[Reversal effect of antisense RNA targeting human papillomavirus 16 (HPV16) E6E7 on malignancy of human cervical cancer cell line SiHa].
OBJECTIVE
The ubiquitinated degradation of p53 can be induced by human papillomavirus type 16 (HPV16) early gene 6 (E6); the phosphorylated inactivation of pRb can be induced by HPV16 E7. They are closely associated with the carcinogenesis and progression of cervical cancer. This study was to investigate the effects of HPV16 E6E7 antisense RNA on the expression of oncogene E6 and E7 and apoptosis of human cervical cancer cell line SiHa harboring HPV16 genome.


METHODS
The antisense sequence of HPV16 E6E7 was cloned into eukaryotic expression vector pEGFP to prepare recombinant plasmid containing E6E7AS, which was transfected into SiHa cells. The mRNA and protein levels of E6 and E7 gene were detected by Western blot and reverse transcription-polymerase chain reaction (RT-PCR). Cell proliferation after transfection was evaluated by MTT assay. The apoptosis of transfected SiHa cells was assessed by flow cytometry (FCM) and confocal laser microscopy.


RESULTS
After transfection of HPV16 E6E7 antisense RNA, the mRNA and protein levels of HPV16 E6 and E7 in SiHa cells were obviously decreased. The proliferation activity of SiHa/E6E7AS cells was significantly lower than that of SiHa/EGFP and SiHa cells (0.50+/-0.05 vs. 1.01+/-0.06, 1.28+/-0.06, P<0.05). The apoptosis rate was significantly higher in SiHa/E6E7AS cells than in SiHa/EGFP and SiHa cells [(59.3+/-11.3)% vs. (9.4+/-1.8)%, (2.1+/-0.4)%, P<0.05]. The apoptotic cells increased noticeably after transfection.


CONCLUSIONS
Down-regulating HPV16 E6 and E7 with antisense RNA induces apoptosis in SiHa cells, and may be useful for HPV-associated malignancy gene therapy.
Sima
Ni
N
Cancer Biology Research Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, 430030, P. R. China.
Wang
Wei
W
Xu
Qian
Q
Tian
Xun
X
Luo
Ai-Yue
AY
Lu
Yun-Ping
YP
Wang
Shi-Xuan
SX
Ma
Ding
D
D017209:Apoptosis; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D005260:Female; D005822:Genetic Vectors; D006801:Humans; D009856:Oncogene Proteins, Viral / Q000096:biosynthesis* / Q000235:genetics; D050725:Papillomavirus E7 Proteins; D010957:Plasmids; D016372:RNA, Antisense / Q000235:genetics*; D012333:RNA, Messenger / Q000378:metabolism; D012097:Repressor Proteins / Q000096:biosynthesis* / Q000235:genetics; D014162:Transfection; D002583:Uterine Cervical Neoplasms / Q000378:metabolism / Q000473:pathology*
C068651:E6 protein, Human papillomavirus type 16; D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins; D016372:RNA, Antisense; D012333:RNA, Messenger; D012097:Repressor Proteins; C059731:oncogene protein E7, Human papillomavirus type 16