| Background and purpose:Condyloma Acuminata (CA) is a benign neoplasm of the skin and mucosa, which is caused by the human papillomavirus (HPV). It mainly occurs in young and middle-aged people, and is one of the most common sexually transmitted diseases. Recently, its morbidity has risen fast. CA has a long incubation period, strong infectivity, a rapid growth rate, a high recurrence rate, and a long treatment time. In some patients, CA begins by growing rapidly in a short period of time and eventually can cover large areas of skin. A few cases of CA also may become malignant. CA can be very damaging to the physical and psychological health of the patients and has a bad effect on society and families. Therefore, it is of vital importance to study its pathogenesis for the prevention and treatment of C A. At present, a large number of researches have confirmed that increased proliferation and abnormal apoptosis of keratinocyte are related to the tumorigenesis and progression of CA. However, the pathogenesis underlying it has not yet been clarified.Caveolae is a 60-80nm invagination of the plasma membrane which mediates cellular internalization and endocytosis. It is also the center of cellular signal transduction and the region where plasma membrane recognize, separate, and transmit signals from outside the cell.Caveolin-1 (Cav-1) is the essential structural and symbolic protein of Caveolae, and it mediates the formation of Caveolae. The caveolin-1 gene is located in human chromosome 7q31.1-31.2. Cav-1 protein’s molecular weight is 22kD and consists of 178 amino acids. The center of the main structure includes highly conserved hydrophobic domains and the two sides contain variable N-terminal, C-terminal regions, which look like a hairpin structure in the plasma membrane. Cav-1 is widely expressed in many kinds of cells, such as epithelial, endotheliocyte, adipocyte, and fibroblast to name a few. The formation of the amino acids of the N-terminal looks like scaffolding, so it is also called the scaffolding domain. The Cav-1 scaffolding domain mediates signal transduction pathways by interacting with a wide range of specific proteins that are localized in lipid rafts and caveolar membranes, such as epidermal growth factor receptor (EGFR) and endothelial nitric oxide synthase (eNOS). And Cav-1 takes an important part in the many physiological, pathological processes by participating in cell proliferation, apoptosis, differentiation, migration, and angiogenesis. Furthermore, it has a close relationship with pathogen infection, diabetes, atherosclerosis, systemic sclerosis, and the stages of cancer. Current research on the relation between Cav-1 and cell proliferation as well as apoptosis indicates that Cav-1 could regulate the proliferation and apoptosis in many kinds of cell, and its altered expression participate in the pathogenesis of many diseases. In some diseases, Cav-1 protein expression is closely correlated to the clinical parameters of the patients. The expression of Cav-1 can be used as a biological marker. At present, there is no research about whether the expression of Cav-1 is altered in CA, whether it takes part in the abnormal proliferation, apoptosis of keratinocyte and the pathogenesis of CA, and whether its expression is correlated with the CA patient’s clinical parameters.Initially, we detected the expression of keratinocytes’Cav-1 in CA, and compared Cav-1 expression in CA and in normal skin. Then we analyzed the relationship between Cav-1 expression in keratinocytes and clinical parameters of CA. We want to know whether Cav-1 takes part in the pathogenesis of CA and whether it can be used as biological marker of CA.To reveal the function of Cav-1 in the pathogenesis of CA, we contrasted the keratinocytes’proliferation index the apoptosis index in both CA and in normal skin, then analyzed the correlation between keratinocytes’Cav-1 expression, proliferation index, and apoptosis index in CA.In order to explain the relationship between Cav-1 and keratinocyte proliferation apoptosis, we decreased the expression of Cav-1 in Hacat cells and detected the influence of down-regulation Cav-1 expression to the proliferation and apoptosis of Hacat cells.Method:1. Collect the tissue samples and clinical data of CA patients. The expression and location of Cav-1 protein in keratinocyte of CA was investigated by immunohistochemistry. We used Image-Pro Plus 6.0 software to measure the expression of Cav-1 protein by analyzing the average absorbance. We used the dispase to segregate the epidermis from the whole skin of CA. Then, western blot was conducted to verify the expression of Cav-1 protein. Next, real-time PCR was performed to check the mRNA quantity of Cav-1 in the keratinocytes of C A. Finally, we analyzed the relationship between keratinocytes’Cav-1 expression and clinical parameters of CA. Normal males’foreskin were taken as a control.2. Immunohistochemistry was used to mark the proliferating cell nuclear antigen and detect the proliferating keratinocytes then, the proliferative index was calculated. TUNEL was performed to detect the apoptotic keratinocytes and the apoptotic index was calculated. Finally, we analyzed the correlation between the expression of Cav-1 and the proliferative index as well as the apoptotic index in keratinocyte. The normal male foreskin were again used as controls.3. Three siRNA targeting to Cav-1 were designed and transfected into Hacat cells using liposomes. Then, fluorescence quantitative PCR was performed to find the most efficient siRNA and the optimum concentration. Hacat cells were divided into three groups:experimental group transfected with Caveolinl-siRNA, negative control group transfected