The Association Of The Expression Of△Np63α And The Differentiation Of The Cervical Cancer Cell

Background:Cervical cancer, one of the most malignant tumors, is a severe threat to women's health. Till now, the pathogenesis is still not fully understood, which is associated with early marriage, early age at first intercourse and multiple sexual partners, early age at first childbirth, too much birth, smoking, poor economic conditions, geographical factors, viral infection and other factors, the most risk factor of them is the persistent infection of the high-risk human papillomavirus (HR-HPV), but the exact mechanisms has not been clearly clarified. Generally, it is believed that the progress of cervical cancer is closely related to the expression of viral oncoprotein E6and E7, which causes the inactivation of p53and Rb protein, abnormal activation of telomerase activity.One of the most important mechanisms of carcinogenesis of cervical epithelial cells is the inactivation of p53protein, and it has been reported that80%of cervical cancer cells with wild-type p53protein. Therefore, an important strategy for the biological treatment of cervical cancer is to restore the expression of wild-type p53. The effects are not shown as expected, as a large number of E6protein expressed in the HR-HPV in cervical cancer cells, the restoration of wild type p53protein is rapidly degradated by E6/E6AP complex.With the discovery of the other p53family's members such as p63and p73, more and more scholars realized that p63, one member of p53family, is closely related to the regulation of the differentiation of epithelial cells, and may play an important and key role in the pathogenesis of epithelial tumors (such as head and neck squamous cell carcinoma).As the p53family members, p63is located at3q27.29. The function of p63has been complicated by the existence of multiple different subtypes, or isoforms of the protein. According to the different N-terminal, and C-side of p63protein, there are the main following six subtypes of p63as TAp63a TAp63β,TAp63γ, ΔNp63a, ΔNp63p, ΔNp63γ. The function of p63has been complicated by the existence of multiple different subtypes, or isoforms of the protein. The current study has reported as follows:①Regulation tissue development. Recent study has shown the most significant difference between p63and p53function lies in the induction of tumor formation by p53mutations; while the development of congenital disorders such as malformations of the limbs and mesodermal dysplasia by p63mutations.②Inhibition of tumor metastasis. p63can bind to Smad to form complexes, and inhibit TGFβ-induced tumor cell metastasis; Furthermore, tumor cells invasion and metastasis-related gene expression, which contributes to the transformation of epithelial cells to mesenchymal cells (Epithelial-mesenchymal transformation, EMT), can downregulate the expression of p63and increase cell migration ability.③Regulation epithelial cell differentiation. Squamous epithelial tissue is originated in the basal cell, which can differentiate into the cells of other layers. Squamous epithelial tissue is mainly composed of the basement membrane, basal cell, sub-basal cell, middle cell, and keratinocytes. The basal cell, also known as reserve cells or the cervical stem cells, has the potential of both differentiation and proliferation. p63, one of the most important regulatory genes, can induce differentiation of the basal cell. Recent studies have found that the carcinogenic mechanism is associated not only with the inhibition of apoptosis and proliferation abnormality but also the blockage of cell differentiation.Compared with the differentiation outcomes of the other types cell, the most distinct outcome of squamous cell differentiation lies in the formation the keratinocytes, which the progress also is known as cornification or keratinization). Besides, there are many similarities in the morphological characteristics of keratinocytes and apoptotic cells; so that keratosis is considered as another form of apoptosis of the squamous cell. As a result, any steps during the squamous cell differentiation are interrupted (such as abnormal expression of p63, the key regulatory genes of differentiation) will lead to cell keratosis obstacles, be away from the death fate, and keep out of control of the cell cycle, which ultimately contributes to cells malignant transformation.④Other roles:p63also plays roles in regulation apoptosis, cell cycle, cell proliferation and so on.Involucrin is the early marker of the terminal differentiation of the epithelial tissue. Involucrin could be detected in the cell membrane, cytoplasm, once the basal cell begins to differentiate, they will become large and form the cell cross-linking. It has been shown that involucrin is expressed in many epithelial tumors, and its expression level is closely connected with tumor cell differentiation. Involucrin is highly expressed in well-differentiated tumor cells, while is significantly reduced in poorly differentiated tumor cells, and can be used as tumor cell differentiation marker.Since p63is a key regulatory gene of the differentiation of epithelial cells, while involucrin is important to