UNASSIGNED: We collected 4,030 cervical exfoliated cell samples for genotyping HPV using HybriBio\'s proprietary flow-through hybridization technique, liquid-based cytology (LBC), colposcopy, and biopsies if indicated. Four plasmids containing E6 and E7 of HPV16 and 58 were constructed and transfected into 293T and U2OS cells. We detected the cell phenotype using Cell Counting Kit 8 (CCK8) assay, Transwell assay, flow cytometry, and apoptosis assay; the expression of retinoblastoma protein (Rb) and phosphorylated Rb (pRb) was determined via Western blot; and the cell activity was determined via a zebrafish model treated with or without roscovitine.
UNASSIGNED: The positive rates of HPV16 and 58 were, respectively, 18.9% and 19.7% in the ≤ low-grade squamous intraepithelial lesion (LSIL) group, 49.5% and 19.6% (P<0.001) in the high-grade squamous intraepithelial lesion (HSIL) group, 65.3% and 9.0% (P<0.001) in the cancer group. In vitro, both 293T and U2OS cells with overexpressed HPV16 E6 and E7 displayed significantly higher cell proliferation, faster cell invasion, decreased cell apoptosis, and accelerated cell cycle from G1 phase to S phase compared to those with overexpressed HPV58 E6 and E7 (all P values <0.05). Rb loss of function was observed in cells with HPV16 E7 overexpression, while a greater level of phosphorylated Rb was observed in cells with HPV58 E7 overexpression. Roscovitine restored Rb expression and decreased the cell activity in zebrafish.
UNASSIGNED: HPV16 possesses a stronger carcinogenic ability than does HPV 58, and the mechanism underlying this effect may be the impairment of the E7-Rb pathway.
■我们收集了4,030个宫颈脱落细胞样品,用于使用HybribBio专有的Fow-through杂交技术对HPV进行基因分型,液基细胞学(LBC),阴道镜检查,和活检,如果指示。构建了含有HPV16和58的E6和E7的四个质粒,并将其转染到293T和U2OS细胞中。我们使用细胞计数试剂盒8(CCK8)检测细胞表型,Transwell分析,流式细胞术,和凋亡测定;通过Westernblot确定视网膜母细胞瘤蛋白(Rb)和磷酸化Rb(pRb)的表达;通过用或不用roscovitine处理的斑马鱼模型确定细胞活性。
■HPV16和58的阳性率分别为,分别,≤低度鳞状上皮内病变(LSIL)组分别为18.9%和19.7%,高度鳞状上皮内病变(HSIL)组分别为49.5%和19.6%(P<0.001),癌症组分别为65.3%和9.0%(P<0.001)。体外,HPV16E6和E7过表达的293T和U2OS细胞均表现出明显更高的细胞增殖,更快的细胞入侵,细胞凋亡减少,与过表达HPV58E6和E7的细胞周期相比,细胞周期从G1期到S期加速(所有P值<0.05)。在HPV16E7过表达的细胞中观察到Rb功能丧失,而在HPV58E7过表达的细胞中观察到更高水平的磷酸化Rb。Roscovitine在斑马鱼中恢复Rb表达并降低细胞活性。
■HPV16比HPV58具有更强的致癌能力,这种作用的潜在机制可能是E7-Rb通路的损害。