【作者】 张娟；

【导师】 张蔚；

【作者基本信息】 武汉大学 ， 妇产科学， 2017， 博士

【摘要】 背景:宫颈癌是常见的妇科恶性肿瘤之一。尽管手术和放化疗治疗有了很大的进步,恶性宫颈癌仍有较高的死亡率。肿瘤的侵袭和转移是影响宫颈癌预后的主要因素,深入研究宫颈癌侵袭转移机制是当下亟待解决的研究课题。CCL20/CCR6是肿瘤微环境中的趋化因子的成员,高表达于肝癌、胰腺癌和乳腺癌等肿瘤,与肿瘤的预后不良相关。AEG-1在肝癌、神经母细胞瘤和乳腺癌等肿瘤侵袭转移中发挥作用。但是CCL20/CCR6和AEG-1在宫颈癌中的作用和机制不太明确。目的:明确CCL20/CCR6和AEG-1在宫颈癌组织中的表达及临床意义,研究CCL20/CCR6对宫颈癌细胞增殖和侵袭的作用,探索AEG-1在CCL20/CCR6促宫颈癌细胞增殖和侵袭中的作用。方法:收集94例原发性宫颈癌患者组织标本、外周血和临床病理资料,同期排除宫颈病变、因子宫体良性疾病切除子宫的50例患者的宫颈组织为对照,同时收集对照组外周血和临床病理资料。应用免疫组织化学技术检测宫颈癌组织中CCL20、CCR6的表达,然后分析其与患者临床病理特征的关系。利用ELISA检测宫颈癌患者和对照组患者外周血CCL20水平。分别用RT-PCR、Western Blot检测宫颈癌细胞HeLa、SiHa、C-33A中CCL20、CCR6的mRNA和蛋白表达水平。将不同浓度CCL20人重组蛋白与宫颈癌细胞HeLa、SiHa共培养,运用MTT法、细胞划痕实验、Transwell观察不同浓度CCL20作用下宫颈癌细胞增殖和迁移能力。利用RT-PCR技术、Western Blot实验检测不同浓度CCL20刺激下,宫颈癌细胞系vimentin、N-cadherin、MMP2的mRNA和蛋白表达。接着利用RNA干扰技术下调宫颈癌细胞HeLa、SiHa中CCL20的表达,然后运用MTT法、Transwell观察宫颈癌细胞HeLa、SiHa细胞增殖和迁移能力。利用免疫组织化学法检测宫颈癌、正常宫颈组织中AEG-1的表达,分析宫颈癌组织中AEG-1的表达与宫颈癌患者临床病理特征的相关性。运用Western Blot实验检测在不同浓度CCL20作用下宫颈癌细胞中AEG-1、p-Erk1/2和p-Akt蛋白表达。利用RNA干扰技术下调宫颈癌细胞中AEG-1的表达后,观察在CCL20刺激条件下,使用Western Blot检测vimentin、N-cadherin、MMP2、p-Erk1/2和p-Akt蛋白及细胞周期相关蛋白Cyclin D1、CDK2蛋白的表达。运用流式细胞仪检测细胞周期的实验方法观察siRNA-AEG-1-HeLa、siRNA-AEG-1-SiHa与空白对照组细胞,计算各细胞周期细胞数,观察细胞增殖情况。结果:CCL20、CCR6在宫颈癌组织中有表达,CCL20和CCR6在宫颈癌组织中的表达与FIGO分期、肿瘤大小、淋巴结转移有相关性(P<0.05),而与年龄、组织分化程度无关。宫颈癌患者血清CCL20表达量高于对照组,且与患者FIGO分期、肿瘤大小、淋巴结转移有相关性(P<0.05)。CCL20和CCR6在宫颈癌细胞系HeLa、SiHa、C-33A中均有表达,其中CCL20和CCR6在HeLa、SiHa中的mRNA和蛋白表达水平相对低于C-33A。用MTT法测Ong/ml、10 ng/ml、20 ng/ml、30 ng/ml和40 ng/ml人重组蛋白CCL20与HeLa细胞和SiHa细胞共培养24h后细胞增殖率,CCL20处理组细胞增值率高于空白对照组,差异有差异统计学意义(P<0.05),细胞增值率随着CCL20作用浓度增加而增加。用细胞划痕实验检测Ong/ml、10 ng/ml、20 ng/ml和40 ng/ml CCL20与宫颈癌细胞共培养24h和48h后,发现Hela组和SiHa组细胞划痕修复率随CCL20作用时间和浓度增加而升高,且高于空白组,差异具有统计学意义(P<0.05)。Transwell检测Ong/ml、10 ng/ml、20 ng/ml和40 ng/ml CCL20与宫颈癌细胞共培养24h后发现Hela组和SiHa组细胞侵袭数随CCL20作用浓度增加而增加,且高于空白组,差异具有统计学意义(P<0.05)。0 ng/ml、5 ng/ml、10 ng/ml、20 ng/ml、30 ng/ml 和 40 ng/ml CCL20 与 HeLa 和 SiHa 细胞共培养 24h 后,vimentin、N-cadherin、MMP2mRNA和蛋白的表达高于空白对照组,差异具有统计学意义(P<0.05),而且呈浓度依赖性升高。下调Hela和SiHa中CCL20表达后,si-CCL20-Hela、si-CCL20-SiHa组细胞增殖率和侵袭到滤膜下层细胞数都比con-RNA-Hela 和 con-RNA-SiHa 低,差异有统计学意义(P<0.05)。AEG-1 在宫颈癌组织中高表达,AEG-1表达量与FIGO分期、肿瘤大小、淋巴结转移有关(P<0.05),与年龄、组织分化程度无关(P>0.05)。0ng/ml、5ng/ml、10 ng/ml、20 ng/ml、30 ng/ml 和 40 ng/ml CCL20 与 HeLa 和 SiHa 细胞共培养 24 小时后,AEG-1、p-Erk和p-Akt蛋白表达高于对照组,差异有统计学意义(P<0.05),呈浓度依赖性增加。下调Hela和SiHa中AEG-1表达后48小时使用30ng/ml CCL20刺激,si-AEG-1-Hela 组和 si-AEG-1-SiHa 组 vimentin、N-cadherin、MMP2、p-Erk1/2、p-Akt蛋白表达均低于con-RNA-Hela和con-RNA-SiHa对照组,差异有统计学意义(P<0.05)。30μMCCL20 与 si-AEG-1-SiHa 和 con-RNA-SiHa 共培养24小时后,si-AEG-1-SiHa组和si-AEG-1-Hela组进入细胞周期S期的数量少于con-RNA-SiHa和con-RNA-Hela对照组,差异有统计学意义(P<0.05)。si-AEG-1-SiHa组中细胞周期相关蛋白Cyclin D1、CDK2蛋白表达量较con-RNA-SiHa低,差异有统计学意义(P<0.05)。结论:CCL20/CCR6、AEG-1在宫颈癌组织中有表达,与宫颈癌FIGO分期、肿瘤大小、淋巴结转移相关。外周血CCL20升高与患者不良预后有关。AEG-1在CCL20/CCR6促进宫颈癌细胞增殖和侵袭中发挥作用。AEG-1通过Erk1/2和Akt通路介导CCL20/CCR6诱导EMT过程促宫颈癌发生侵袭,CCL20/CCR6-AEG-1-Erk1/2-EMT 和 CCL20/CCR6-AEG-1-Akt-EMT 通路有望成为抗宫颈癌进展的有效靶点,有助于宫颈癌临床治疗。更多还原

【Abstract】 Background:Cervical cancer is one of the most common gynecologic malignancies.In spite of the advancement in surgical and chemotherapeutic techniques,the mortality of malignant cervical cancer still has a high level。