【作者】 刘娟；

【导师】 赵蔚明；

【作者基本信息】 山东大学 ， 病原生物学， 2011， 博士

【摘要】 宫颈癌是一种威胁妇女生命健康的常见恶性肿瘤,在世界范围内,每年约有45万女性发病,20多万死于宫颈癌。特别是在发展中国家,宫颈癌在女性恶性肿瘤中发病死亡率仅次于乳腺癌。我国每年新发现的病例为13-15万,每年大约有2万多名妇女死于这种疾病。目前分子生物学研究已经确认宫颈癌致病首要因素是人乳头瘤病毒(Human papillomavirus, HPV)。人乳头瘤病毒是一种双链DNA病毒,根据是否能引起恶性病变分为低危型HPV和高危型HPV两种。低危型HPV主要引起皮肤良性增生,而高危型HPV的长期感染与宫颈癌的发生密切相关。特别是HPV16、18型,两者在宫颈癌组织中阳性率的总和可达90%。研究表明HPV致宫颈癌的机制主要与病毒癌蛋白E6、E7有关。高危型HPV的长期感染使病毒基因组有机会整合入宿主细胞基因组中,病毒基因组在整合状态下持续高表达两种病毒癌蛋白E6、E7。E6、E7蛋白是参与细胞恶性转化的重要因子。HPV16、18的E6、E7可以分别抑制p53及视网膜母细胞瘤蛋白pRb的功能,使这两种蛋白失活,然后通过一系列的蛋白分子相互作用,使宿主细胞蛋白表达发生变化,正常细胞的信号转导受到扰乱,使细胞脱离正常增殖周期,最终发生细胞转化。虽然HPV诱导宫颈癌的基本模型已知,但是病毒基因组整合后E6、E7蛋白持续表达引起宿主细胞内哪些蛋白表达发生变化,这种变化影响的细胞信号途径尚未完全明确。所以近年来有关E6、E7新的分子靶点及其改变的细胞信号途径的报道不断出现。HPV持续感染引起的细胞内的变化仍然是近年来宫颈癌致癌机制领域的研究热点。Cancerous inhibitor of protein phosphatase 2A (CIP2A)是一种新发现的具有癌蛋白性质的人类细胞蛋白。据文献报道,CIP2A可抑制PP2A对c-Myc 62位丝氨酸(S62)的去磷酸化作用,阻止c-Myc的降解,从而增加细胞中c-Myc的蛋白水平。CIP2A能够促进细胞的生长增殖以及小鼠体内的肿瘤生长,和ras共同转染可使细胞发生转化。研究发现,CIP2A在很多人类恶性肿瘤中过表达,例如胃癌、结肠癌、乳腺癌、非小细胞肺癌、前列腺癌、食道癌、卵巢癌等。此外,CIP2A的过表达还与乳腺癌、肺癌、卵巢癌的预后不良密切相关。那么,CIP2A在宫颈癌中的表达情况如何?CIP2A在宫颈癌中的表达与HPV的癌蛋白E6、E7有无关系,是否影响某些细胞信号途径?本论文就这些问题做了初步的探讨。一、CIP2A在宫颈癌中的表达我们在山东省肿瘤医院收集了15例宫颈癌和8例正常宫颈组织的新鲜冰冻标本,利用RT-PCR分析CIP2A mRNA的表达水平。结果表明,在mRNA水平上,15例宫颈癌组织中有11例(73.3%)表达CIP2A,而8例正常宫颈组织无一例表达CIP2A。同时,我们在山东大学齐鲁医院收集了15例正常宫颈、21例C1NⅠ、25例CINⅡ、24例CINⅢ、72例宫颈癌的石蜡包埋组织块,利用免疫组化分析CIP2A的蛋白表达。结果显示CIP2A在15例正常组织、CINⅠ、CINⅡ组织中无一表达,在24例CINⅢ组织中有3例(12.5%)CIP2A阳性,而在72例宫颈癌组织中有38例(52.8%)有CIP2A的表达。统计学分析CIP2A表达和各种临床指标的关系的结果显示CIP2A的表达与病人年龄、临床分期、分化程度、淋巴转移、肿瘤尺寸以及Ki67的表达无相关性。这部分结果表明CIP2A在宫颈正常组织和程度较轻的宫颈上皮内瘤变中(CINⅠ、CINⅡ)不表达,在CINⅢ中有部分表达,而在宫颈癌组织中是过表达的。由此推测,CIP2A有可能通过某种途径参与高程度宫颈上皮内瘤变向宫颈癌转化的过程。二、CIP2A表达对宫颈癌细胞的影响为检测CIP2A表达对宫颈癌细胞的生长增殖有无促进作用,我们用CIP2A特异的小干扰RNA和通用阴性对照siRNA转染宫颈癌细胞系HeLa、SiHa、Caski。RT-PCR和wesntern blot分析表明CIP2AsiRNA转染后各种细胞系中CIP2A的表达量均显著下降。成功干扰了CIP2A的表达之后,我们检测到细胞内c-Myc蛋白表达水平明显降低。随后我们用CCK-8试剂盒和克隆形成实验检测了各种细胞系的增殖活性,利用软琼脂克隆形成实验检测了细胞的锚定非依赖的生长能力。CCK-8检测表明,转染CIP2AsiRNA的HeLa、SiHa、Caski细胞与对照组相比,生长增殖活性显著下降；在克隆形成试验中,转染CIP2A siRNA的各细胞与对照组相比均表现出克隆形成能力的明显下降；同样,CIP2A siRNA转染组的软琼脂克隆形成能力也显著低于转染control RNA的细胞。为了阐明干扰CIP2A表达后宫颈癌细胞的生长增殖能力下降的原因,我们用P-gal染色方法检测了细胞的衰老,PI单染、PI-Annexin V双染流式细胞仪分别检测了细胞周期和细胞凋亡。在各宫颈癌细胞系中,与对照组相比,CIP2A干扰组未出现明显的细胞衰老现象,细胞周期也没有明显变化。在凋亡检测中,虽然Caski和HeLa的CIP2A干扰组出现了比对照组高6-12%的细胞凋亡,但是差别并不显著,凋亡细胞群和正常细胞群也没有明显的分界。这部分的结果显示,干扰CIP2A之后宫颈癌细胞的生长繁殖能力和恶性表型显著下降,说明CIP2A促进了宫颈癌细胞系的生长增殖,但是这种对宫颈癌细胞生长增殖的促进作用并不是通过改变细胞周期的或者抑制凋亡来实现的。三、CIP2A的表达与HPV16 E7的关系以及对Wnt信号途径的影响众所周知,宫颈癌的形成与高危型HPV的持续感染及其癌蛋白的表达密切相关。高危型HPV的癌蛋白E6、E7能够作用于宿主细胞内的一些关键分子,使细胞蛋白表达谱发生变化,从而诱导肿瘤细胞产生。为了分析HPV癌蛋白E7和CIP2A的表达有无相关性,在利用免疫组化分析宫颈癌组织中CIP2A蛋白表达的同时,我们也检测了HPV16 E7蛋白的表达。结果显示在宫颈癌样本中HPV16 E7和CIP2A的表达不论在数量上或者是位置上都有高度的统一性,同时统计学分析说明E7和CIP2A的表达有显著相关性。为了进一步验证CIP2A和HPV16 E7表达的关联性,我们利用HPV16 E7特异性的小干扰RNA转染SiHa细胞,RT-PCR检测表明转染后E7的表达量显著下降,同时CIP2AmRNA水平也明显降低。Western bolt检测显示E7被干扰后CIP2A的蛋白水平也显著降低。接下来我们又利用人原代角质细胞(PHK)和稳定转染了HPV16 E7以及HPV58 E7的PHK细胞检测了CIP2A的表达情况,发现不论在mRNA水平或者是蛋白水平,稳定表达HPV16 E7的PHK细胞(PHK 16E7)中的CIP2A的表达显著高于PHK细胞。稳定转染HPV58 E7的PHK细胞也得到了同样的结果。最近Wnt/β-catenin途径在宫颈癌中的作用逐渐成为本领域的研究热点。我们利用western blot检测了干扰CIP2A以后Wnt途径的关键蛋白beta-catenin的表达及核转位的情况。