[Inhibitory effect of RNA interference on expression of HPV16 E6 oncogene in cervical cancer cell line CaSki].
OBJECTIVE
Tumorigenesis and progression of cervical cancer closely relate with human papilloma virus (HPV) E6 and E7 oncogenes. Ribozyme and antisense oligonucleotides had been used to inhibit the expression of HPV E6 or E7 oncogenes to treat cervical cancer, but problems, including low efficiency, short-period maintenance, hard work, and high costs, still exist. This study was to evaluate the specific inhibitory effect and time-efficiency of RNA interference (RNAi) on HPV16 E6 gene in cervical cancer cell line CaSki.


METHODS
The specific small interfering RNA (siRNA) of HPV16 E6 modified by fluorescein was synthesized, and transfected into CaSki cells. The transfection efficiency of siRNA was evaluated by calculating the ratio of fluorescent cells to total cells. Cell apoptosis was evaluated by flow cytometry (FCM). The mRNA level of HPV16 E6 before and after siRNA transfection was measured by RT-PCR, and protein level of HPV16 E6 was measured by Western blot and FCM.


RESULTS
The transfection efficiency of siRNA was 81%. Apoptosis rates of CaSki cells at 1, 2, 5, and 9 d after transfection were 7.7%, 11.8%, 37.4%, and 12.6%, respectively. The mRNA level of HPV16 E6 at 1, 2, 5, and 9 d after transfection reduced by 77%, 83%, 59%, and 41%, respectively, but the mRNA level of beta-actin, as internal control, had no change. The inhibition rates of HPV16 E6 protein at 1, 2, 5, and 9 d after transfection were 79.7%, 80.4%, 71.3%, and 57.4%, respectively, but the protein level of Lamin A/C, as internal control, had no change at each time point.


CONCLUSIONS
RNAi exists in CaSki cells, and has specific high efficiency on HPV16 E6 gene.
Niu
Xiao-Yu
XY
Obstetrics and Gynecology Department, West China Second Hospital, Sichuan University, Chengdu, Sichuan 610 041, P.R. China.
Peng
Zhi-Lan
ZL
Wang
He
H
D000199:Actins / Q000378:metabolism; D017209:Apoptosis; D045744:Cell Line, Tumor; D005260:Female; D006801:Humans; D034904:Lamin Type A / Q000378:metabolism; D009856:Oncogene Proteins, Viral / Q000096:biosynthesis / Q000235:genetics*; D027383:Papillomaviridae / Q000235:genetics; D034622:RNA Interference; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics*; D012097:Repressor Proteins / Q000096:biosynthesis / Q000235:genetics*; D014162:Transfection; D002583:Uterine Cervical Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology
D000199:Actins; C068651:E6 protein, Human papillomavirus type 16; D034904:Lamin Type A; D009856:Oncogene Proteins, Viral; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D012097:Repressor Proteins