【作者】 贺晓琪；

【导师】 王泽华；

【作者基本信息】 华中科技大学 ， 妇产科学， 2008， 博士

【摘要】 研究背景和目的:宫颈癌是妇科最常见的恶性肿瘤,发病率在我国妇科恶性肿瘤中居首位。宫颈癌的转移是影响患者预后的重要因素。肿瘤的恶性进展涉及到细胞的异常增殖与凋亡,对细胞外基质的粘附、侵袭以及迁移等过程。Rho GTP酶是一组鸟核苷酸调节蛋白,其介导的细胞运动可能在恶性肿瘤的侵袭和转移中发挥重要作用。近年来研究发现,RhoC与Rho家族其它成员相比,与其下游效应蛋白ROCK有更强的结合力,因而认为RhoC对细胞骨架介导的细胞运动具有更强的调节能力。RhoC的作用可能贯穿于肿瘤转移的全过程,是肿瘤转移过程中的一个重要“开关”基因,值得深入研究。此外,在肿瘤细胞的上皮间质转化过程中也发现有细胞骨架的改变,同时伴随着上皮细胞标记性蛋白E-cadherin的表达下降,而细胞内出现间质细胞标记性蛋白Vimentin,细胞具有更强的迁移和侵袭能力。宫颈癌来源于上皮组织,有研究证实E-cadherin表达减少与宫颈癌的转移密切相关。从而推测上皮间质转化在宫颈癌的转移中起一定作用。由于EMT的表现与RhoC效应的诸多相似性,这种相似性是因为两者之间具有一定的关联,还是彼此独立,值得进一步研究。因此本实验研究目的在于:1.在组织学水平探讨RhoGTP酶与宫颈癌恶性进展的关系。2.在细胞水平和分子水平,比较RhoC在不同宫颈癌细胞株中的表达,分析表达差异与粘附、侵袭和转移的关系。采用RNA干扰技术对RhoC进行沉默,研究RhoC对人宫颈癌SiHa细胞增殖、凋亡、粘附、侵袭等转移相关生物学行为的影响。体外模拟肿瘤恶性进展中,细胞表型转换过程,即上皮-间质转化过程,分析RhoC在此过程中的表达及活性的变化,并探讨沉默RhoC对该过程的影响。方法:1.研究RhoC在宫颈癌组织和细胞株中的表达。利用免疫组化方法和半定量RT-PCR方法检测正常宫颈组织、CIN组织和宫颈癌组织以及宫颈癌SiHa、HeLa和C33a细胞株RhoC蛋白和mRNA的表达,利用体外基质胶粘附实验、Transwell体外侵袭实验和迁移实验比较不同细胞之间粘附率、侵袭能力和迁移能力的差异,初步研究RhoC的表达水平与宫颈癌恶性进展以及与细胞体外粘附、侵袭和迁移能力的关系。2.研究RhoC对宫颈癌SiHa细胞增殖、凋亡和侵袭能力的影响。将RhoC siRNA转入SiHa细胞中,在RhoC基因沉默前后,分别用MTT细胞增殖实验检测细胞增殖活性,Annexin V-PI染色检测细胞凋亡率,体外基质胶粘附实验、Transwell体外侵袭实验和迁移实验比较细胞粘附率、侵袭能力和迁移能力的改变。3.分析上皮间质转化(EMT)对RhoC表达及活性的调控作用以及RhoC表达下调对上皮间质转化的影响。利用TGFβ1体外诱导宫颈癌细胞株SiHa发生上皮间质转化,以上皮表型的标志蛋白E-cadherin和间质表型的标志蛋白Vimentin免疫荧光染色和western blot方法检测的蛋白含量作为监测EMT的指标。western blot和Pull down实验检测EMT前后RhoC表达和活性的改变,同时利用体外侵袭实验比较SiHa细胞侵袭能力的变化。在TGFβ1刺激细胞前后,分别在细胞内转入RhoC siRNA,western blot检测E-cadherin和Vimentin的表达变化,观察EMT进程的变化。结果:1.RhoC在正常宫颈组织、CIN组织和宫颈癌组织中的表达逐渐增高,在有淋巴结转移的宫颈癌组织中的表达明显高于无淋巴结转移组织。在SiHa细胞和HeLa细胞中的表达明显高于C33a细胞,前两者的粘附率、侵袭能力和迁移能力明显高于后者,而在SiHa细胞和HeLa细胞之间,以上指标均没有明显差异。2.RhoC的沉默可以明显降低SiHa细胞的体外粘附能力和侵袭、迁移能力,但对SiHa的增殖和凋亡没有明显影响。3.在TGFβ1诱导的EMT过程中,SiHa细胞的侵袭能力明显增强,同时,RhoC的蛋白表达及活性明显增强,沉默RhoC可明显降低SiHa细胞的侵袭力,EMT通过提高RhoC的表达及活性增强细胞侵袭能力。抑制RhoC的表达可以阻滞EMT过程,但不能逆转EMT过程。结论:1.宫颈癌中RhoC的表达与宫颈癌的进展和远处转移有关。RhoC主要参与调控宫颈癌细胞的粘附、侵袭和迁移等过程。2.RhoC能够介导TGFβ1诱导的EMT过程中的细胞侵袭能力增强。抑制RhoC的表达能够阻滞EMT的发生。更多还原

