| BackgroundCervical cancer is the most common malignancy in female reproductive system,which pose a serious threat to women’s reproductive health.Up till now, the pathogenesis of Cervical cancer has not been elucidated, which may be associated with poor economic conditions, smoking, early age at first childbirth, too much birth, geographical factors, high-risk human papillomavirus (HR-HPV) and other factors, especially the chronic persistent infection of the HR-HPV is the most risk factor of development of cervical neoplasia,suchas HPV-16,HPV-18,but the concrete and exact mechanisms has not been elucidated. In general, many scholars believe that the occurrence and progress of cervical cancer are closely linked with the expression of viral oncoprotein E6, which causes the inactivation of p53protein to promote the occurrence and progress of cancer.p53is not an indispensable protein for cell function. It is activated when the cell is damaged by various factors stimulation and repair the damage by means of inhibition of the progression of cell cycle and promotion of cell apoptosis. After the infection of HPV, cervical epithelial cells will express oncoprotein E6to mediate ubiquitination of p53and its degradation.Which ultimately cause damaged cells to enter the cell cycle and promote cell proliferation and induce the occurrence of tumor. Therefore, increased expression of p53is still an important research direction for molecular targeted therapy of cervical cancer. For example, there is an treatment in tumor cells by means of injecting a recombinant p53adenovirus injection (rAd-p53).With the deepening of the research about the family of p53, a growing number of scholars began to realize that p63, new member of p53family, is closely related to the regulation of cell differentiation of epithelial cell, may play an important role in the process of invasion, metastasis and recurrence in tumor cells of epithelial origin.As the p53family members, p63has a high homo logy with p53in sequence and structure. Because p63has different promoters and splicing modes of introns, p63encodes many kinds of isoforms, and these subtypes play various roles in different tissues and biological functions. But about excat function of p63has not been clearly clarified. Currently reports about its function were reported as follows:(1) Involving in the development of the organization;(2) Regulation of cell cycle and apoptosis. For example, making cell stable expression of TAp63by transfected virus showed that TAp63could block the cell cycle and induce apoptosis. Interestingly, there are some opposite results in the research about△Np63. At the same time, the results showed that TAp63or△Np63α in p53-/-cells can regulate downstream target genes of p53.(3) The role in tumor metastasis and invasion. On the one hand, it could inhibit the epithelial-mesenchymal transition (EMT) and invasion and metastasis in tumor cell through downregulating the expression of the zinc finger transcription factor which is closed related to EMT, such as Snail, Slug and Zebl. On the other hand, p63can regulate the metastasis of tumor cells which is regulated by the TGF β through combining with Smad complexes.(4) Regulation of epithelial cell differentiation. p63activate various differentiation gene promoter of human keratin cells, which control the differentiation of cells.(5) Other biological functions. p63also play many important roles in cell proliferation and tumor stem cells and drug resistance. The subtype△Np63a is dominating express in cervical tissue. Due to the relatively short half-life of TAp63, it is difficult to be detected in cervical tissue. So in this research we used the antibody of p63a to take the place of the antibody of△Np63a detect the expression level of△Np63a in cervical cancer tissue. More than90%of cervical cancer is squamous cell carcinomas, so this research mainly focuses on the study of squamous cell carcinomas. Firstly, we will make it clear that the expression of the ANp63α proteins in squamous carcinoma of the cervix and its significance. Secondly, we will clarify that△Np63α regulate the mechanisms of EMT in cervical squamous carcinoma.This research was divided into two parts:Part Ⅰ:The expression of p63α in cervical squamous carcinoma tissues and its relationship with EMT.Part Ⅱ:Construction of recombined△Np63α lentivirus and its suppression to EMT in cervical cancer cell. Part ⅠThe expression of p63a in cervical cancerous tissue and its association with EMTObjective:1. To explore epithelial-mesenchymal transition in cervical squamous cell carcinoma.2. To detect the expression levels of p63a protein in normal and cancerous cervical squamous cell tissue.3. To explore the association of the expression of p63protein with EMT in cervical squamous cell carcinoma.4. To explore the clinical