目的研究特异小干扰RNA(sm all interfering RNA,siRNA)对宫颈癌HeLa细胞中人乳头瘤病毒(hum an pap-illom avirus,HPV)18型E6基因的抑制及其对细胞凋亡的影响。方法针对HPV18E6基因设计siRNA序列,经PCR方法体外扩增,得到含有U6启动子以...目的研究特异小干扰RNA(sm all interfering RNA,siRNA)对宫颈癌HeLa细胞中人乳头瘤病毒(hum an pap-illom avirus,HPV)18型E6基因的抑制及其对细胞凋亡的影响。方法针对HPV18E6基因设计siRNA序列,经PCR方法体外扩增,得到含有U6启动子以及siRNA序列的PCR产物,利用L ipofectam ineTM2000脂质体转染HeLa细胞,在U6启动子的作用下于细胞内转录siRNA。针对转染后不同时间点采用四唑盐(MTT)比色法测定细胞活力,流式细胞仪PI染色法检测细胞凋亡率,RT-PCR测定HPV18E6 mRNA变化。结果转染siRNA后细胞活力受到显著抑制(P<0.05),光镜下出现明显的凋亡形态,72 h的凋亡率达到55.8%。RT-PCR结果显示,细胞转染24、48和72 h后HPV18E6 mRNA分别减少了57%、78%和40%,而siRNA阴性对照与未转染细胞相比差异不显著。结论siRNA可特异有效的干扰宫颈癌HeLa细胞内HPV18E6基因的表达,从而可诱导肿瘤细胞凋亡。展开更多
By using a yeast two-hybrid system,a yeast two-hybrid bait vector was constructed and identified for screening of the HPV18 E6-interacting proteins,and its effects on the growth of yeast cells and the activation of re...By using a yeast two-hybrid system,a yeast two-hybrid bait vector was constructed and identified for screening of the HPV18 E6-interacting proteins,and its effects on the growth of yeast cells and the activation of reporter genes were investigated.Total mRNA extracted from Hela cells was reversely transcribed into cDNA.Fragment of HPV18 E6 cDNA was amplified using RT-PCR and directly ligated to the pGBKT7 vector.The recombinant plasmid was confirmed by restriction endonuclease analysis and DNA sequencing.Th...展开更多
[目的]探讨HPV18致癌基因E6选择性剪接在宫颈癌细胞中的潜在作用。[方法]HPV18感染或过表达HPV18致癌基因E6后,MTT法检测宫颈癌细胞C-33 A的增殖水平、逆转录PCR和琼脂糖凝胶电泳检测E6选择性剪接产物表达水平、免疫印迹检测E7蛋白表达...[目的]探讨HPV18致癌基因E6选择性剪接在宫颈癌细胞中的潜在作用。[方法]HPV18感染或过表达HPV18致癌基因E6后,MTT法检测宫颈癌细胞C-33 A的增殖水平、逆转录PCR和琼脂糖凝胶电泳检测E6选择性剪接产物表达水平、免疫印迹检测E7蛋白表达水平。高通量测序HPV18感染或不感染的C-33 A细胞后调控E6选择性剪接的关键分子。[结果]过表达E6并感染HPV18的C-33 A细胞的增殖水平(2.35±0.37 vs 1.27±0.28)显著上升,且高于未过表达E6(1.27±0.28 vs 0.68±0.11)(P<0.05)。HPV18感染和过表达SRSF3后,选择性剪接产物E6*Ⅰ水平上升(0.25±0.03 vs 0.65±0.13)、E7蛋白表达水平(0.20±0.04 vs 0.77±0.18)上升。敲低SRSF3和过表达miR-1208时,C-33 A细胞的增殖水平(1.30±0.18 vs 0.65±0.13,1.75±0.27 vs 0.75±0.13)显著下降(P<0.05)。与只过表达E6相比,E6与SRSF3共表达、E6过表达同时干扰miR-1208均能够显著促进C-33 A细胞的增殖水平(0.65±0.13 vs 1.65±0.40,0.59±0.11 vs 1.70±0.24)(P<0.05)。敲低SRSF3后,选择性剪接产物E6*Ⅰ水平下降(0.15±0.02 vs 0.57±0.15)、E7蛋白表达水平下降(0.20±0.04 vs 0.77±0.18)(P<0.05)。