【Abstract】 Background and purpose:Cervical cancer is the most common malignant tumor in gynecology. The incidence of cervical cancer has become the first place among the female malignant tumors. Metastasis is an important facet that influences the patients’prognosis. Malignant progression of tumor involves cellular abnormal proliferation and apoptosis, adhesion to extracellular matrix, invasion and migration. Rho GTPases, a group of guanine nucleotide binding proteins, may play a pivotal role in tumor invasion and metastasis by mediating cell motility. Moreover, some recent studies showed that, compared to other members of Rho GTPase superfamily, RhoC has stronger binding affinity to its downstream effector ROCK. Thus, RhoC may have stronger ability in regulating cytoskeleton-mediated cell motility. RhoC might be involved in the whole tumor metastasis process, and function as a critical“switch”gene. In addition, during the tumor cells’epithelial-mysenchymal transition progress, cytoskeleton is also reorganized, accompany with decreased expression of epithelial phenotype mark protein, E-cadherin, meanwhile, the tumor cells that express the mesenchymal phenotype mark protein, Vimentin, show stronger migratory and invasive capability. Cervical caner stems from epithelial tissue. Previous research works demonstrated that reduced expression of E-cadherin and metastasis of cervical cancer is closely related. Therefore, we hypothesized that epithelial-mesenchymal transition may affect the metastasis of cervical cancer. The resemblance between EMT (epithelial- mesenchymal transition) manifestation and RhoC effect leads us to further explore whether these two phenomena are closely related or are independent of each other?Objective:1. To explore the relationship between RhoC and cervical carcinoma malignant progress at histopathological level.2. To study the expression of RhoC in different cervical cancer cell lines SiHa, HeLa and C33a and to compare the adhesive rate,invasive and migratory capability. To study the effect of down-regulated RhoC on SiHa (human cervical cancer cells) biological behaviour, including proliferation, apoptosis, adhesion and invasion, by using RNAi to silence RhoC expression at cellular and molecular level; To analyze RhoC expression level and activity variation during in vitro tumor malignant progress, and in vitro cellular phenotype transition (epithelium-mesenchymal transition); To examine the effect of down-regulation of RhoC on tumor malignant progress, and on in vitro cellular phenotype transition.Methods1. Studying RhoC expression level in cervical cancer tissues and cell line. The level of RhoC mRNA and RHOC protein in normal cervical tissue, CIN tissue, cervical cancer tissue, and cervical cancer cell lines, including SiHa, Hela and C33a, were measured by immunohistochemistry and semi-quantitative RT-PCR. We also compared the adhesive rate, invasive capability and migration capability between different tissues and cells by using Matrigel adhesive assay, transwell invasive assay and migration assay. This study is an initial study on exploring the relationship between RhoC expression level and cervical malignant progress, in vitro cellular adhesion, invasion and migration ability.2. Studying the effects of RhoC on SiHa cells proliferation, apoptosis and invasion. In order to down-regulate or silence RhoC expression, we introduced RhoC siRNA into SiHa cells. Then we compared the following indications before and after RhoC siRNA treatment in SiHa cells: cellular proliferation activity by using MTT cell proliferation assay, cell apoptosis rate via Annexin V-PI staining, cell adhesive rate by using Matrigel adhensive assay, and cell invasive capability and migration capability by using Transwell invasive assay and migration assay respectively. 3. Analyzing the effects of epithelial-mesenchymal transition (EMT) on RhoC expression and activity, and vice versa. We used TGFβ1 to induce epithelial-mesenchymal transition in SiHa cells. Then, using immunofluorescent staining, we detected the levels of E-cadherin and Vimentin. The corresponding protein levels were measured by western blot. Also, using western blot and Pull-down assay, we compared the RhoC expression level and activity before and after EMT. At the same time, we compared the SiHa cell invasive capability before and after EMT by using in vitro invasion assay. Furthermore, before and after TGFβ1 stimulated , we introduced RhoC siRNA into SiHa cells, and compared the expression level of E-cadherin and Vimentin protein with western blot. EMT processes were observed.Results:1. The expression level of RhoC was gradually increased in normal cervical tissue, CIN tissue, and cervical cancer tissue. Moreover, the expression level of RhoC was significantly higher in cervical cancer tissue with lymph node metastasis than that without metastasis. RhoC was expressed higher in SiHa and HeLa cells than in C33a cells. The adhesive rate, invasive capability and migration capability in SiHa and HeLa cells were obviously higher that those in C33a cells. But, no significant differences between SiHa and HeLa cells were observed.2. Down-regulation or silence of RhoC was able to dramatically decrease the SiHa cells in vitro adhesive ability, invasive capability and migration capability, but no significant effect on SiHa cells proliferation and apoptosis.3. During TGFβ1-induced EMT progress, the invasive capability of SiHa cells was enhanced. Meanwhile, the expression level and activity of RHOC protein were increased. Down-regulation of RhoC can dramatically decrease SiHa cell invasive capability, while EMT can enhance invasive capability through increasing RhoC expression and activity. Therefore, inhibition of RhoC expression can block EMT process, instead of reversing it.Conclusion:1. The expression level of RhoC is related to cervical cancer progress and distant metastasis. RhoC is mainly involved in cervical cancer cells adhesion, invasion and metastasis. 2. RhoC is capable of mediating TGF-β1-induced EMT progress to promote cells invasion capability. Thus, the conclusion that we can draw here is inhibition of RhoC expression is able to block the occurrence of EMT.更多还原