significance of p63a expression in cervical squamous cell carcinoma.Methods:Neoplastic specimens were collected from128patients diagnosed with cervical squamous cell carcinoma from2000to2003.The tissue samples were obtained from the Department of Pathology, Anhui Provincial Hospital, which is affiliated with Anhui Medical University, Hefei, China. The age rang22-24years old and the median age is45years, the SCC disease stage of each patient was determined based on the International Federation of Gynecology and Obstetrics (FIGO)1994classicatioa All patients with clinical diagnosis of cervical squamous cell carcinoma by two senior gynecologist reference20001FIGO classication for diagnosis, and based on the postoperative pathological diagnosis of squamous cell carcinoma diagnosis. None of the patients had received radiotherapy or adjuvant chemotherapy and follow-up data is integrated. The clinicopathological features of the SCC patients are listed in Tablel. Immunohistochemistry was performed the following research:①To detect the expression of β-catenin、Vimentin in normal squamous cell tissue and cervical cancerous squamous cell tissue, and correlation analysis confirmed the existence of the EMT in cervical squamous cell carcinoma.②To detect the expression levels of p63a in normal and cancerous cervical squamous cell tissue.③Statistical analysis confirmed the relationship between p63a expression in cervical squamous cell carcinomas and EMT.④To analyze the clinical significance of p63a expression in cervical squamous cell carcinoma.Results:1. The expression of β-catenin and Vimentin in normal and cancerous cervical squamous cell tissueIn normal cervical epithelium, distinct expression of β-catenin was observed in the membrane. Among the40cases normal cervical epithelium,33cases (82.9%) showed high expression of β-catenin,5cases (12.5%) showed low expression of P-catenin. In cancerous issue, β-catenin was expressed in cell membrane or cytoplasm. Among the128SCC samples,46cases (35.9%) showed high expression of β-catenin,55cases (43.0%) with low expression of β-catenin. The expression of β-catenin protein in cervical squamous cell carcinoma was significantly lower than normal cervixtissue. The expression of Vimentin was focused in the cytoplasm of mesenchymal cells, but it is negative in normal cervical squamous epithelium.The negative expression rate of Vimentin in cervical is87.5%, which is significantly higher than the negative expression rate in squamous cell carcinoma of the cervix (P<0.05).2. Correlation between the expressions of β-catenin and VimentinSpearman correlation identifies statistically significant between the expression of β-catenin andVimentin, rs=-0.277, P=0.002. Furthermore, we divided the SCC tissues into three groups:Group Ⅰ (24cases):SCC tissues with high expression of β-catenin and negative expression of Vimentin. Group Ⅱ(38cases):SCC tissues with low expression of β-catenin and low expression of vimentin; Group Ⅲ (18cases):SCC tissues with negative expression of β-catenin and high expression of vimentin.5. The expression of p63a protein in cervical squamous cell tissueThe nuclear expression of p63a was determined in normal cervical and SCC tissues, among the40cases normal cervical epithelium,75.0%(30/40) showed high expression of p63a,15.0%(6/40) showed low expression of p63a; In contrast, among the128SCC samples,33.6%(43/128) showed high expression of p63a and46.9% (60/128) showed low expression. There was significant difference in p63a expression in cervical squamous carcinoma tissue and normal cervical tissue (P <0.05).6. The association of the expression of p63a with EMT in cervical squamous cell carcinomaP63a and β-catenin expression levels were inversely associated with P-catenin expression in128SCC samples, whereas the expression levels of p63a was directly associated with β-catenin expression (all P-values<0.05). There was significant difference in p63a expression of the SCC tissues with occurrence of EMT and the SCC tissues without occurrence of EMT (P<0.05). Showing the increase expression of p63a might contribute to the onset of EMT in cervical cancers. We also found that there was significantly difference for the p63a expression among Group Ⅰ,Ⅱ,Ⅲ (all P-values<0.05). Above results indicate that p63a downregulation in cervical squamous carcinoma tissues is closely related to the occurrence of EMT.7. The Clinical Significance of p63a expression levels in the patients’clinical variablesThe expression of P-catenin and Vimentin significantly were closely related to differentiations, lymphatic metastases and postoperative recurrence of the patients with SCC,(all P-values<0.05). There was no difference in expression of p63a expression for the ages, histo logical grade or clinical stages of the patients with SCC. However, nodal metastasis