干扰miR-1208后,选择性剪接产物E6*Ⅰ水平上升(1.12±0.25 vs 2.56±0.33)、SRSF3(0.15±0.03 vs 0.75±0.15)和E7蛋白表达水平上升(0.65±0.11 vs 0.98±0.20);过表达miR-1208后,选择性剪接产物E6*Ⅰ水平下降(1.85±0.34 vs 1.15±0.20)、SRSF3(1.33±0.14 vs 0.20±0.05)和E7蛋白表达水平下降(0.88±0.13 vs 0.50±0.10)(P<0.05)。此外,miR-1208靶向SRSF3 mRNA的3′端非编码区。[结论]miR-1208靶向SRSF3 mRNA的3′端非编码区并减少SRSF3的蛋白表达水平。SRSF3能够促进HPV18致癌基因E6的选择性剪接和E7蛋白的表达。HPV18感染后操纵miR-1208/SRSF3/E6/E7调控轴促进宫颈癌细胞的增殖。展开更多
基金supported by grants from National Natural Sciences Foundation of China(No.30672227,30600668)"973"Program of China(No.2009CB521800)Joint Research Fund for Overseas Chinese,Hong Kong and Macao Young Scholars(No.30628029)
文摘By using a yeast two-hybrid system,a yeast two-hybrid bait vector was constructed and identified for screening of the HPV18 E6-interacting proteins,and its effects on the growth of yeast cells and the activation of reporter genes were investigated.Total mRNA extracted from Hela cells was reversely transcribed into cDNA.Fragment of HPV18 E6 cDNA was amplified using RT-PCR and directly ligated to the pGBKT7 vector.The recombinant plasmid was confirmed by restriction endonuclease analysis and DNA sequencing.Th...
文摘[目的]探讨HPV18致癌基因E6选择性剪接在宫颈癌细胞中的潜在作用。[方法]HPV18感染或过表达HPV18致癌基因E6后,MTT法检测宫颈癌细胞C-33 A的增殖水平、逆转录PCR和琼脂糖凝胶电泳检测E6选择性剪接产物表达水平、免疫印迹检测E7蛋白表达水平。高通量测序HPV18感染或不感染的C-33 A细胞后调控E6选择性剪接的关键分子。[结果]过表达E6并感染HPV18的C-33 A细胞的增殖水平(2.35±0.37 vs 1.27±0.28)显著上升,且高于未过表达E6(1.27±0.28 vs 0.68±0.11)(P<0.05)。HPV18感染和过表达SRSF3后,选择性剪接产物E6*Ⅰ水平上升(0.25±0.03 vs 0.65±0.13)、E7蛋白表达水平(0.20±0.04 vs 0.77±0.18)上升。敲低SRSF3和过表达miR-1208时,C-33 A细胞的增殖水平(1.30±0.18 vs 0.65±0.13,1.75±0.27 vs 0.75±0.13)显著下降(P<0.05)。与只过表达E6相比,E6与SRSF3共表达、E6过表达同时干扰miR-1208均能够显著促进C-33 A细胞的增殖水平(0.65±0.13 vs 1.65±0.40,0.59±0.11 vs 1.70±0.24)(P<0.05)。敲低SRSF3后,选择性剪接产物E6*Ⅰ水平下降(0.15±0.02 vs 0.57±0.15)、E7蛋白表达水平下降(0.20±0.04 vs 0.77±0.18)(P<0.05)。干扰miR-1208后,选择性剪接产物E6*Ⅰ水平上升(1.12±0.25 vs 2.56±0.33)、SRSF3(0.15±0.03 vs 0.75±0.15)和E7蛋白表达水平上升(0.65±0.11 vs 0.98±0.20);过表达miR-1208后,选择性剪接产物E6*Ⅰ水平下降(1.85±0.34 vs 1.15±0.20)、SRSF3(1.33±0.14 vs 0.20±0.05)和E7蛋白表达水平下降(0.88±0.13 vs 0.50±0.10)(P<0.05)。此外,miR-1208靶向SRSF3 mRNA的3′端非编码区。[结论]miR-1208靶向SRSF3 mRNA的3′端非编码区并减少SRSF3的蛋白表达水平。SRSF3能够促进HPV18致癌基因E6的选择性剪接和E7蛋白的表达。HPV18感染后操纵miR-1208/SRSF3/E6/E7调控轴促进宫颈癌细胞的增殖。
