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背景与目的:新疆是宫颈癌高发区,该地区宫颈癌高发与人乳头瘤病毒16型(human papillomavirus type 16,HPV-16)感染密切相关。该研究旨在分析新疆地区妇女宫颈病样组织中HPV-16上游调控区(upstream regulatory region,URR)的突变及其功能。方法:以新疆妇女子宫颈上皮非典型增生(cervical intraepithelial neoplasia,CIN)和宫颈癌病样组织标本DNA为模板,PCR扩增HPV-16 URR片段,PCR产物经测序比对,筛选代表性的URR突变体构建至p GL3-Basic载体,将其转染Vero细胞,48 h后检测荧光素酶活性,分析URR突变体启动子活性。结果:采用聚合酶链反应(polymerase chain reaction,PCR)获得了55个HPV-16 URR DNA片段,测序及序列分析发现44个突变位点,其中nt7192(G→T)、nt7433(-→T)、nt7435(C→G)和nt7863(A→-)4个位点的突变为所有序列共有,nt7520(G→A)位点的突变存在于54个样品中,剩余39个位点的突变存在于不同样品中。根据突变的位置、频率和程度,筛选出9个URR突变体分别克隆至p GL3-Basic中荧光素酶基因前并转染Vero细胞。荧光素酶活性分析表明,不同URR突变体的启动子活性差异较大,来源于宫颈癌的URR突变体启动子活性显著高于来源于CIN的URR突变体(P<0.01),部分宫颈癌URR突变体的启动子活性显著高于Si Ha和Caski细胞来源的URR参照序列的启动子活性。结论:新疆地区分离的HPV-16URR发生多位点突变,其中部分突变增强了URR内部启动子的活性,导致HPV-16致癌活性增强。
BACKGROUND & OBJECTIVE: Xinjiang is a high incidence of cervical cancer. The high incidence of cervical cancer in this area is closely related to the infection of human papillomavirus type 16 (HPV-16). The aim of this study was to analyze the mutation and function of HPV16 upstream regulatory region (URR) in women with cervical disease in Xinjiang. Methods: HPV-16 URR fragments were amplified by polymerase chain reaction (PCR) from samples of cervical intraepithelial neoplasia (CIN) and cervical cancer-like tissue from Xinjiang women. The PCR products were sequenced and compared to screen the representative URR The mutant was constructed into pGL3-Basic vector and transfected into Vero cells. After 48 h, luciferase activity was assayed and URR mutant promoter activity was analyzed. Results: Fifty-five HPV-16 URR DNA fragments were obtained by polymerase chain reaction (PCR). Sequencing and sequence analysis revealed 44 mutation sites, of which nt7192 (G → T), nt7433 (- → T) , Nt7435 (C → G) and nt7863 (A → -) were shared by all the sequences. The mutation of nt7520 (G → A) was found in 54 samples and the remaining 39 mutations were found In different samples. According to the location, frequency and degree of the mutations, nine URR mutants were selected and cloned into pGL3-Basic before luciferase gene and transfected into Vero cells. The analysis of luciferase activity showed that the promoter activity of URR mutant varied greatly. URR mutant derived from cervical cancer had significantly higher promoter activity than URIN mutant derived from CIN (P <0.01), and some URR The promoter activity of the mutant was significantly higher than that of the URR reference sequence derived from Si Ha and Caski cells. CONCLUSIONS: HPV-16URR isolated in Xinjiang region undergoes multi-locus mutation. Some mutations enhance the activity of URR promoter and result in the enhancement of oncogenic activity of HPV-16.