and postoperative recurrence were significantly correlated with p63a expression.8. Survival analysis associationt with p63α, β-catenin, Vimentin protein expressionFollow-up results of128cases of cervical squamous cell carcinoma patients showed that23patients were dead, and5years cumulative survival rate was90%,10-year cumulative survival rate was81%. The mortality rate of patients with high and low expression of p63a was9.3%(4/43) and15.0%(9/60), respectively. However, the mortality rate with negative expression of p63a was40.0%(10/25) The mortality rate of patients with high and low expression of β-catenin was8.7% (4/46) and14.5%(8/55), respectively. However, the mortality rate with negative expression of β-catenin was40.7%(11/27).In contrast,the mortality rate for patients with high expression of Vimentin was41.7%(15/36). However, the mortality rate of patients with low and negative expression of Vimentin was10.2%(5/49) and7.0%(3/43), respectively. Statistical analysis found that the expression of p63a, P-catenin, Vimentin were related to survival rate of SCC patients (P=0.000; P=0.001; P=0.002respectively).9. Univariate Cox regression and Multivariate Cox regression analysis of overall survivalThe univariate cox proportional hazard regression model analysis shown in table7, differentiation degree, lymphatic metastasis and recurrence, the expression of p63a, β-catenin and Vimentin were significantly related to overall survival. Univariate Cox regression survival analysis showed that a low degree of cervical squamous cell differentiation, lymph node metastasis, recurrence and the expression of Vimentin were risk factors for survival, while the expression of p63a and β-catenin were protective factors affect survival (all P-values<0.05). Multiariable Cox regression survival analysis showed that high lymphatic metastasis and relapse, Vimentin protein expressed as independent risk factors affecting survival (all P-values<0.05).Conclusion1. EMT may represent an important molecular alteration during the progression and metastasis in carcinoma of the cervix.2. The expression of p63a in normal squamous cervical epithelial tissue is significantly higher than expression of p63a in squamous cell carcinoma of the cervix. And its expression is closely related to lymphatic metastasis and recurrence of cervical cancer.3. Down-regulation of p63a is closely related to the occurrence of EMT in cervical squamous cell carcinomas.4. The expression of p63α are closely associated with the prognosis of cervical squamous cell carcinoma PartⅡConstruction of re combined△Np63a lentivirus and its suppression to EMT in cervical cancer cellsObjective:1. To explore the predominant subtype of p63a in cervical cancer tissues and somecervical cells;2. Construction of recombined ANp63a lentivirus;3. To confirm the relations hip of ANp63αwith EMT in cervical cancer cell;4. To analyze the mechanism of ANp63a modulation on EMT.Methods:We observed the effects of ANp63a modulation on EMT by constructing of recombined ANp63a lentivirus and infecting C33A and SiHa cell with ANp63a construct (ANp63a) virus.Results1.The expression of p63a, ANp63and TAp63is of or ms in normal and cancerous cervical squamous cell tissueAmong the40cases normal cervical epithelium,30cases showed high expression of p63a,6cases showed low expression of p63a; In contrast, among the128cases SCC samples,43cases showed high p63α expression and60cases showed low expression of p63a. There are28cases with high expression of ANp63and7cases with low expression in40cases normal tissue, while39cases with high ANp63expression and63cases with low expression among the128cases SCC samples, and no case showed high expression of TAp63in normal and cancerous tissue, only22cases showed low expression of TAp63, the other is negative. Among the128cases SCC samples,10cases with high, low and negative expression of p63a were randomly obtained, respectively. We compared the staining ofp63α, ANp63, TAp63in these serial sections. The same staining results were obtained from p63α and△Np63, 30cases showed negative expression of TAp63.2. The expression of△Np63a in HaCaT cell and some cervical cancer cellsThe detection of mRNA fragment of p63isoforms, including p63a.