HPV16 E6-295T/G和E6-350T/G变异位点影响IFNκ、MHC-Ⅰ、STAT1和IRF9蛋白质表达的研究

Study on the influence of HPV16 E6-295T/G and E6-350T/G variation sites on the protein expression of IFNκ、MHC-Ⅰ、STAT1 and IRF9

目的探讨HPV16(Human Papillomavirus Type 16)E6-295T/G和E6-350T/G变异位点对IFNκ、MHC-Ⅰ、STAT1和IRF9蛋白质表达的影响。方法在宫颈癌细胞C33A(HPV阴性)中分别转染真核表达载体295G/350G-GV230、295T/350G-GV230和295T/350T-GV230作为实验组,以NC-GV230作为对照组。通过免疫组织化学法、Western blot方法检测HPV16 E6295T/G、350T/G不同变异位点对IFNκ、MHC-Ⅰ、STAT1和IRF9蛋白质表达的影响。通过IBM SPSS Statistics 26软件对实验结果进行统计学分析,P<0.05为差异具有统计学意义,免疫组织化学法定量采用image J软件进行阳性细胞计数。结果Western blot结果显示,免疫分子干扰素κ(Interferon Kappa,IFNκ)的表达,HPV16 E6-295G/350G变异组较HPV16 E6-295T/350T原型组和HPV16 E6-295T/350G组降低(P<0.01,P<0.05)。HPV16 E6-295G/350G变异组的免疫分子主要组织相容性复合体I类(Major Histocompatibility Complex Class I,MHC-I)的表达显著低于HPV16 E6-295T/350T原型组和HPV16 E6-295T/350G组(P<0.05)。HPV16 E6-295G/350G变异组的信号转导子和转录激活子1(Signal Transducer and Activator of Transcription 1,STAT1)表达显著低于HPV16 E6-295T/350T原型组和HPV16 E6-295T/350G组(P<0.05,P<0.01)。变异组HPV16 E6-295G/350G与HPV16 E6-295T/350T原型组和HPV16 E6-295T/350G变异组相比,干扰素调节因子9(Interferon Regulatory Factor 9,IRF9)表达显著降低(P<0.05,P<0.01)。结论HPV16病毒E6蛋白质可以降低IFNκ、MHC-Ⅰ、STAT1和IRF9的蛋白质表达水平,E6基因T295G/T350G变异降低这些分子的表达作用最强。

Objective To explore the effect of variation sites E6-295T/G and E6-350T/G on the protein expression of IFNκ、MHC-Ⅰ、STAT1 and IRF9.Methods Cervical cancer cells C33A were transfected with eukaryotic expression vectors 295G/350G-GV230,295T/350G-GV230,295T/350T-GV230 as the experimental group and NC-GV230 as the control group.Immunohistochemistry and Western blot methods were used to detect the effects of HPV16 E6295T/G and 350T/G variation sites on IFNκ、MHC-Ⅰ、STAT1 and IRF9.IBM SPSS Statistics 26 software was used for statistical analysis of the experimental results.P<0.05 was considered statistically significant.Immunohistochemical method was used to quantitatively count positive cells by image J software.Results The results of Western blot showed that the expression of IFNκwas decreased in the HPV16 E6-295G/350G variant group compared with the HPV16 E6-295T/350T prototype group and HPV16 E6-295T/350G(P<0.05,P<0.01).The expression of MHC-I in the HPV16 E6-295G/350G variants was significantly lower than that of HPV16 E6-295T/350T prototype group and HPV16 E6-295T/350G(P<0.05).The expression of STAT1 was significantly lower in HPV16 E6-295G/350G variant group than in HPV16 E6-295T/350T prototype group and HPV16 E6-295T/350G(P<0.01,P<0.05).IRF9 in HPV16 E6-295G/350G was significantly decreased than that of HPV16 E6-295T/350T prototype group and HPV16 E6-295T/350G(P<0.05,P<0.01).Conclusion HPV16 E6 protein can reduce the expression of IFNκ,MHC-Ⅰ,STAT1 and IRF9,and E6 gene 295G/350G variation has the strongest effect on reducing the expression of these molecules.