HPV18E6蛋白与蛋白激酶R对核因子NF—κBp65表达及磷酸化调控作用比较

Comparing the regulation effects on expression and phosphorylation of nuclear factor NF-κB p65 by HPV18E6 protein and protein kinase R

目的比较人乳头状瘤病毒（humanpapillomavirus，HPV）18型E6蛋白与蛋白激酶R（PKR）对核因子NF-κBp65表达及磷酸化的调控作用。方法分别构建靶向HPV18E6及PKR基因的短发夹结构RNA干扰序列的重组质粒载体，转染HeLa细胞，以实时荧光定量PCR法检测各组细胞HPV18E6、PKR及NF-κBp65mRNA的表达，以WesternBlot法检测各组细胞HPV18E6、PKR、NF—κBp65蛋白及后两者磷酸化型蛋白的表达，采用SPSS15．0软件包单因素方差分析及Newman-Keuls-q检验比较上述指标在各组间的表达差异。结果在沉默HPV18E6基因表达的基础上，pSilencer—RNAi-E6转染组PKRmRNA、蛋白及磷酸化型蛋白的相对表达量均高于pSilencer—NC转染组及空白对照组（P〈0．05），而NF-κBp65mRNA、蛋白及磷酸化型蛋白的相对表达量均低于pSileneer—NC转染组及空白对照组（P〈0．05）；在沉默PKR基因表达的基础上，pSileneer—RNAi-PKR转染组NF-κBp65mRNA、蛋白及磷酸化型蛋白的相对表达量与pSileneer-NC转染组及空白对照组比较差异无统计学意义（P〉0．05）。结论HPV18E6蛋白是HeLa细胞核因子NF—κBp65表达及磷酸化的主要调控者；PKR的表达及磷酸化均受到抑制，对NF—κBp65的调控作用弱。

Objective To Compare the regulation effects on expression and phosphorylation of nuclear factor NF-κB p65 by human papilloma virus 18 subtype E6 （HPV18E6） protein and protein kinase R （PKR）. Methods Constructed two recombinant plasmid vectors which contained shRNA interfering sequence aiming at the targets of HPV18E6 oneogene and PKR gene, and transfected them respectively into HeLa cell,the expression of mRNA of HPV18E6,PKR and NF-κB p65 were determined with real-time quantitative PCR, the expression of protein HPV18E6, PKR, NF-κB p65 and phosphating PKR （P-PKR）, phosphating NF-κB p65 （P-NF-κB p65 ）were determined with Western Blot. The expression differences of these indicators among every group were compared with one-factor analysis of variance and Newman-keuls-q test of statistical software of SPSS15.0 edition. Results Based on interfering the expression of HPV18E6 oncogene, the expression of PKR mRNA and protein and phosphating protein in pSilencer-RNAi-E6 transfection group were higher than those in pSilencer-NC transfeetion group and blank control group（ P 〈 0. 05 ） , but the expression of NF-κB p65 mRNA and protein and phosphating protein in pSilencer-RNAi-E6 transfeetion group were lower than those in pSilencer-NC transfection group and blank control group（ P 〈 0. 05 ）. Based on interfering the expression of PKR gene, the expression of NF-κB p65 mRNA and protein and phosphating protein in pSilencer-RNAi-PKR transfection group, pSilencer-NC transfection group and blank control group had no significant difference（ P 〉0. 05）. Conclusions HPV18E6 protein was the main regulator of expression and phosphorylation of nuclear factor NF-κB p65 in HeLa cell. The expression and phosphorylation of PKR were both suppressed,so PKR regulated NF-κB p65 weakly in HeLa cell.