△Np63, by qRT-PCR in HaCaT cell and five cervical cell lines. The results showed the expression of p63a and△Np63is relatively high in HaCaT and ME180cells, while the rest of cervical cancer cells express relatively low level. Further detection of the mRNA level of△Np63、TAp63in6cell line demonstrated that△Np63is the main subtypes of p63isoforms in cervical cancer cells. In addition, the high expression of p63were tested in HaCaT, MS751, CaSki,ME180cells, while low expression of p63in SiHa, C33A cells using Western bloting method to detect p63protein. Further study by detecting the p63antibody isoform, such as p63α、△Np63、TAp63, found that p63α、△Np63is the main subtypes of p63in HaCaT, MS751, CaSki, ME180cells. The above results showed the same results with results of immunohistochemistry, further validate that ANp63a protein is the main isoforms of p63a isoforms in cervical cancer.3. PCR to obtain the target gene fragmentObtaining1761bP gene fragment by agarose gel electrophoresis, which is in conformity with the expected size of TP63transcript variant4(amplification bands around Marke1500bp on a bit of location), showed that the TP63gene has been successfully amplified out.4.Double enzyme digestion successfully constructed recombinantUsing XhoI and BamHI double digestion TP63PCR recycled product and pLVX-IRES-ZsGreenl vector to construct recombinant pLVX-IRES-ZsGreenl+TP63transcript variant4. Digestion products and extraction of plasmid TP63transcript variant4were Separated by1%agarose gel electrophoresis containing ethidium bromide (EB) and a target band appeared in the corresponding position of UVP gel imaging system imaging which is consistent with the theoretical value of DNA fragments of constructed fusion proteins.200ul recombinant plasmid extracted and packed were sent to golden wisdom gene sequencing company and the sequencing results by BLAST analysis showed that gene TP63transcript variant4has been successfully cloned to pLVX IRES-ZsGreenl carrier, which is fully consistent with NCBI BLAST known sequences.The above results show that the recombinant pLVX IRES-ZsGreenl+TP63transcript variant4has been built successfully, which can be used in subsequent experiment.5. The lentivirus was produced successfullyThe lentivirus was produced successfully by293T cells and the titer could reach3.7×108TU/ml TU/ml if concentrating was carried out.6. Expression of△ANp63a after infected with construction of recombined ANp63alentivirusThe successful construction of recombinant lentivirus pLVX-IRES-ZsGreenl+△Np63a were successfully infecteded into C33A and SiHa cells, respectively, then monoclonal screening to obtain stable expression of△Np63a cell line. Detection of lentivirus transfection efficiency, using qRT-PCR method, showed:the level of mRNA of p63in C33A and SiHa cells were significantly increased after infected with construction of recombined ANp63a lentivirus7.△Np63a inhibition of cell invasion, migrationThe C33A and SiHa△Np63a-overexpressing cells became less invasive and migration than the corresponding empty vector infected controls.8.△Np63a suppress EMTPhotos taken under a bright field microscope show changes in cell morphology when△Np63a expression is modulated in SiHa cell. While cell morphology of C33A with△Np63a over-expression cells without obvious morphological changes. qRT-PCR and Western bloting results showed that the mRNA and protein expression levels of Vimentin in both SiHa and C33A cells with over-express ion ANp63a was significantly decreased, β-catenin mRNA was obvious increased in SiHa cell and slightly increased in C33A cell. Similarly, Western bloting results showed the same results with the qRT-PCR except the expression level of β-catenin protein was without any change. qRT-PCR results also showed that SNAI2and ZEB1mRNA was decreased in SiHa, C33A△Np63a-overexpressing cells. In addition, Western bloting results showed that the expression level of Slug protein was decreased significantly and ZEB1protein expression level was slightly decreased in SiHa, C33A cells infected with construction of recombined△Np63a lentivirus, respectively9. The miRNA-205is△Np63a targets for Inhibition of EMT induction in cervical cancer cellsqRT-PCR results showed that the mRNA expression of miRNA-205were increased significantly in SiHa, C33A cells (over expression of ANp63a). To further verify the experimental results, the mRNA expression of Vimentin, SNAI2, ZEB1were decreased significantly and the mRNA expression of β-catenin were increased in C33A、SiHa cells transiently transfected with miRNA-205minic, which are consistent with the discovery in SiHa, C33A cells whit stable high expression of ANp63a. After further transiently transfected miRNA-205antagomir into SiHa, C33A cells with stable high△Np63α expression, it is found that the inhibition of miRNA-205weaken the regulation effect of△Np63a to β-catenin, Vimentin, SNAI2, and the expression of ZEB1increased significantly. The above results suggest that△Np63a inhibits EMT in cervical cancer cells:role of miR-205.Conclusions1.△Np63a was predominant subtype of p63a and showed low or negative expression in squamous cell carcinoma of the cervix and cervical cancer cells2. Successfully constructing the lentivirus recombined of ANp63a;3.△Np63a suppress EMT in cervical cancer cells;4.△Np63a inhibits EMT in cervical cancer cells:role of miR-205. |
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