁组织中增殖与凋亡情况2.1 ESCC组织中呈现非常明显的高增殖指数,平均为60.40%,而癌旁组织增殖指数为11.90%。ESCC和癌旁组织的增殖指数差异具有统计学意义(p(27)0.05)。2.2 ESCC组织中凋亡指数约为29.60%,癌旁组织中凋亡指数约为42.20%,两者相比ESCC组织的凋亡指数较低,两者差异具有统计学意义(p(27)0.05)。3 Emi1、UBCH10、Cyclin B1蛋白及m RNA表达与ESCC增殖和凋亡的关系3.1 Emi1、UBCH10、Cyclin B1蛋白表达与增殖指数呈现明显的正相关性(r=0.4561、r=0.4082、r=0.4300;p=0.0009、p=0.0033、p=0.0018);Emi1、UBCH10、Cyclin B1 m RNA表达与增殖指数呈现明显的正相关性(r=0.5326、r=0.5764、r=0.6794;p<0.0001、p<0.0001、p<0.0001),伴随着Emi1、UBCH10、Cyclin B1蛋白及m RNA高表达,肿瘤组织中细胞的增殖状态越活跃。3.2 Emi1、UBCH10、Cyclin B1蛋白表达与凋亡指数呈现明显的负相关性(r=-0.5737、r=-0.4178、r=-0.4939;p<0.0001、p=0.0025、p=0.0018),Emi1、UBCH10、Cyclin B1 m RNA表达与凋亡指数呈现明显的负相关性(r=-0.4614、r=-0.3450、r=-0.4742;p=0.0007、p=0.0141、p=0.0005),伴随着Emi1、UBCH10、Cyclin B1蛋白及m RNA高表达,肿瘤组织中细胞的凋亡状态越弱。3.3 Emi1、UBCH10蛋白及m RNA表达呈正相关(r=0.5418、r=0.4181;p<0.0001、p=0.0025);Emi1、Cyclin B1蛋白及m RNA表达呈正相关(r=0.5539、r=0.7357;p<0.0001、p<0.0001);UBCH10、Cyclin B1蛋白及m RNA表达呈正相关(r=0.6020、r=0.5997;p<0.0001、p<0.0001)。结论1 Emi1、UBCH10和Cyclin B1是ESCC肿瘤进程中的重要参与者,能够影响肿瘤分化、淋巴结转移和病理分期。2 Emi1、UBCH10和Cyclin B1促进ESCC的肿瘤增殖,抑制ESCC的肿瘤凋亡,且三者在ESCC中的表达具有正相关性,提示三者协同参与ESCC肿瘤增殖和凋亡过程。更多还原

【Abstract】 Background and objectiveEsophageal cancer is one of the most common malignant tumors in the world,and about 300,000 people die of this disease every year.According to the 2020 Global Cancer Report,the incidence of esophageal cancer ranks seventh among all tumors,and the mortality rate ranks sixth.The incidence of esophageal cancer has significant regional differences.China is a high-incidence area of esophageal cancer.Its incidence ranks fifth among all types of tumors in China,and its mortality ranks fourth among malignant tumors.The main type of esophageal cancer in China is esophageal squamous cell carcinoma(ESCC),accounting for about 95% of esophageal cancer patients,which seriously threatens human health.ESCC is a highly aggressive malignant tumor with insidious onset and rapid progression.Radical resection is the first choice for the treatment of esophageal cancer,but the high local recurrence rate leads to a poor prognosis and a low five-year survival rate.So far,a large number of studies have attempted to elucidate the basic molecular mechanism and biological behavior of ESCC,but the molecular mechanism of the specific occurrence and development is still unclear.Finding new genes and proteins that may play an important role in the occurrence and development of ESCC and elucidating their related molecular mechanisms will be of great significance in the diagnosis and treatment of ESCC.Early mitoticinhibitor 1(Emi1)belongs to the F-box family of proteins and participates in the formation of Skp1-Cul1-F-box-protein(SCF)ubiquitin ligase,which mediates the ubiquitination and degradation of proteins.Emi1 is upregulated in many malignancies,and its overexpression leads to mitotic defects that may contribute to tumorigenesis.A large number of studies have confirmed that Emi1 is closely related to the occurrence of various tumors,but the expression level and function of Emi1 in ESCC are rarely reported and need to be studied.Ubiquitin-conjugating enzyme 10(UBCH10),also known as ubiquitin-conjugating enzyme E2C(UBE2C),is a member of the ubiquitin-conjugating enzyme E2 family.It is an important regulator of cell cycle regulation(mitosis,spindle formation,etc.).UBCH10 is highly expressed in non-small cell lung cancer,meningioma,colon cancer,breast cancer,osteosarcoma and