中文摘要：

背景与目的：丝氨酸／苏氨酸蛋白激酶31（serine，threoninekinases31，STK31）基因在人类多种癌症中扮演重要角色，且STK31基因的表达受其启动子及第一外显子区甲基化状态的影响；病毒感染与肿瘤组织中某些抑癌基因启动子区高甲基化有关。本研究旨在探讨宫颈癌细胞系中HPV16E6、E7及E6／E7癌基因对STK31基因甲基化状态及表达的影响，以及不同种类甲基转移酶（DNAmethyltransferases，DNMTs）基因在STK31基因甲基化中的潜在作用。方法：构建外源性HPV16E6、E7以及E6／E7基因共表达慢病毒，分别感染人乳头瘤病毒humanpapillomavirus，HPV）阴性宫颈癌细胞系HT-3及C33A，获得稳定转染的细胞系；采用亚硫酸氢盐基因组测序法（bisulfitegenomicsequencing，BGS）和甲基化特异性PCR（methylation—specificPCR，MSP）检测3种HPV阳性宫颈癌细胞系HeLa、SiHa和CasKi以及HPV阴性宫颈癌细胞系HT-3和C33A转染前后STK31基因的甲基化状态；RT—PCR及蛋白[质]印迹法（Westernblot）检测上述宫颈癌细胞系中STK3l基因的表达以及DNMT1、DNMT2、DNMT3a、DNMT3b和DNMT3L基因在HPV16转染前后宫颈癌细胞系及HPV阳性、HPV阴性宫颈癌组织中的表达情况。结果：外源一~HPV16E6、E7以及E6／E7基因可在HPV阴性宫颈癌细胞系中稳定表达。3种HPV阳性细胞系HeLa、SiHa和CasKi中STK31基因呈低甲基化状态，STK31mRNA及蛋白质表达阳性；2种HPV阴性细胞系HT-3、C33A中STK31基因则表现为高甲基化状态，STK31mRNA及蛋白质表达缺失；与未感染慢病毒HT-3和C33A细胞系比较，外源性HPV16E7以及E6／E7表达的HT-3和C33A细胞系STK31基因甲基化程度降低，其mRNA及蛋白质重新表达。DNMT1、DNMT3a和DNMT6基因在HT-3E6，E7和C33AE6／E7细胞系中mRNA水平分别高于HT-3空载细胞系和C33A空载细胞系，差异有统计学意义（P〈0．001）。DNMT1、DNMT3a和DN-MT3b基因的mRNA水平在HPV16阳性宫颈癌组?

英文摘要：

Background and purpose： Studies have proved that the serine/threonine kinases 31 （STK31） gene plays important roles in human cancers. The STK31 gene expression was demonstrated to be regulated by the methylation status of its promoter/exonl region. Viral infection was revealed to he associated with the hypermethylation of some tumor suppressor genes in some tumor samples. The purposes of this paper were to study the roles of HPV16 E6, E7, or E6/ E7 oncogenes in methylation status and expression of the STK31 gene, and potential effects of DNA methyltransferases （DNMTs） on STK31 gene methylation status. Methods： Ectopically-expressed HPV16 E6, E7, or E6/E7 cells were estab- lished by transfecting HPV16 E6, E7, or E6/E7 oncogenes with lentivirus vectors into HPV-negative cervical cancer cell lines HT-3 and C33A. Bisulfite genomic sequencing PCR （BGS） combined with TA clone and MSP （methylation-specific PCR） were used to analyze methylation status of the STK31 gene promoter/exonl region in HPV-positive cervical cancer cell lines （HeLa, SiHa, CaSki）, HPV-negative cervical carcinoma cell lines （C33A, HT-3） and the transfected ceils. The mRNA and protein expression of STK31, DNMT1, DNMT2, DNMT3a, DNMT3b and DNMT3L were detected by RT-PCR and Western blot. Results： Transfection efficiency was tested by Western blot, which showed that the transfected cells successfully expressed E6, E7, or E6/E7 proteins, respectively. The STK31 gene promoter/exonl was hypomethylated in HPV-positive cell lines HeLa, SiHa and CasKi resulting in detection of mRNA and protein expression. STK31 gene promoter/exonl showed hypermethylation leading to silenced expression in the two HPV-negative cervical cancer cells HT-3 and C33A. Compared with primary HT-3 and C33A cells, the methylation status of STK31 promoter/exonl was down-regulated that led to expression of STK31 in the eetopically-expressed HPV16 E7 and E6/E7 cells. Expressions of DNMT1, DNMT3a and DNMT3b genes at the level of transcription were higher in C33