【作者】 张欣；

【导师】 王传新；

【作者基本信息】 山东大学 ， 临床检验诊断学， 2012， 博士

【摘要】 研究背景宫颈癌(Uterine cervical cancer, UCC)是妇科常见恶性肿瘤之一,全球每年新增病例49.3万例,死亡病例27.4万例。虽然由于细胞学筛查策略的广泛应用,UCC的发病率有明显下降,但其仍未女性第二大高发恶性肿瘤。近年来随着UCC发病明显趋于年轻化以及宫颈腺癌发生比例的明显增加,对UCC的诊治又提出了新的问题。因此,寻找一种科学、可行的途径来提高加对UCC的早期诊断和预后判断,从而实现对UCC的早期发现,早期治疗,已成为当前研究的热点问题。目前认为高危型人乳头瘤病毒(Human papillomavirus, HPV)的感染是导致UCC发生主要的因素,流行病学资料显示高危型HPV DNA在UCC中的检出率达95%～100%。然而,在感染高危型HPV的妇女中,只有少数发展为UCC,提示可能还有其他因素参与肿瘤的发生。B细胞特异性莫洛尼氏白血病毒插入位点1(B cell-specific MLV integrationsite-1, Bmi-1)基因是PcG基因家族的重要成员(PcG),于1991年由荷兰癌症中心作为一种原癌基因与另一种癌基因c-myc共同导致B细胞非霍奇金淋巴瘤的发病而发现的。Bmi-1通过抑制p16Ink4a和p14Arf两种抑癌蛋白的表达,能够抑制p53和pRB生长调节途径诱导的凋亡和衰老。并且,Bmi-1能够激活端粒酶逆转录基因(hTERT),从而能够延长生命复制周期,使细胞发生无限增殖。最近,Bmi-1发现在血液恶性肿瘤和部分实体瘤中呈过表达。由此可见,Bmi-1在肿瘤的发生、发展中发挥着重要作用。然而目前有关Bmi-1在UCC中的表达情况以及Bmi-1在UCC发生、发展中的作用研究国内外报道较少。基于此,本研究目的是对Bmi-1在UCC组织和外周血中的表达进行检测,并通过RNAi技术抑制UCC细胞系中Bmi-1基因的表达,观察Bmi-1在UCC组织和外周血中的表达情况及其对细胞增殖、凋亡及侵袭能力的影响,探讨Bmi-1在UCC早期诊断和预后判断中的价值及Bmi-1在UCC发生发展、侵袭转移等过程中的作用机制,为寻找UCC诊断及治疗新靶点提供依据。第一部分Bmi-1蛋白在宫颈癌组织中的表达及其与临床病理特征和预后的关系目的：观察Bmi-1在宫颈癌(UCC)中的表达情况,探讨Bmi-1与UCC临床病理特征和预后的关系。方法：采用Western blot方法检测Bmi-1在宫颈正常上皮细胞(NCEC)和四种UCC细胞系(Hela, SiHa, CasKi和C33A)中的表达。利用免疫组织化学方法检测152例UCC和30例癌旁正常石蜡包埋组织中的Bmi-1表达情况。结果：Western blot分析显示,Bmi-1在四种人UCC细胞系(Hela, SiHa, CasKi和C33A)中过表达,而在NCEC中呈弱表达。在152例UCC石蜡包埋组织切片中,Bmi-1在27例中呈阴性表达(-),29例中呈弱阳性表达(+),55例中呈中度表达(++)和41例中呈强表达(+++)。Bmi-1在UCC组织中的过表达率为63.2%(Bmi-1++或+++)。然而,在30例癌旁正常对照组织中,Bmi-1只发现阴性或弱阳性表达。Bmi-1的过表达率与肿瘤大小(P=0.046)、临床分期(P=0.021)和局部淋巴结转移(P=0.010)密切相关,而与患者年龄(P=0.645)、间质浸润深度(P=0.428)、组织类型(P=0.367)和细胞分化程度(P=0.247)无关。生存曲线分析显示,Bmi-1的过表达与患者差的整体生存期(OS)明显相关(P=0.021)。Cox比例风险模型分析显示,Bmi-1过表达是OS的独立预后因素。结论：Bmi-1在UCC中过表达,并且与UCC负面临床特征和预后不良相关,提示Bmi-1参与了UCC的发生、发展过程,其可以作为恶性表型和预后不良的一项预测指标。第二部分血浆循环Bmi-1mRNA的检测及其对宫颈癌诊断和预后价值的评价目的：评估血浆循环Bmi-1mRNA对宫颈癌(UCC)的潜在诊断和预后价值。方法：采用实时荧光定量PCR方法检测109例UCC患者、138例宫颈上皮内瘤变患者(CIN)和80例健康对照者血浆中循环Bmi-1mRNA水平。采用受试者工作曲线分析循环Bmi-1mRNA对UCC的诊断效能。采用Cox比例风险模型分析Bmi-1mRNA对UCC的预后价值。结果：GAPDH和EEF1A1mRNAs在247例(91.8%)宫颈病变患者和80例(94.1%)健康对照者中检测到,两组比较差异无统计学意义(P>0.05)。UCC组血浆循环Bmi-1mRNA的中位水平明显高于CIN各组和健康对照组(P均<0.001)。CIN2组、C1N3组血浆循环Bmi-1mRNA的中位水平明显高于CIN1组、CIN2组和健康对照组(P均<0.05)。UCC组血浆SCC、CA125的中位水平明显高于CIN3组、CIN2组、CIN1组和健康对照组(P均<0.01)。血浆循环Bmi-1mRNA水平与UCC患者临床分期(P<0.001)和淋巴结转移相关(P=0.002)。循环Bmi-1mRNA诊断UCC的ROC曲线下面积(AUC)为0.881,最佳临界值为0.057,诊断敏感性为69.7%,特异性为95.9%。SCC-Ag和CA125诊断UCC的敏感性分别为56.0%和33.9%。循环Bmi-1mRNA对UCC的诊断效能明显高于SCC-Ag(0.740,95%CI=0.689～0.787,P=0.035)和CA125(0.689,95%CI=0.636～0.739, P<0.001)单独检测。Kaplan-Meier曲线分析显示,高水平的循环Bmi-1mRNA与较低的无病生存期(DFS)(P=0.001)和整体生存期(OS)(P=0.015)明显相关。Cox比例风险模型分析显示,循环Bmi-1mRNA是DFS和OS的独立预后因素。结论：循环Bmi-1mRNA水平在UCC血浆中明显升高,并且与患者负面临床病理特征和预后不良相关。循环Bmi-1mRNA可以作为UCC诊断和预后判断的一项潜在的无创性指标。第三部分Bmi-1对宫颈癌SiHa细胞的生物学效应研究目的：通过siRNA干扰宫颈癌(UCC)SiHa细胞中Bmi-1的表达,分析沉默Bmi-1的表达对SiHa细胞增殖、凋亡及侵袭能力的影响,进一步分析Bmi-1在UCC发生、发展中的作用。方法：根据Bmi-1基因序列,设计合成干扰Bmi-1的四条siRNA序列(siRNA-1、 siRNA-2、siRNA-3和siRNA-4),分别转染SiHa细胞,同时设立阴性对照组(转染阴性对照siRNA, NC组)、转染试剂对照组(加入与实验组等量的转染试剂,Mock组)和空白对照组(对细胞不做任何处理,Blank组)。RT-qPCR方法检测转染后SiHa细胞中Bmi-1mRNA的表达,应用CCK8实验、Annexin V-FITC/PI双标记实验和Transwell实验分析转染后细胞的增殖、凋亡及侵袭能力的变化。结果：siRNA-1、siRNA-2、siRNA-3、siRNA-4组转染SiHa细胞24h、48h后Bmi-1mRNA水平较NC组、Mock组和Blank组明显降低(P<0.05),其中siRNA-4组的Bmi-1mRNA下降最为明显。与NC组、Mock组、Blank组比较,siRNA-4转染组细胞在24、48、72h、96h4个时段里生长速度明显减缓,差异均有统计学意义(P<0.05)。siRNA转染SiHa细胞48h后,siRNA-4组的凋亡率为(62.43±9.86)%,明显高于NC组的(10.17±5.15)%、Mock组的(8.56±4.27)%和Blank组的(8.12±3.89)%,差异均有统计学意义(P<0.05)。侵袭试验中,siRNA-4组的穿膜细胞数为(253.29±68.72)明显少于NC组(350.21±98.62)、Mock组(406.38±113.51)和Blank组(418.46±119.78)。结论：siRNA可特异性抑制SiHa细胞中Bmi-1基因的表达,从而抑制肿瘤细胞的增殖,诱导细胞发生凋亡,减弱其侵袭能力,进一步证实Bmi-1在UCC中的作用,同时该研究也为应用RNAi进行UCC的基因治疗提供了实验依据。更多还原