Invasion and metastasis of tumor are the main factors affecting the prognosis of cervical cancer.Further research on the mechanism of invasion and metastasis in cervical cancer is an urgent research subject.CCL20/CCR6 is a member of chemokines in tumor microenvironment,which is highly expressed in liver cancer,pancreatic cancer and breast cancer and associated with poor prognosis.AEG-lplay a role in invasion and metastasis of hepatocellular carcinoma,neuroblastoma and breast cancer.But the functions and mechanisms of CCL20/CCR6 and AEG-1 in cervical cancer are not clear.Objective:To detect and investigate the expression and the clinical significance of CCL20/CCR6 and AEG-1 in cervical cancer.To study the effect of CCL20/CCR6 on the proliferation and invasion of cervical cancer cells.To explore the role of AEG-1 in CCL20/CCR6 in promoting the proliferation and invasion of cervical cancer cells.Methods:The specimens,peripheral blood and clinicopathological data of 94 patients with primary cervical cancer were collected.During the same period,50 patients with uterine benign disease were divided into control group.The normal cervical tissues,the peripheral blood and clinical data of the control group were collected.Immunohistochemical technique was used to detect the expression of CCL20 and CCR6 in cervical cancer tissues.The level of CCL20 in peripheral blood of patients with cervical cancer and control group was detected by ELISA.The expression of mRNA and protein of CCL20 and CCR6 in HeLa,SiHa and C-33A were detected by RT-PCR and Western Blot respectively.The recombinant plasmids of CCL20 were co-cultured with HeLa and SiHa.Wound healing assay,MTT and Transwell were used to observe the effects of different concentrations of CCL20 on the proliferation and migration capacity of cervical cancer cells.The expression of mRNA and protein of vimentin,N-cadherin and MMP2 in cervical cancer cells were detected by RT-PCR and Western Blot under the different concentrations of CCL20.The expression of CCL20 in cervical cancer cells HeLa and SiHa was down-regulated by RNA interference technique,then we observe the proliferation and migration ability of HeLa and SiHa cells by MTT and Transwell.The expression of AEG-1 in cervical cancer and normal cervical tissues was detected by immunohistochemistry.We discussed the relatioinship between the expression of AEG-1 in cervical cancer and the clinicopathological features of cervical cancer.The expression of AEG-1,p-Erkl/2 and p-Akt protein in cervical cancer cells were detected by Western Blot assay at different concentrations of CCL20.The level of vimentin,N-cadherin,MMP2 p-Erkl/2,p-Akt,Cyclin D1 and CDK2 were detected by Western Blot under the condition of CCL20 stimulation after down-regulating the expression of AEG-1 in cervical cancer cells by RNA interference.The number of cells in per cell cycle was calculated of siRNA-AEG-1-HeLa、siRNA-AEG-1-SiHa and control si-RNA groups by flow cytometry.Results:CCL20 and CCR6 were expressed in cervical cancer tissues.The expression of CCL20 and CCR6 in cervical cancer were correlated with FIGO staging,tumor size and lymph node metastasis(P<0.05),but wasn’t with the age and histological differentiation(P>0.05).The level of CCL20 in cervical cancer patients’serum was higher than that in the control group,and the level was correlated with FIGO staging,tumor size and lymph node metastasis(P<0.05),but wasn’t with the age and histological differentiation(P>0.05).The CCL20 and CCR6 were expressed in HeLa,SiHa and C-33A.The proliferation rates were measured by MTT assay after HeLa and SiHa co-cultured with human recombinant protein CCL20 at the concentration of Ong/ml,10 ng/ml,20 ng/ml,30 ng/ml and 40 ng/ml for 24 hours.The proliferation rates of HeLa and SiHa treated with CCL20 cells were higher than that of the control group,and increased in a concentration-dependent manner,the differences were statistically