慢病毒介导的靶向沉默HPV16 E7-shRNA对SiHa细胞DNA甲基转移酶表达的影响

Effects of lentivirus-delivered short hairpin RNA targeting human papillomavirus 16 E7 gene on the expression of DNA methyltransferases in SiHa cells

的 探讨慢病毒携带的靶向人乳头瘤病毒16（HPV16）E7基因的短发夹干扰RNA（shRNA）对HPV16阳性宫颈癌SiHa细胞中4种甲基转移酶（DNMT1、DNMT3A、DNMT3B、DNMT3L）表达水平的影响。方法 构建HPV16 E7基因靶序列的shRNA重组质粒，用BLOCK-iT^TM Lentiviral RNAi Expression System试剂盒进行慢病毒包装，分别转染293T细胞48、72 h后收集病毒上清液。分别用含靶向HPV16 E7-shRNA重组质粒的病毒上清液与完全培养基（1∶1）的混合液（shRNA组）、含空白质粒（即不含靶向HPV16 E7-shRNA序列）的病毒上清液与完全培养基（1∶1）的混合液（阴性对照组）、完全培养基（空白对照组）培养SiHa细胞。感染0、48、96 h后，用实时荧光定量PCR（qRT-PCR）检测3组SiHa细胞中HPV16 E7和4种甲基转移酶mRNA的表达，Western印迹检测4种甲基转移酶的蛋白表达。结果 感染0 h时，shRNA组、阴性对照组、空白对照组SiHa细胞HPV16 E7、DNMT1、DNMT3A、DNMT3B、DNMT3L mRNA相对表达量比较，差异无统计学意义（均P 〉 0.05）；感染48、96 h时，3组细胞间HPV16 E7和4种DNMT mRNA相对表达量比较，差异有统计学意义（均P 〈 0.05）。shRNA组HPV16 E7、DNMT1、DNMT3A、DNMT3B和DNMT3L mRNA表达水平在48 h时的沉默效率分别为71.13%、50.53%、13.72%、46.27%和17.92%，96 h时分别为83.50%、74.2%、47.8%、64.7%和48.9%；而阴性对照组和空白对照组各时间点表达水平差异无统计学意义（均P 〉 0.05）。慢病毒感染48、96 h时，3组细胞间上述4种DNMT蛋白表达量差异有统计学意义（均P 〈 0.01）。shRNA组4种DNMT蛋白表达水平随感染时间的延长而逐渐下降，感染48 h时DNMT1、DNMT3A、DNMT3B和DNMT3L蛋白表达抑制率分别为84%、37.2%、59.8%、49.3%，96 h时分别为73.1%、68.7%，55.5%、65.5%。结论 靶向沉默HPV16阳性宫颈癌SiHa细胞E7基因能够干扰DNMT1、DNMT3A、DNMT3B、DNMT3L mRNA及蛋白的表达。

Objective To evaluate the effects of lentivirus-delivered short hairpin RNA （shRNA） targeting human papillomavirus 16 （HPV16） E7 gene on the of 4 kinds of DNA methyl-transferases （DNMTs）, including DNMT1, DNMT3A, DNMT3B and DNMT3L, in HPV16-positive cervical cancer cell line SiHa. Methods The recombinant plasmid containing HPV16 E7 gene-targeting shRNA was constructed firstly. Then, the BLOCK-iTTM lentiviral RNAi system kit was used to package the lentiviral vector, which was transfected into 293T cells. The lentivirus-containing supernatants were collected at 48 and 72 hours after transfection. The SiHa cells were divided into 3 groups to be cultured with lentiviral supernatant containing HPV16 E7 gene-targeting shRNA recombinant plasmids mixed with complete medium at a ratio of 1∶1 （shRNA group）, lentiviral supernatant containing empty plasmids mixed with complete medium at a ratio of 1∶1 （negative control group）, and complete medium alone （blank control group）, respectively. Real-time fluorescence-based quantitative PCR （qRT-PCR） was performed to measure mRNA of HPV16 E7 and 4 kinds of DNMTs in the above 3 groups at 0, 48, 96 hours after infection, and Western blot analysis to determine protein of the 4 DNMTs at 48, 96 hours after infection. Results There were no significant differences in the mRNA of HPV16 E7 and the 4 DNMTs among the shRNA group, negative control group and blank control group at 0 hour after infection （all P 〉 0.05）. At 48, 96 hours after infection, the mRNA of HPV16 E7 and the 4 DNMTs decreased significantly in the shRNA group compared with the negative control group and blank control group （all P 〈 0.05）, but did not differ between the negative control group and blank control group （all P 〉 0.05）. Additionally, E7, DNMT1, DNMT3A, DNMT3B and DNMT3L gene-silencing efficiencies in the shRNA group were 71.13%, 50.53%, 13.72%, 46.27% and 17.92% at 48 hours, and 83.50%, 74.2%, 47.8%, 64.7% and 48.9% at 96 hours after infection, respectively. Western blot analysis showed that the protein of the 4 DNMTs significantly decreased in the shRNA group compared with the negative control group and blank control group at 48, 96 hours after infection （all P 〈 0.01）. Moreover, the protein of DNMT1, DNMT3A, DNMT3B and DNMT3L in the shRNA group gradually decreased over time, and was inhibited by 84%, 37.2%, 59.8% and 49.3% at 48 hours respectively, and by 73.1%, 68.7%, 55.5% and 65.5% at 96 hours after infection respectively. Conclusion Targeted silencing of E7 gene in HPV16-positive SiHa cells can interfere with the mRNA and protein of DNMT1, DNMT3A, DNMT3B and DNMT3L.