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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siR-NA2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。
Objective To screen siRNAs targeting HPV16 E7 gene and investigate its effect on the expression of HPV16 E7 mRNA and cell surface HLA-Ⅰ molecules in HaCaT-E7 cells. Methods Three HPV16 E7 specific siRNAs were chemically synthesized and transfected into HaCaT-E7 cells using Lipofectamine2000. The expression of HPV16 E7 mRNA was detected by real-time fluorescence quantitative PCR (RT-PCR) (FCM) was used to detect the expression of HLA class I molecules on the cell surface. Results All three siRNAs could effectively inhibit the transcriptional expression of HPV16 E7 in HaCaT-E7 cells. The effect of siR-NA2 was most obvious with the inhibition rate of 75%. The expression of HLA class I molecules on the cell membrane was significantly up-regulated. The average fluorescence intensity MFI) was 130.18 ± 1.07, which was higher than that of control group (100.32 ± 3.01) and non-specific siRNA control group (100.82 ± 2.87). Conclusion The chemically synthesized HPV16 E7 specific siRNA can effectively inhibit the expression of E7 mRNA in HaCaT-E7 cells and up-regulate the expression of HLA class I molecules on the cell surface.