with the nonspecific siRNA, and normal control group without any treatment. Western blot and immunofluorescence were conducted to detect the protein expression levels of Caveolin-1 in Hacat cells. MTS assay was performed to evaluate the proliferation of the cells at 24,48, and 72 hours after transfection. Flow cytometry was used to detect cell apoptosis at 48 hours after transfection.4. Statistical analysis:All statistical analyses were performed using SPSS 20.0. All data were expressed as mean ± standard error of the mean. The statistical significance of the difference in the expression of Cav-1, PI, and AI between normal foreskin and CA patients was assessed using two independent sample t-test. Correlation coefficients of Cav-1 expression with PI, AI were determined using Pearson correlation analysis. The correlation between Cav-1 expression and clinical parameters was evaluated using two independent category sample t-tests or a one-way ANOVA test (the categories were above 2 kinds). The comparison of the 3 cell group adopted a one-way ANOVA test (the multiple comparison between 2 groups adopted LSD method). A two tailed test was performed, and statistical significance was defined as P<0.05.Result:1. The expression of Cav-1 in CA keratinocyte and its relationship with clinical parameters:1.1 The result of the Cav-1 immunohistochemistry:Cav-1 protein is expressed in the cytoplasm and cytomembrane of the keratinocyte. Cav-1 was detected in the keratinocyte of both CA and normal skin. In CA tissues, Cav-1 staining was mainly detected in the basal layer, while few Cav-1 proteins were expressed in spinous and granular layers. However, Cav-1 was strongly expressed in all epidermal layers in normal foreskin. The average absorbance of Cav-1 in the keratinocyte of CA and normal skun were (0.042±0.021) and (0.181±0.075), a statistically significant difference (t= 5.843, P<0.001).1.2 The result of the western blot:The expression of Cav-1 proteins were much lower in CA than normal foreskin, a statistically significant difference (t=7.533,P< 0.05).1.3 The result of real-time PCR. The expression of Cav-1 mRNA was much lower in CA than normal foreskin, a statistically significant difference(tF=4.135,P< 0.05).1.4 The correlation between Cav-1 expression of keratinocyte and clinical parameters in CA:No significant correlations were found between Cav-1 expression and the patients’gender, age, onset, location, course, HPV type, and relapse (all P> 0.05).2. The correlation between Cav-1 expression and PI, AI in CA keratinocytes:2.1 The result of PCNA immunohistochemistry:Prolifered keratinocytes were detected in all epidermal layers in CA, and the proliferation index was (63.63±19.86)%. Prolifered keratinocytes were detected in basal and down-spinous layers, and the proliferation index was (23.51±4.00)%, a statistically significant difference(t= 12.44, P<0.001). The correlation analysis showed that PI was correlated negatively with Cav-1 expression(r=-0.798, P<0.05).2.2 The result of TUNEL:Apoptotic keratinocytes were detected in all epidermal layers in CA, and the apoptosis index was (24.12±10.86)%. Apoptotic keratinocytes were detected in the up-spinous layer, and the apoptosis index was (3.13±1.12)%, a statistically significant difference (t= 11.97, P<0.001). The correlation analysis showed that AI was correlated positively with Cav-1 expression(r=0.825, P<0.05).3. The impact to proliferation and apoptosis of the Hactat by down-regulating Cav-1 expression:3.1 The result of real-time PCR screening:Among the three siRNA targeting Cav-1, siRNA-2 at the 25nM concentration was the most efficient, which cut Cav-1 expression 84.32%.3.2 The result of the western blot:After transfected with the siRNA, the protein level of Caveolin-1 significantly decreased in the experiment group when compared to the normal control group and the negative control group(P<0.05).3.3 The result of the immunofluorescence:the fluorescence intensity in the experiment group was significantly lower when compared to the normal control group and the negative control group3.4 The result of MTS:the proliferation levels (represented as the absorbance value at 490 nm) of Hacat cells were (0.563±0.013,0.628±0.006, and 0.811±0.018) in the experimental groups at 24,48, and 72 hours after transfection, respectively, These results were significantly higher than in the normal control group (0.541±0.009, 0.575±0.012, and 0.722±0.004) and negative control group (0.536±0.006, 0.571±0.015 and 0.719±0.002), all P<0.05. No significant differences were observed in the proliferation level between the normal control group and the negative control group (P>0.05).3.5 The result of the flow cytometry:After transfected 48h, the apoptosis rate in the experimental group was (8.90%±0.06%), significantly lower than in the normal control group (20.59%±0.87%) and the negative control group (20.64%±0.09%), both P<0.05. No significant differences were observed in the apoptosis rate between the normal control group and negative control group (P>0.05).Conclusions:The expression of Cav-1 protein and Cav-1 mRNA in keratinocyte were significantly down-regluated in CA when compared to normal skin. No significant correlation was found between Cav-1 expression and the clinical parameters of CA patients; Cav-1 exprsssion of keratinocytes in CA was negatively correlated with PI and positively correlated with AI. Down-regulating Cav-1 expression enhanced the proliferation and supressed the apoptosis in Hacat cells. Decreased Cav-1 expression of keratinocyte in CA may lead to high proliferation and low apoptosis of keratinocytes, which contribute to the pathogenesis of CA. |
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