identify the differentiation of epithelial cells and epithelial tumor cells, the aim of this paper is to make out the predominant isoform of p63expressed in normal cervical epithelial tissue and cervical cancer tissue, and then clarify the expression levels of the identified isoform of p63in the cervical cancer tissue, together with the association of p63with cervical cancer cell differentiation.This study is divided into two parts:Part Ⅰ:The subtype of p63expression in the normal and cancerous cervical tissue;Part Ⅱ:The association of the expression levels of p63with the differentiation of cervical squamous cell carcinoma and its clinical significance. The subtype of p63expression in the normal and cancerous cervical tissueObjective:1. To explore the subtype, expression levels, local sites of p63protein in normal cervical tissue;2. To explore the subtype, expression levels, local sites of p63protein in cervical cancer tissue;3. To explore the subtype, expression levels of p63protein in cervical cancer cell lines.Methods:A total of134cervical tissue samples were used in this study at the Anhui Provincial Hospital, Hefei, China. A total of104cases of squamous epithelial carcinomas consisted of type la (21patients), Ⅰb (28patients) and Ⅱa (55patients). Of the104cases that were analysed,29cases were diagnosed as well differentiated squamous cell cancer,29cases were diagnosed as moderately differentiated squamous cell cancer, and46cases were diagnosed as poorly differentiated squamous cell cancer. A total of30cases of adenocarcinoma consisted of type la (6patients), Ⅰb (8patients) and Ⅱa (16patients). Of the30cases that were analysed,6cases were diagnosed as well differentiated squamous cell cancer,7cases were diagnosed as moderately differentiated squamous cell cancer, and17cases were diagnosed as poorly differentiated squamous cell cancer. Additionally,79cases of normal cervical tissue from patients who underwent hysterectomy for reasons other than neoplasias of the cervix or endometrium were collected and used as normal controls in this study. human cervical cancer cell lines included HeLa, SiHa, CaSki, C-33A, ME-180, and MS751. To explore the following studies with p63subtype-specific detection of antibodies:①immunohistochemical detection of the subtype, expression levels, local sites of p63in normal cervical tissue and cervical cancer tissue;②Western blot detection of the subtype, expression levels of p63in cervical cancer cell lines;③RT-PCR detection of the subtype, expression levels of p63in cervical cancer cell lines. Results:1. The expression of p63in the normal cervical epithelium and its predominant isoforms.p63protein is specifically expressed in the basal and sub-basal cells of the normal cervical squamous epithelium, while is negative expressed in normal cervical glandular epithelium, connective tissue, middle cells and keratinocytes of the cervical epithelial tissue in all of79cases of samples by detection with4A4antibody which can recognise all subtypes of p63.10cases of79samples were used for further study. The different antibodies against peptides representing AN terminal region, a terminal region, TA terminal region, and y terminal region of p63were used to explore the predominant isoforms expressed in normal cervical epithelium by immuno-histochemstry. The antibodies specific for ΔN and a isoforms can detect p63-specific expression in the basal and sub-basal cells of the normal cervical epithelium.2. ΔNp63a is expressed in the cervical squamous cell carcinoma tissue, not in the cervical adenocarcinoma tissue.134cases of cervical cancer samples were used in this study, we found that p63was expressed in35cases, lowly expressed in39cases, and negative expressed in30cases of cervical squamous cancer; while p63is negative expressed in all of the30cases of cervical adenocarcinoma.10cases of104samples were used for further study. The different antibodies against peptides representing AN terminal region, a terminal region, TA terminal region, and γ terminal region, and all subtypes of p63were used for detection the expression of p63in cervical cancer tissues. We found that ΔN and a antibody for detection p63were positive in the cervical squamous cell carcinoma.3. The expression of ΔNp63a is reduced in cervical cancer cell linesThe TA, ΔN, α, γ subtypes of p63mRNA fragments can be detected in these6cervical cancer cell lines, and ΔN and a subtype were the predominant isoforms of p63expressed in these cells. p63was expressed in MS751, ME-180in CaSki cells, decreased in SiHa cells, and negative expressed in HeLa and C33-A cells by Western Blot detection with4A4antibody. In further study, we found that ΔN and a subtype were the predominant isoforms of p63expressed in MS751, ME-180in CaSki cells.Conclusion:1. ΔNp63a is the predominant isoform of p63protein expressed in normal cervical squamous epithelium, cervical squamous cell cancer tissue, and cervical cancer cell lines.2. p63is not expressed in the normal cervical glandular epithelium and the cervical adenocarcinoma. The association of the expression levels of p63with the differentiation of cervical squamous cell carcinoma and its clinical significanceObjective:1. To investigate