分别提取转染CIP2A siRNA和control siRNA的细胞总蛋白,细胞质蛋白和细胞核蛋白。Wenstern blot分析表明在总蛋白,胞质蛋白和核蛋白中,干扰CIP2A表达后beta-catenin的水平均显著增加,说明CIP2A的降低激活了Wnt/β-catenin途径。这部分的实验结果说明,CIP2A是HPV16 E7的靶分子,HPV16 E7能上调CIP2A的表达。此结果可部分解释CIP2A在宫颈癌组织中过表达的原因。在宫颈癌前病变中,HPV多处于游离状态,E6、E7的表达受到HPV早期蛋白E2的抑制；而在宫颈癌中,HPV基因组多数处于整合状态,此时HPV基因组自E2处断裂,从而使E7表达失控,E7表达的上升提高了CIP2A在宫颈癌中的表达水平。此外,CIP2A的减少可促进β-catenin的表达增加以及核转位,显示了宫颈癌细胞中的Wnt/β-catenin途径的激活。综上所述,本研究首次发现了HPV16 E7的一个新的靶分子——CIP2A,并在宫颈癌细胞系中对其做了较为详细的功能学研究,初步探讨了CIP2A的调控机制及其参与的细胞途径。结果显示,癌蛋白CIP2A在宫颈癌组织中过表达并且其表达与HPV16 E7蛋白密切相关,在宫颈癌细胞系中HPV16 E7可上调CIP2A的表达。CIP2A能够促进宫颈癌细胞的生长增殖并且有助于稳定宫颈癌细胞系的恶性表型。CIP2A表达的变化还能够影响Wnt信号途径中的关键蛋白beta-catenin的表达和核转位,说明CIP2A可能参与Wnt途径的调节。这些发现进一步完善和补充了HPV致宫颈癌的发病机制,对于发展宫颈癌的诊断标志,寻找新的药物靶点以及治疗策略具有重要意义。更多还原

【Abstract】 Cervical carcinoma (CC) is one of the most common cancers and a leading cause of death among women worldwide. The number of patients who died from this disease each year was estimated at 200,000. An important percentage of cases occur in the developing world, where in some countries the mortality of CC is next only to that of breast cancer. In our country, there are 130,000 to 150,000 new cases of CC each year among which about 20,000 women die from this disease.At present it has been well established that the infection of Human papillomavirus (HPV) is the main etiology of cervical cancer. HPV is a kind of double-strand DNA virus. HPV can be classified into two groups on the basis of their ability of causing malignant disease:High-risk HPV and Low-risk HPV. Low-risk HPVs produce localized benign warts that do not undergo malignant progression even if left untreated, whereas persistant infection of High-risk HPVs is associated with cervical cancer. Especially HPV 16 and HPV 18 are almost detected in up to 90% of cervical cancers. Till now, the mechanism of by which HPV infection induces cervical cancer is not clarified. It is well-known that persistant infection of HPV give the virus chances to integrate viral DNA into the genome of the host cells. The integration of viral DNA leads to uncontrolled expression of HPV oncoprotein E6 and E7 which is essential for cell transformation. E6, E7 of HPV16 and HPV18 can inhibit the function of p53 and pRb respectively. The inactivation of p53 and pRb result in differential protein expression profile in host cells and disturb the cell signaling pathway. The apoptosis and senescence are repressed, finally the cell transformation happen.Though the skeleton model of HPV infection of HPV inducing cervical cancer has been established, the expression changes of protein in host cells as a result of high levels of E6 and E7 expression after the viral DNA integration are not completely clear. Therefore, new targets of E6 and E7 protein have been continuously identified over the past decade.Cancerous inhibitor of protein phosphatase 2A (CIP2A) is a new-identified oncoprotein which inhibits PP2A activity toward c-Myc serine 62 (S62), and thereby prevents c-Myc proteolytic degradation. In addition