Abstract:

Xiong Hao, Li Qian, Liu Qingxiu, Chen Pingjiao, Chen Minghua, Zhang Jing, Gao Yan, Zeng Kang Department of Dermatology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China Corresponding author: Zeng Kang, Email: npfkzk@163.com 【Abstract】 Objective To investigate the of caveolin-1 in keratinocytes of condylomata acuminatum （CA） skin lesions, and to explore its correlation with keratinocyte proliferation and apoptosis. Methods Tissue specimens were obtained from lesions of 40 patients with CA （CA group） and normal skin of 10 healthy human controls （control group）, then fixed with formalin and embedded with paraffin. An immunohistochemical analysis was done using the EnVision system to measure the s of caveolin-1 （expressed as absorbance value） and proliferating cell nuclear antigen （PCNA） in keratinocytes, and terminal deoxynucleotidyl transferase dUTP nick end labeling （TUNEL） assay to evaluate apoptosis of keratinocytes in these tissue specimens. The proliferative index and apoptotic index of cells were calculated. Independent samples t test was conducted to compare these parameters between the two groups, and Pearson correlation analysis to assess the relationship of caveolin-1 with proliferative index and apoptotic index. Three small interfering RNAs （siRNAs） were designed targeting the caveolin-1 gene, and transfected into HaCaT cells separately by using liposomes. Then, fluorescence-based quantitative PCR （qPCR） was performed to detect the of caveolin-1, so as to find the most efficient siRNA. Cultured HaCaT cells were divided into 3 groups: experimental group transfected with caveolin-1 siRNA, negative control group transfected with nonspecific siRNA, and blank control group receiving no treatment. Western blot analysis and immunofluorescence assay were carried out to quantify the protein of caveolin-1 in HaCaT cells at 48 hours after transfection, MTS assay was performed to evaluate proliferative activity of HaCaT cells at 24, 48 and 72 hours, and flow cytometry （FCM） to detect cell apoptosis at 48 hours. Results Compared with the control group, the CA group showed significantly decreased caveolin-1 （0.042 ± 0.021 vs. 0.181 ± 0.075, t = 5.843, P < 0.001）, but increased proliferative index （65.63% ± 19.86% vs. 23.51% ± 4.00%, t = 12.440, P < 0.001） and apoptotic index （24.12% ± 10.86% vs. 3.13% ± 1.12%, t = 11.970, P < 0.001）. The of caveolin-1 was negatively correlated with proliferative index （r = -0.798, P < 0.05）, but positively correlated with apoptotic index （r = 0.825, P < 0.05） in CA lesions. qPCR showed that siRNA-2 at the concentration of 25 nmol/L had the strongest inhibitory effect on caveolin-1 . Western blot analysis and immunofluorescence assay both revealed a significant decrease in the protein of caveolin-1 in the experimental group compared with the blank control group and negative control group at 48 hours after transfection （both P < 0.05）. The proliferative activity of HaCaT cells was significantly higher in the experimental group than in the blank control group and negative control group after transfection （0.563 ± 0.013 vs. 0.541 ± 0.009 and 0.536 ± 0.006 at 24 hours, 0.628 ± 0.006 vs. 0.575 ± 0.012 and 0.571 ± 0.015 at 48 hours, 0.811 ± 0.018 vs. 0.722 ± 0.004 and 0.719 ± 0.002 at 72 hours, all P < 0.05）, while the apoptosis rate at 48 hours was significantly lower in the experimental group than in the blank control group and negative control group （8.90% ± 0.06% vs. 20.59% ± 0.87% and 20.64% ± 0.09%, both P < 0.05）. In addition, no significant differences were observed in the proliferative activity or apoptosis rate between the blank control group and negative control group at any of the above time points （all P > 0.05）. Conclusion The of caveolin-1 is decreased in keratinocytes of CA skin lesions, which may be associated with accelerated proliferation and decreased apoptosis of keratinocytes.