Jonson Amy L, Rogers Lisa M, Ramakrishnan Sundaram, Downs Levi S
University of Minnesota Medical School, Department of Obstetrics, Gynecology and Women's Health, 420 Delaware Street S.E., MMC 395, Minneapolis, MN 55455-0374, USA.
Gynecol Oncol. 2008 Nov;111(2):356-64. doi: 10.1016/j.ygyno.2008.06.033. Epub 2008 Aug 27.
Selective silencing of HPV oncogenes using short interfering RNA (siRNA) blocks E6/E7 expression and restores normal p53 and Rb function. Our objective was to determine if siRNA targeting E6/E7 would inhibit the growth of established tumors in a mouse model of cervical cancer.
In vitro studies were performed using unique siRNA sequences to confirm their ability to target and reduce E6/E7 mRNA and restore functioning p53. Next, siRNA targeting lamin was injected daily for three days into tumors established from HPV 16 positive CaSki human cervical cancer cells. Immunohistochemistry and branched DNA gene quantification were used to determine distribution and duration of activity of these siRNA. For our therapeutic studies tumors were directly injected with siRNA targeting E6/E7, non-targeting control siRNA, or saline. In preliminary experiments injections were daily or every three days for a total of three doses. A second therapeutic experiment utilized every three day dosing for 35 days. Tumor volume, growth curves and E7 mRNA levels were assessed.
The two most active siRNA sequences resulted in a 67% and 71% reduction in E6/E7 mRNA. Fluorescent lamin siRNA was visualized up to 120 h after the initial tumor injection and was evenly distributed throughout the tumors. IHC showed lamin expression to be inhibited by 68% and 75% when compared to controls at 54 and 120 h respectively. In our preliminary therapeutic intervention experiments there was no significant difference in tumor growth between the treatment groups when mice were treated with three daily injections (p=0.41). However, when treated every third day for three injections final tumor volume was less in animals injected with siRNA sequences A (78% reduction; p<0.0001) and G (60% reduction; p=0.005) compared to saline injection. Tumors showed a corresponding decrease in E6/E7 mRNA. Extended treatment with siRNA completely or nearly eradicated tumors in 70% of the animals.
Therapeutic siRNA targeting E6/E7 significantly inhibits tumor growth in this mouse model of cervical cancer. Further investigation is needed to determine optimal dosing and route of delivery.
使用小干扰RNA（siRNA）选择性沉默人乳头瘤病毒（HPV）致癌基因可阻断E6/E7表达，并恢复正常的p53和Rb功能。我们的目的是确定靶向E6/E7的siRNA是否会抑制宫颈癌小鼠模型中已形成肿瘤的生长。
使用独特的siRNA序列进行体外研究，以确认其靶向和降低E6/E7 mRNA以及恢复p53功能的能力。接下来，将靶向核纤层蛋白的siRNA每天注射到由HPV 16阳性的CaSki人宫颈癌细胞形成的肿瘤中，持续三天。采用免疫组织化学和分支DNA基因定量法来确定这些siRNA的活性分布和持续时间。在我们的治疗研究中，将靶向E6/E7的siRNA、非靶向对照siRNA或生理盐水直接注射到肿瘤中。在初步实验中，每天或每三天注射一次，总共注射三剂。第二项治疗实验采用每三天给药一次，持续35天。评估肿瘤体积、生长曲线和E7 mRNA水平。
两个活性最高的siRNA序列使E6/E7 mRNA分别降低了67%和71%。最初肿瘤注射后长达120小时均可观察到荧光核纤层蛋白siRNA，且其在肿瘤中均匀分布。免疫组织化学显示，与对照组相比，在54小时和120小时时核纤层蛋白表达分别被抑制了68%和75%。在我们的初步治疗干预实验中，当小鼠每天注射三次时，各治疗组之间的肿瘤生长没有显著差异（p=0.41）。然而，当每三天注射一次，共注射三次时，与注射生理盐水相比，注射siRNA序列A（降低78%；p<0.0001）和G（降低60%；p=0.005）的动物最终肿瘤体积较小。肿瘤的E6/E7 mRNA相应减少。用siRNA进行延长治疗可使70%的动物肿瘤完全或几乎完全消除。
在该宫颈癌小鼠模型中，靶向E6/E7的治疗性siRNA可显著抑制肿瘤生长。需要进一步研究以确定最佳给药剂量和给药途径。
