Abstract:

Wang Yongfang, Li Xinyu, Song Shasha, Sun Yang Pharmacal Research Laboratory, Institute of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical College, Nanjing 210042, China Corresponding author: Li Xinyu, Email: xinyusli609@163.com 【Abstract】 Objective To investigate mRNA expressions of apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 family （APOBEC3s or A3s） as well as expressions and subcellular distribution of major A3 proteins in HaCaT keratinocytes carrying the genome of human papillomavirus type 11 （HPV11.HaCaT）, and to evaluate regulatory effects of exogenous interferon-alpha （IFN-α） on the expressions of A3s. Methods The basal levels of A3A, A3B, A3C and A3H mRNA expressions were measured by real-time fluorescence-based quantitative PCR （qRT-PCR） in HPV11.HaCaT cells and normal HaCaT cells. Cultured HaCaT, HPV11.HaCaT and Hela cells were treated with recombinant human IFN-α 2b （rhIFN-α2b） at concentrations of 104, 105 and 106 IU/ml for 6, 24 and 48 hours separately, and those receiving no treatment served as the normal control groups. Then, qRT-PCR was performed to measure mRNA expressions of A3A, A3B, A3C and A3H in these cells. Immunofluorescence staining was conducted to observe the expression and distribution of A3A protein in cells after the treatment with rhIFN-α2b for 6 hours. Results As qRT-PCR showed, the basal levels of A3A, A3B and A3C mRNA expressions were all significantly higher in HPV11.HaCaT cells than in normal HaCaT cells （all P < 0.05）. After stimulation, the mRNA expressions of the four A3 members increased to different extents with the increase in rhIFN-α2b concentrations, and the increase in A3A mRNA was the most significant. Compared with corresponding normal control groups, the mRNA expression of A3A was significantly increased in HaCaT cells （35.77 ± 5.01 vs. 1.00 ± 0.05, P < 0.05）, HPV11.HaCaT cells （15.34 ± 2.14 vs. 0.99 ± 0.01, P < 0.05） and Hela cells （24.60 ± 5.45 vs. 0.97 ± 0.03, P < 0.05） after the treatment with rhIFN-α2b at 106 IU/ml for 6 hours, while the increase in A3B, A3C and A3H mRNA expressions was no more than 9-fold in these cell lines after that. Enhanced staining for A3A was observed in nuclei and cytoplasm of the 3 cell lines after the treatment with rhIFN-α2b at 106 IU/ml for 6 hours. Conclusions HPV11 transfected into HaCaT cells can activate intracellular A3s, especially A3A. IFN-α may play an immunoregulatory role by inducing high levels of A3A expression.