慢病毒介导的靶向沉默HPV16E7-shRNA对SiHa细胞DNA甲基转移酶表达的影响

摘要

目的 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-iTTM 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HPV 16 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0h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 expression of 4 kinds of DNA methyltransferases (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 expression 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 expression 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 expression of the 4 DNMTs at 48,96 hours after infection.Results There were no significant differences in the mRNA expression 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 expression 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 expression 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 expression 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 expression of DNMT1,DNMT3A,DNMT3B and DNMT3L.