the expression levels of p63in cervical squamous cell carcinoma, and its correlation with the clinical staging and cell differentiation.2. To investigate the positive correlation of p63and cervical squamous cell differentiation by using involucrin, one of squamous cell differentiation markers as the control.Methods:A total of134cervical tissue samples were used in this study at the Anhui Provincial Hospital, Hefei, China. A total of104cases of squamous epithelial carcinomas consisted of type la (21patients), Ib (28patients) and Ila (55patients). Of the104cases that were analysed,29cases were diagnosed as well differentiated squamous cell cancer,29cases were diagnosed as moderately differentiated squamous cell cancer, and46cases were diagnosed as poorly differentiated squamous cell cancer. A total of30cases of adenocarcinoma consisted of type Ⅰa (6patients), Ⅰb (8patients) and Ⅱa (16patients). Of the30cases that were analysed,6cases were diagnosed as well differentiated squamous cell cancer,7cases were diagnosed as moderately differentiated squamous cell cancer, and17cases were diagnosed as poorly differentiated squamous cell cancer. Additionally,79cases of normal cervical tissue from patients who underwent hysterectomy for reasons other than neoplasias of the cervix or endometrium were collected and used as normal controls in this study. human cervical cancer cell lines included HeLa, SiHa, CaSki, C-33A, ME-180, and MS751. To explore the following studies with p63all subtype detection of antibody (4A4):①immunohistochemical detection of the expression levels of p63in the high, middle, low differentiated cervical squamous cancer tissue; And SPSS13.0analysis the correlation of the expression levels of p63and the differentiation and clinical stages of the cervical squamous cancer tissue.②immunohistochemical detection of the expression levels of p63and involucrin in the high, middle, low differentiated cervical squamous cancer tissue;③Western blot detection of the expression levels of p63and involucrin in cervical cancer cell lines;④Immunohistochemical detection of the expression of p63and involucrin in serial slides of the pathology specimens with both normal cervical squamous epithelial tissue and cervical squamous cell carcinoma differentiated from G1to G3.Results:1. The association of the expression level of p63and clinical characteristics of the patients and tissue differentiation gradesBy detection with4A4antibody which can recognize all subtypes of p63, we found that p63was100%(79/79) expressed in all cases of samples; while p63was33.65%(35/104) expressed in104cases of cervical squamous cancer, suggesting that there was significant difference between the normal cervical tissue and the cervical squamous carcinoma (p<0.001). To analyze the104cases of cervical squamous cancer, we found that p63was29/29(100%),5/29(17.24%),1/46(2.17%) expressed in high differentiation, middle differentiation, poor differentiation tumor tissue respectively; p63was13/21(61.90%,10/28(35.71%),12/55(21.82%) expressed in the tumor tissues at Ⅰa,Ⅰb,Ⅱa stages respectively.2. The expression of p63,involucrin in the normal and cancerous tissues, cervical cancer cell linesIn all of the79cases of the normal cervical squamous epithelium, involucrin is mainly expressed in the middle and surface of squamous epithelial cells, which are mature differentiated epithelial cells, while p63is expressed in the basal and sub-basal cells. The expression of both p63and involucrin was significantly reduced in poorly differentiated tissues. There is positive correlation for the expression of p63and involucrin in the cervical cancer cells; to analyze the104cases of cervical squamous cancer, we found that involucrin, p63was100%expressed in the29cases of the high differentiation tissues, lowly expressed in the29cases of the middle differentiation tissues, and negatively expressed in the45cases of all the46cases of the poor differentiation tissues. The expression of p63and involucrin is detected and decreased gradually in ME-180, MS751, and CaSki cells.3. The expression of p63,involucrin in the cervical squamous cancerous tissue with both normal cervical squamous epithelial tissue and cervical squamous cell carcinoma differentiated from G1to G3As for the advantages of the pathological slides in the observation of the progress of cell malignant transformation, we detected the expression of p63and involucrin in serial slides of the pathology specimens with both normal cervical squamous epithelial tissue and cervical squamous cell carcinoma differentiated from G1to G3, and found that the expression of p63was gradually down-regulated, and positively related with that of involucrin during the invasion and the reduction of cell differentiated status of the cervical squamous cancer in all of the10cases of representative specimen.Conclusions:1. The expression of p63in cervical squamous cancer tissue is significantly decreased, and the expression levels of p63were associated with the grades and TNM stage of the tumors.2. The expression of p63is positively correlated with the grades of cervical squamous cancer, and is another molecular marker of cervical squamous cell differentiation status.