【Abstract】 BackgroundUterine cervical cancer (UCC) is one of the most common malignancies, with an estimated493,000new cases and274,000UCC related deaths each year. Although incidence and mortality are decreasing owing to the implementation of routine screening, UCC still remains the second most common malignancy in women worldwide. Recently, the incidence of UCC has been rising in younger women and there has been a rise in the relative incidence of cervical adenocarcinoma, and these made the diagnosis and treatment of UCC face the new problems. Therefore, a scientific and feasible way to improve the early diagnosis and prognosis judgment of UCC, in order to achieve the early detection and treatment of UCC, has become a hot issue for the current study.High risk human papillomavirus (HPV) infection has been determined as the main risk factor for UCC, and the epidemiological data indicate high risk types of HPV DNA have been detected in95%～100%of UCC patients. However, only a few women with HPV infection have developed into UCC, indicating other unidentified genetic alterations are also involved.B cell-specific moloney murine leukemia virus integration site1(Bmi-1) gene was one member of polycomb group (PcG), originally identified as an oncogene that cooperated with c-myc in murine lymphomas in1991. Bmi-1acts as a transcriptional repressor by deregulating p16Ink4a and p14Arf tumor suppressor proteins, which can inhibit the apoptosis and senescence induced by p53and pRB growth regulatory pathways. Moreover, Bmi-1also plays an important role in the activation of human telomerase reverse transcriptase gene (hTERT), which can extend the replicative lifespan and immortalize cells. Recently, some researches have demonstrated that Bmi-1is overexpressed in hematological malignancies and a variety of solid cancers. All these suggested that the Bmi-1might play an important role in cell proliferation and tumor progression. However, the role of Bmi-1protein in human UCC has not been investigated so far.Base on these, our research aims to detect the expression of Bmi-1in UCC tissues and peripheral blood, and inhibit the expression of Bmi-1in UCC cell lines using RNAi method, and observe the expression of Bmi-1in UCC tissues and peripheral blood and its affection on cell proliferation, apoptosis and invasion, to discuss the value of Bmi-1in early diagnosis and prognosis judgment of UCC and the mechanism of UCC development, invasion and metastasis, in order to provide evidences for searching for new target of UCC diagnosis and treatment. Part1Overexpression of Bmi-1in uterine cervical cancer and its correlation with clinicopathology and prognosisObjective:To investigate the expression of Bmi-1protein in human uterine cervical cancer (UCC) and explore its associations with clinicopathological factors and prognosis.Methods:Western blot was used to detect the expression of Bmi-1in a normal cervical epithelial