| 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. |
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