特异性siRNA抑制HPV16 E7基因表达的实验研究

目的筛选针对HPV16 E7基因有效的siRNA，探讨其对HaCaT E7 细胞中HPV16 E7 mRNA及细胞表面HLA Ⅰ类分子表达的影响。方法采用化学法合成3条HPV16 E7特异性siRNA，应用转染试剂Lipofectamine2000将其转染入HaCaT E7细胞，采用实时荧光定量PCR（RT PCR）检测HPV16 E7 mRNA的表达，采用流式细胞术（FCM）检测细胞表面HLA Ⅰ类分子的表达。结果3条siRNA均能有效抑制HaCaT E7细胞中HPV16 E7的转录表达，以siRNA2作用效果最明显，抑制率达75%，细胞膜表面HLA Ⅰ类分子的表达明显上调，平均荧光强度(MFI)为130.18±1.07，高于空转染对照组（100.32±3.01）和非特异性siRNA对照组（100.82±2.87）。 结论化学合成的HPV16 E7特异性siRNA能有效抑制HaCaT E7细胞中E7 mRNA的表达，同时使细胞表面HLA Ⅰ类分子表达上调。

Specific siRNA inhibits HPV16 E7 gene expression: an experimental study

Objective To screen an effective HPV16 E7 specific siRNA and investigate its effect on expression of HPV16 E7 mRNA and cell surface HLA class Ⅰ antigen in HaCaT-E7 cells. Methods Three HPV16 E7 specific siRNAs were chemically synthesized and transfected into HaCaT-E7 cells by Lipofectamine2000.Expression of HPV16 E7 mRNA was determined by real-time PCR while expression of cell surface HLA class Ⅰ antigen was determined by flow cytometry(FCM). Results All three siR-NAs effectively inhibited expression of HPVI6 E7 mRNA in HaCaT-E7 cells. Of the siRNAs, siRNA2 was the most effective and its inhibitory rate reached 75%. Expression of cell surface HLA class Ⅰ antigen was obviously up-regulated. Post-transfected aver-age mean fluorescence intensity (MFI) of HaCaT-E7 cell surface HLA class Ⅰ antigen was 130.18 ± 1.07, which was significantly higher than that in the mock control group (100.32 ± 3.01) and the non-specific siRNA control group (100.82 ± 2.87). Con-clusion HPV16 E7 specific siRNA can effectively inhibit expression of E7 mRNA in HaCaT-E7 cells and induces up-regulation of cell surface HLA class Ⅰ antigen.