cell line (NCEC) and four human cervical cancer cell lines (Hela, SiHa, CasKi and C33A). In addition,152UCC and30adjacent normal cervical paraffin-embedded samples were collected to detect Bmi-1expression by immunohi stochemistry.Results:Western blot analysis showed Bmi-1was overexpressed in four human UCC cell lines (Hela, SiHa, CasKi and C33A) but not in the NCEC. Among of152paraffin-embedded archival UCC tissues,27cases showed negative expression (-),29cases showed weak expression (+),55cases showed moderate expression (++), and41cases showed strong expression (+++). Bmi-1was overexpressed in63.2%UCC tissues (Bmi-1++or+++). However, only negative or weak immunohistochemical staining was detected in the30adjacent normal cervical tissues. The overexpression of Bmi-1protein was significantly correlated with tumor size (P=0.046), clinical stage (P=0.021) and regional lymph nodes metastasis (P=0.010). However, no significant associations were found between Bmi-1protein overexpression and age (P=0.645), depth of stromal invasion (P=0.428), histological type (P=0.367), and cell differentiation (P=0.247). Survival analysis showed a significant difference between Bmi-1protein overexpression and poor overall survival (OS)(P=0.021). Cox proportional hazards risk analysis indicated that Bmi-1protein overexpression was an independent prognostic factor for OS.Conclusions:Bmi-1is overexpressed in UCC and correlated with adverse clinical characteristics and poor prognosis, which suggests that the Bmi-1might participate in the development and progression of UCC and have clinical potential not only as a useful predictor of aggressive phenotype but also a promising prognostic predictor. Part2Detection of circulating Bmi-1mRNA in plasma and its potential diagnostic and prognostic value for uterine cervical cancerObjective:To assess the diagnostic and prognostic potential of plasma circulating Bmi-1mRNA in uterine cervical cancer (UCC)Methods:The levels of circulating Bmi-1mRNA in plasma were detected by reverse transcription quantitative real-time PCR in109UCC patients,138cervical intraepithelial neoplasia (CIN) patients, and80healthy volunteers. Receiver operating characteristic (ROC) curve was established to discriminate the subjects with or without UCC. Cox model was employed to identify the independent prognostic factors.Results:GAPDH and EEF1A1mRNAs were detected in the plasma of247(91.8%) patients with cervical lesions and80(94.1%) healthy controls. No significant difference was observed between patients with cervical lesions and healthy controls (P>0.05). Median circulating Bmi-1mRNA levels were significantly higher in UCC compared with CINs and healthy controls (all at P<0.001). The levels of circulating Bmi-1mRNA were significantly higher in CIN2and CIN3patients than those in CIN1patients and healthy controls (all at P<0.05, respectively). The levels of SCC-Ag and CA125were significantly higher in UCC compared with CIN3, CIN2, CIN1and healthy controls (all at P<0.01, respectively). The high level of Bmi-1mRNA correlated significantly with advanced clinical stage (P<0.001) and lymph nodes metastasis (P=0.002). The area under the ROC curve (AUC) was0.881, and the optimal cut-off value