other tumors and is positively correlated with tumor stage,indicating that UBCH10 may be a potential oncogene.However,there is little research on the expression of UBCH10 in ESCC,and there is no clear research result.Cyclin B1 is a key protein that regulates the G2 phase and is essential for the progression of the entire cell cycle.Cyclin B1 is abnormally expressed in various tumors,such as breast cancer,liver cancer,nasopharyngeal cancer,gastric cancer,etc.In-depth study of the link between Cyclin B1-mediated cell cycle dysregulation in ESCC and disordered proliferation characteristics in tumor tissues can provide a new target for ESCC therapy.Emi1 can regulate the ubiquitination and degradation of target proteins by anaphase-promoting complex/cyclosome(APC/C).Cyclin B1 is an important target protein of APC/C,and UBCH10 is required for APC/C-mediated degradation of Cyclin B1 protein.Some studies have pointed out that Emi1 can antagonize the D-box binding domain in APC,hinder the binding of UBCH10 to the substrate of APC/C,and finally inhibit the degradation of Cyclin B,the target protein of APC/C.The combined detection of Emi1,UBCH10,Cyclin B1 in ESCC and their relationship with tumor proliferation and apoptosis have not been reported in the literature.In this study,the expressions of Emi1,UBCH10,Cyclin B1 in ESCC tissues and normal esophageal mucosa tissues were detected by immunohistochemistry and in situ hybridization,and their correlations were explored.The apoptosis and proliferation of cells in ESCC tissues were further analyzed by TUNEL method and immunohistochemical detection of Ki67 index,and the relationship between the expression of Emi1,UBCH10,Cyclin B1 and the apoptosis and proliferation of ESCC was investigated.It is hoped to provide a new theoretical basis for improving the therapeutic effect of ESCC and improving the prognosis.Methods1 Immunohistochemical methods were used to detect the protein expressions of Emi1,UBCH10,Cyclin B1 in 50 normal esophageal mucosal tissues and 50 ESCC tissues,and to explore the relationship between protein expression and the development of ESCC.2 In situ hybridization was used to detect the m RNA expressions of Emi1,UBCH10,Cyclin B1 in 50 normal esophageal mucosal tissues and 50 ESCC tissues,and to explore the relationship between m RNA expression and the development of ESCC.3 The expression of Ki-67 protein in 50 normal esophageal mucosa tissues and50 ESCC tissues was detected by immunohistochemistry,and the relationship between the expressions of Emi1,UBCH10,Cyclin B1 and the proliferation of ESCC was investigated.4 TUNEL method was used to detect apoptosis in 50 normal esophageal mucosal tissues and 50 ESCC tissues,and to explore the relationship between the expressions of Emi1,UBCH10,Cyclin B1 and ESCC apoptosis.Results1 Relationship between Emi1,UBCH10,Cyclin B1 and ESCC1.1 The positive expression rates of Emi1 protein and m RNA in ESCC tissues were both 86.00%,which were significantly higher than those in adjacent tissues(18.00% and 12.00%),and the differences in the positive expression rates of Emi1 protein and m RNA between the two groups were statistically significant(p<0.05).1.2 The positive expression rates of UBCH10 protein and m RNA in ESCC tissues were both 88.00%,which were significantly higher than those in adjacent tissues(14.00% and 16.00%),and the differences in the positive