to its function in c-Myc stabilization, CIP2A promotes anchorage independent cell growth and in vivo tumor formation. The oncogenic activity of CIP2A is demonstrated by transformation of human cells by overexpression of CIP2A. Importantly, CIP2A is overexpressed in several human malignancies, such as gastric cancer, colon cancer, breast cancer, non-small cell lung cancer, prostatic cancer, esophageal cancer and ovarian cancer. CIP2A is associated with prognosis of breast cancer, lung cancer and ovarian cancer. Whether CIP2A is overexpressed in cervical cancer? Whether its expression is associated with HPV E6 and E7 protein? Is there any cell signaling pathway involved? These questions are still unclear. This study focused on these questions and abtained some preliminary data.Ⅰ. Overexpression of CIP2A in cervical caner We used 23 tissue samples (15 cervical cancer and 8 normal cervical samples) obtained from surgical patients at Shandong Cancer Hospital for mRNA extraction and consequently RT-PCR.We obtained 157 paraffin-embedded tissue blocks (72 cervical cancer,21 CINⅠ,25 CINⅡ,24 CINⅢand 15 normal cervical tissues) from Qilu Hospital for immunohistochemistry staining.CIP2A mRNA expression was found in 11 of 15 (73.3%) cervical cancer samples but none of 8 normal cervical tissues. CIP2A protein was absent in normal, CIN I and CIN II tissues but was present in 3 of 24 (12.5%) CIN III samples and was present in 38 of 72 (52.8%) cervical cancer samples. Meanwhile, we analyzed the correlation of CIP2A and some clinical characteristics. CIP2A protein expression was not associated with age, tumor size, differentiation status, node metastasis or clinical stage.The data of this part showed that CIP2A expression significantly differed among normal, CINⅠ, CINⅡ, CINⅢand cervical cancer samples. The overexpression of CIP2A in cervical cancer indicates that CIP2A may play an important role in driving cells from the precancerous to invasive cancer stage.II. CIP2A promotes c-Myc expression and growth of cervical cancer cells To determine whether CIP2A contributes to the proliferation of cervical cancer cells, we introduced CIP2A-specific siRNA to knock down CIP2A expression in HeLa, SiHa and Caski cells. Efficient depletion of CIP2A protein expressionwas verified by western blot analysis. In agreement with recent findings, CIP2A depletion in these cervical-cancer. CIP2A depletion with siRNA transfection downregulates the expression of c-Myc protein and impairs cell proliferation and cell growth (analyzed by CCK-8 kit) of HeLa, SiHa and Caski cells. Further more, as compared with control siRNA treatment, CIP2A siRNA treatment significantly decreased the formation of foci and colony size as well as the growth in soft agar of these cervical cancer cell lines.For clarifying why CIP2A siRNA treatment impaired the growth of cervical cancer cells, we usedβ-Galactosidase Staining kit to detect cell senescence, flow cytometry propidium iodide stain to analyzed cell cycle, flow