Inhibition of HPV 16 E6 oncogene expression by RNA interference in vitro and in vivo
Inhibition of HPV 16 E6 oncogene expression by RNA interference in vitro and in vivo
复制标题
DOI：
10.1111/j.1525-1438.2006.00384.x
复制
发表时间：
2006-03-01
影响因子：
4.8
通讯作者：
Wang, P
中科院分区：
文献类型：
--
作者：
Niu, XY;Peng, ZL;Wang, P
大量研究表明，宫颈癌的发生和发展与人乳头瘤病毒（HPV） E6和E7癌基因密切相关。采用核酶或反义寡核苷酸抑制HPV E6、E7癌基因表达的治疗策略虽有一定效果，但仍存在效率低、维持周期短、成本高等问题。本研究旨在探讨HPV 16e6小干扰RNA （hpv16e6 siRNA）在体外和体内对宫颈癌细胞系CaSki细胞的影响。合成了HPV 16e6特异性siRNA，并通过脂质体转染CaSki细胞。转染前后分别采用流式细胞术、逆转录聚合酶链反应和Western blot检测凋亡细胞数、HPV 16 E6 mRNA水平和E6蛋白表达。建立裸鼠宫颈癌，将siRNA直接注射到裸鼠腹膜腔或皮下肿瘤中。通过肿瘤体积变化、HPV 16e6蛋白表达和肿瘤细胞凋亡来评价siRNA的效果。转染siRNA后第1、2、5、9天CaSki细胞的凋亡率分别为7.7%、11.8%、37.4%、12.6%。同一时间点HPV 16e6 mRNA水平分别降低77%、83%、59%和41%。而作为内控的β -肌动蛋白mRNA水平无明显变化。转染后第1、2、5、9天E6蛋白合成抑制率分别为79.7%、80.4%、71.3%、57.4%，而作为内控的Lamin A/C蛋白水平没有变化。在体内，与对照组相比，E6 siRNA给药组在抑制肿瘤生长、抑制E6蛋白表达、诱导肿瘤坏死和凋亡方面表现出明显的效果。在皮下肿瘤中直接注射siRNA抑制肿瘤的效果与经腹腔注射相似，在宫颈癌CaSki细胞中进一步注射效果更好。存在RNA干扰，且对HPV 16e6的干扰在体外和体内均具有特异性和高效率。
Substantial studies have demonstrated that the initiation and progression of cervical cancer were closely associated with human papillomavirus (HPV) E6 and E7 oncogenes. The therapeutic strategy with ribozyme or antisense oligonucleotides to inhibit the expression of HPV E6 or E7 oncogenes showed effect to some degree, but problems such as low efficiency, short-period maintenance, and high cost still remain. The aim of this study was to investigate in vitro and in vivo the effect of HPV 16 E6 small interfering RNA (HPV 16 E6 siRNA) on cervical cancer cell line CaSki cells. The specific siRNA of HPV 16 E6 was synthesized and transfected into CaSki cells by liposome. The number of apoptotic cells, HPV 16 E6 messenger RNA (mRNA) level, and E6 protein expression were measured before and after the transfection by flow cytometry, reverse transcriptase-polymerase chain reaction, and Western blot, respectively. Cervical cancer in nude mice was established, and siRNA was injected directly into the nude mice peritoneal cavity or subcutaneous tumor. The efficiency of siRNA was evaluated by tumor volume change, HPV 16 E6 protein expression, and apoptosis of tumor cells. Apoptosis rate of CaSki cells at days 1, 2, 5, and 9 after siRNA transfection were 7.7%, 11.8%, 37.4%, and 12.6%, respectively. The mRNA level of HPV 16 E6 at the same time points were reduced by 77%, 83%, 59%, and 41%, respectively. But the mRNA level of beta-actin, as an internal control, showed no significant change. The inhibition rates of E6 protein synthesis at days 1, 2, 5, and 9 after the transfection were 79.7%, 80.4%, 71.3%, and 57.4%, respectively, whereas the protein levels of Lamin A/C, as internal control, had no change. In vivo, E6 siRNA administration groups showed a dramatic effect in inhibiting tumor growth, suppressing expression of E6 protein, and inducing tumor necrosis and apoptosis as compared with the control group. Direct injection of siRNA into subcutaneous tumor resulted in tumor suppression effect similar to that via the peritoneal cavity, and with additional injection better results could be achieved in cervical cancer CaSki cells. RNA interference exists, and the interference to HPV 16 E6 is specific and highly efficient both in vitro and in vivo.