expression rates of UBCH10 protein and m RNA between the two groups were statistically significant(p<0.05).1.3 The positive expression rates of Cyclin B1 protein and m RNA in ESCC tissues were 86.00% and 82.00%,which were significantly higher than 12.00% and14.00% in adjacent normal tissues.There were statistically significant differences in the positive expression rates of Cyclin B1 protein and m RNA between the two groups(p<0.05).1.4 The expression of Emi1,UBCH10,Cyclin B1 m RNA and protein in ESCC tissue was related to tumor tissue grade,lymph node metastasis and pathological stage(p<0.05),but not to gender,age,tumor diameter and depth of invasion(p>0.05).It is suggested that Emi1,UBCH10 and Cyclin B1 are important players in ESCC tumor process,which can affect tumor differentiation,lymph node metastasis and pathological stage.1.5 Correlation analysis showed that Emi1,UBCH10,Cyclin B1 m RNA and their corresponding protein expressions were positively correlated in ESCC tissues(r=0.6077、r=0.4595、r=0.5484;p(27)0.0001、p(27)0.0008、p(27)0.0001).2 Proliferation and apoptosis in ESCC and adjacent tissues2.1 ESCC tissue showed a very obvious high proliferation index,with an average of 60.40%,while the adjacent tissue proliferation index was 11.90%.The difference in proliferation index between ESCC and paracancerous tissue was statistically significant(p<0.05).2.2 The apoptosis index in ESCC tissue was about 29.60%,and the apoptosis index in adjacent tissue was about 42.20%.The apoptosis index in ESCC tissue was lower,and the difference was statistically significant(p<0.05).3 The relationship between Emi1,UBCH10,Cyclin B1 protein and m RNA expression and ESCC proliferation and apoptosis3.1 Emi1,UBCH10,Cyclin B1 protein expression and proliferation index showed a significant positive correlation(r=0.4561、r=0.4082、r=0.4300;p=0.0009、p=0.0033 、 p=0.0018);Emi1,UBCH10,Cyclin B1 m RNA expression and proliferation index showed a significant positive correlation(r=0.5326、r=0.5764、r=0.6794;p<0.0001、p<0.0001、p<0.0001).With the high expression of Emi1,UBCH10,Cyclin B1 protein and m RNA,the proliferation of cells in tumor tissue is more active.3.2 The expression of Emi1,UBCH10,Cyclin B1 protein was significantly negatively correlated with the apoptosis index(r=-0.5737、r=-0.4178、r=-0.4939;p<0.0001、p=0.0025、p=0.0018);Emi1,UBCH10,Cyclin B1 m RNA expression was significantly negatively correlated with the apoptosis index(r=-0.4614、r=-0.3450、r=-0.4742;p=0.0007、p=0.0141、p=0.0005).With the high expression of Emi1,UBCH10,Cyclin B1 protein and m RNA,the apoptosis state of cells in tumor tissue was weaker.3.3 Emi1,UBCH10 protein and m RNA expression were positively correlated(r=0.5418、r=0.4181;p<0.0001、p=0.0025);Emi1,Cyclin B1 protein and m RNA expression were positively correlated(r=0.5539、r=0.7357;p<0.0001、p<0.0001);UBCH10,Cyclin B1 protein and m RNA expression were positively correlated(r=0.6020、r=0.5997;p<0.0001、p<0.0001).Conclusions1 Emi1,UBCH10 and Cyclin B1 are important players in ESCC tumor progression and can affect tumor differentiation,lymph node metastasis and pathological staging.2 Emi1,UBCH10 and Cyclin B1 promote the tumor proliferation of ESCC and inhibit the tumor apoptosis of ESCC,and the expressions of the three in ESCC are positively correlated,suggesting that the three are synergistically involved in the process of ESCC tumor proliferation and apoptosis.更多还原