cytometry PI/Annexin V stain to analyzed cell apoptosis. To our surprise, as compared with control siRNA treatment, there was no change in cell cycle and no more noticeable senescence and apoptosis in the CIP2A siRNA treatment cells.The data of this part showed that the growth and malignant phenotype of cervical cancer are significantly decreased after depletion of CIP2A, which indicates that CIP2A promote the growth and proliferation of cervical cancer cells while such an promotion is not achieved by changing cell cycle or repressing apoptosis.Ⅲ. CIP2A was upregulated by HPV16 E7 and inhibition of CIP2A activated Wnt signaling pathwayRecent report showed that the main oncoprotein of Helicobactor pylori Cag A upregulated CIP2A in gastric cancer cell lines. Cervical cancer is similar to gastric cancer which is associated with pathogen infection. Further more, HPV also have two important oncoprotein E6 and E7. To determine whether HPV oncoproteins have the ability of regulating CIP2A, we analyzed HPV16 E7 in cervical cancer tissues by using immunohistochemistry. Interestingly, CIP2A and HPV 16 E7 immunoreactivity showed a significantly positive correlation. CIP2A and HPV 16 E7 immunoreactivity was uniform both in location and immunointensity on the same specimens, which suggested an association of HPV16 E7 and CIP2A expression.To investigate the possible association of HPV16 E7 and CIP2A expression, we introduced HPV16 E7-specific siRNA into SiHa cells.we found both mRNA and protein levels of CIP2A significantly decreased after depletion of HPV16 E7. In addition we compared the CIP2A expression of primary human keratinocytes (PHK) and HPV16 E7 stable-transfected PHK (PHK 16E7). Compared to PHK, CIP2A showed higher expression both in mRNA and protein levels.The role of Wnt/β-catenin signaling pathway in cervical cancer attracts many reseachers’attention. In this study, we detected beta-catenin, the key protein of Wnt/β-catenin signaling, in total, cytoplasmic and nuclear protein using western blot assay. We found that beta-catenin increased in all the three protein extract after depletion of CIP2A, which indicated the activation of Wnt/β-catenin signaling.The data in this part showed that HPV16 E7 upregulates the expression of CIP2A. This finding was first reported in our study. Therefore, CIP2A is a newfound target of HPV16 E7. Furthermore, CIP2A may play a role in the regulation of Wnt/β-catenin signaling.In conclusion, we show that CIP2A is overexpressed in cervical cancer, which might result from the regulatory role of HPV16 E7 on CIP2A expression. Moreover, CIP2A plays an important role in the carcinogenesis of cervical cancer by promoting cell proliferation and cell growth. These findings help to understand the regulatory mechanism of CIP2A and untangle the novel function of HPV16 E7. The investigation of CIP2A expression in clinical tissues provides information on its possible use as a candidate biomarker and therapeutic target of cervical cancer.更多还原