RNA干扰沉默食管癌细胞株ECA109中人乳头瘤病毒18型E6、E7基因对人类白细胞抗原Ⅱ类分子表达的影响

Down-regulation of human papillomavirus type 18 E6 and E7 expression attenuates human leukocyte antigens-Ⅱ antigens in esophageal carcinoma cells

目的应用RNA干扰（RNAi）技术，使人乳头瘤病毒18型（HPVl8）E6、E7基因表达下调或缺失，观察下调或缺失后的HPVl8阳性食管癌细胞株ECAl09细胞内的人类白细胞抗原（HLA）-Ⅱ类分子表达。方法．将合成并包装好的含有E6、E7基因片段的短发卡RNA（shRNA）腺病毒载体（shpAd—E6-eGFP、shpAd—E7-eGFP）感染食管癌细胞株，并用实时荧光定量聚合酶链反应（FQ—PCR）[E6siRNA组HLA—IImRNA表达量是空白组的1．27倍，E7siRNA组HLA—IImRNA表达量是空白组的1．66倍（P〉0．05]和Westernblot技术[各组ACTIN蛋白条带灰度基本-致，shRNA—E6、shRNA—E7、无关序列和阴性对照组蛋白表达分别为0．5543、0．7246、0．4452、0．4365，siRNAE6、E7实验组灰度稍高于空白组（P〉0．05）及无关序列组]以β—actin为内参照，在基因和蛋白水平上检测E6、E7和胞内HLA-Ⅱ类分子的表达。结果2条含有E6、E7基因片段的shRNA腺病毒载体（shpAd—E6-eGFP、shpAd—E7-eGFP）能有效的抑制ECAl09细胞中HPVl8E6、E7的转录及表达，抑制率分别为92％和89％，细胞内HLA-11类分子的表达上调，分别是空转染组及无关序列组的1．27和1．66倍，无关序列组和空白对照组HPVl8E6、E7及HLA—II的mRNA和蛋白表达差异无统计学意义（P〉0．05）。结论HLA-Ⅱ类抗原表达的缺失可能与食管癌中的HPV感染及作用明显相关。

Objective To investigate the effect of down-regulation of human papillomavirus type 18 （HPV18） E6/E7 by short hairpin RNA （shRNA） on human leukocyte antigens lI （HLA-Ⅱ ） antigens in esophageal carcinoma ceils. Methods The recombinant adenovirus vectors including E6 shRNA, and E7 shRNA were constructed, and transfected into ECA109 cells. The expression of E6, E7 and intracellu- lar HLA- Ⅱ antigen on gene and protein levels was detected respectively by using real-time quantitative pol- ymerase chain reaction （RT-PCR） （ HLA- Ⅱ mRNA expression level in the E6 siRNA group was 1.27 times that of the blank group, and that in the E7 siRNA group was 1.66 times that of the blank group （ P 〉 0. 05） and Western blotting [ the gray scale of actin protein bands in all groups is basically similar, the protein expression in shRNA-E6, shRNA-E7, un-related sequence and negative control groups was 0. 5543, 0. 7246, 0. 4452, 0. 4365 respectively, and the gray scale in siRNA-E6/E7 experimental groups was slightly higher than in blank group （ P 〉 0. 05, P 〈 0. 05 ） and un-related sequence group ]. Results The expression levels of E6 and E7 mRNA were significantly reduced after transfection of E6 shRNA （92%）, and E7 mRNA （89%）. HLA-Ⅱexpression was up-regulated to 1.27 times （E6 shRNA） and 1.66 times （E7 shRNA） of that in the unrelated sequence group and control group. The expression of HPV18 E6, E7 andHLA-II in un-related sequence group and control group had no significant change. Conclusion Downregula- tion of HLA-Ⅱantigen expression may be associated with HPV infection in esphageal cancer.