was0.057, providing a sensitivity of69.7%and a specificity of95.9%. However, the levels of SCC-Ag and CA125were increased above the upper limits of normal in56.0%and33.9%of UCC patients, respectively. The AUC of Bmi-1mRNA was significantly larger than that for SCC-Ag (0.740,95%CI=0.689to0.787, P=0.035) or CA125(0.689,95%CI=0.636to0.739, P<0.001). Kaplan-Meier analysis showed a significant correlation between increased circulating Bmi-1mRNA level and reduced disease-free survival (DFS)(P=0.001) and overall survival (OS)(P=0.015). Cox analysis indicated that circulating Bmi-1mRNA was an independent prognostic factor for DFS and OS. Conclusion:Circulating Bmi-1mRNA is increased in plasma of UCC and correlated with adverse clinical characteristics and poor prognosis. Circulating Bmi-1mRNA can be served as a potential noninvasive molecular marker for diagnosis and prognosis of UCC. Part3Effects of Bmi-1expression on the biological behavior of uterine cervical cancer cell line SiHa cellsObjective:To analyze the affection on cell proliferation, apoptosis and invasion after silencing the Bmi-1expression in uterine cervical cancer (UCC) cell line SiHa cells with siRNA method, and further discuss the function of Bmi-1in genesis and development of UCC.Methods:The4sequences for siRNAs against Bmi-1were designed and synthesized according to Bmi-1gene sequence, then the siRNAs were transfected into UCC cell line SiHa cells as experiment groups, and transfected with negative control siRNA as negative group (NC group), transfected with transfection reagent only as transfection reagent control group (Mock group) and no treatment of the cells as blank group (Blank group). The expression of Bmi-1mRNA in SiHa cells after transfection was detected by RT-qPCR method. CCK8assay, Annexin V-FITC/PI double labeling experiments and transwell assay were used to examine the changes of cell proliferation, apoptosis and invasion, respectively.Results:Compared with NC group, Mock group and Blank group, the levels of Bmi-1mRNA in siRNA-1group, siRNA-2group, siRNA-3group and siRNA-4group after transfection24h and48h were significantly decreased (P<0.05), of which the infection efficiency of siRNA-4group was best. Compared with NC group, Mock group and Blank group, the proliferation ability in siRNA-4group was decreased after transfection24h,48h,72h and96h. After transfection48h, apoptosis rate in siRNA-4group was (62.43±9.86)%, which significantly higher than those in NC group with (10.17±5.15)%, Mock group with (8.56±4.27)%and Blank group with (8.12±3.89)%, respectively (P<0.05, respectively). In invasion experiment, the number of penetrating cells in siRNA-4group was (253.29±68.72), which significantly lower than those in NC group with (350.21±98.62), Mock group with (406.38±113.51) and Blank group with (418.46±119.78), respectively (P<0.05, respectively).Conclusion:siRNA can specifically inhibit the expression of Bmi-1gene in UCC cell line SiHa cells, thus inhibit the cell proliferation, induce cell apoptosis, and weaken cell invasion capabilities, which further demonstrate the function of Bmi-1in UCC and supply experimental evidences for gene therapeutic strategy in management of human cancers using RNAi method.更多还原