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目的研究人乳头瘤病毒16 E5(HPV16 E5)蛋白对角质形成细胞生长因子(KGF)诱导的细胞自噬的影响。方法构建HPV16 E5蛋白真核表达载体p CI-neo-E5,采用LipofectamineTM2000转染至Ha Ca T人角质形成细胞,实时定量PCR检测HPV16 E5 mRNA转录水平。转染p CI-neo-E5并以KGF刺激Ha Ca T细胞后,实时定量PCR检测自噬相关蛋白beclin 1、自噬相关基因5(ATG5)、ATG7及LC3B mRNA的水平,Western blot法检测LC3BⅡ蛋白的表达量,免疫荧光技术检测LC3B的分布。结果成功构建HPV16 E5真核表达载体p CI-neo-E5并可有效表达。角质形成细胞转染p CI-neo-E5,自噬相关蛋白mRNA的水平显著降低,抑制KGF诱导的LC3BⅡ表达水平升高和LC3B的聚集。结论 HPV16 E5蛋白通过下调自噬相关蛋白的表达,抑制KGF诱导的细胞自噬。
Objective To study the effect of human papillomavirus 16 E5 (HPV16 E5) on autophagy induced by keratinocyte growth factor (KGF). Methods HPV16 E5 protein eukaryotic expression vector p CI-neo-E5 was constructed and transfected into HaCa T human keratinocytes by LipofectamineTM2000. The transcription level of HPV16 E5 mRNA was detected by real-time quantitative PCR. The expression of autophagy-related gene beclin 1, ATG5, ATG7 and LC3B mRNA was detected by real-time quantitative PCR after transfection of p CI-neo-E5 and KGF-stimulated HaCa T cells. Western blot was used to detect the expression of LC3BⅡ Protein expression, immunofluorescence assay LC3B distribution. Results HPV16 E5 eukaryotic expression vector p CI-neo-E5 was successfully constructed and expressed efficiently. Keratinocytes transfected with p CI-neo-E5 significantly reduced the level of autophagy-related protein mRNA and inhibited the KGF-induced LC3BⅡ expression and LC3B accumulation. Conclusion HPV16 E5 can inhibit KGF-induced autophagy by down-regulating the expression of autophagy-related proteins.