significant(P<0.05).The wound healing rates of HeLa and SiHa at 24 hours and 48 hours after co-cultured with CCL20 at the concentration of 0 ng/ml,10 ng/ml,20 ng/ml and 40 ng/ml were detected by wound healing assay.The healing rates at 24 hours and 48 hours of Hela and SiHa that treated with CCL20 were higher than the blank group,the difference was statistically significant(P<0.05),and the increase was in a concentration-dependent and time-dependent manner.The number of invasive cells of HeLa and SiHa at 24 hours after co-cultured with CCL20 at the concentration of 0 ng/ml,10 ng/ml,20 ng/ml and 40 ng/ml were detected by transwell.The number of invasive cells at 24 hours of Hela and SiHa that treated with CCL20 were higher than the blank group,the difference was statistically significant(P<0.05),and the increase was in a concentration-dependent manner.CCL20 at the concentration of 0 ng/ml,5 ng/ml,10 ng/ml,20 ng/ml,30 ng/ml and 40ng/ml was co-cultured with HeLa and SiHa for 24 hours.The expression of vimentin,N-cadherin and MMP2 in HeLa and SiHa treated with CCL20 were higher than that of the blank control group,the difference was statistically significant(P<0.05),and the increase was in a concentration-dependent manner.The cell proliferation rate and the number of cells penetrating into the lower layer of the cells were significantly lower than those of the con-RNA-Hela and con-RNA-SiHa cells after the expression of CCL20 of HeLa and SiHa was down-regulated by RNA interference,the difference was statistically significant(P<0.05).AEG-1 was highly expressed in cervical cancer tissues.The expression of AEG-1 was correlated with FIGO staging,tumor size and lymph node metastasis(P<0.05),but wasn’t with age and histological differentiation(P>0.05).CCL20 at the concentration of 0 ng/ml,5 ng/ml,10 ng/ml,20 ng/ml,30 ng/ml and 40ng/ml was co-cultured with HeLa and SiHa for 24 hours.The expression of AEG-1,p-Erk1/2 and p-Akt in HeLa and SiHa treated with CCL20 were higher than that of the blank control group,the difference was statistically significant(P<0.05),and the increase was in a concentration-dependent manner.HeLa and SiHa were transfected with si-control or si-AEG-1 for silencing of AEG-1.At 48 h post-transfection,all the cells were pre-treated with 30 ng/ml of CCL20.The expressions of vimentin,N-cadherin,MMP2,p-Erk1/2 and p-Akt in si-AEG-1-Hela and si-AEG-1-SiHa were lower than con-RNA-Hela and con-RNA-SiHa,the differences were statistically significant(P<0.05).After co-culture with 30 μM CCL20,the number of cells enter into S phase of cell cycle of si-AEG-1-SiHa and si-AEG-1-Hela were higher than the con-RNA-Hela and con-RNA-SiHa,the differences were statistically significant(P<0.05).The expression of Cyclin D1 and CDK2 in si-AEG-1-SiHa groups were lower than those in con-RNA-SiHa,the differences were statistically significant(P<0.05).Conclusion:CCL20/CCR6 and AEG-1 are expressed in cervical cancer tissues and are associated with FIGO staging,tumor size,lymph node metastasis.The increase in CCL20 of peripheral blood is associated with poor prognosis.AEG-1 plays a role in CCL20/CCR6 promoting the proliferation and invasion of cervical cancer cells.AEG-1 mediates CCL20/CCR6-induced EMT of cervical cancer via both Akt and Erk1/2 signaling pathway.CCL20/CCR6-AEG-1-Erk1/2-EMT and CCL20/CCR6-AEG-1-Akt-EMT pathway could be suggested as a prospective target to antagonize the progression of cervical cancer,which can be beneficial to the patients with cervical cancer in the clinical practice.更多还原