Antisense targeting human papillomavirus type 16 E6 and E7 genes contributes to apoptosis and senescence in SiHa cervical carcinoma cells.
OBJECTIVE
Human papillomavirus type 16 (HPV-16) is a high-risk DNA tumor virus involved in the development of cervical carcinomas. Substantial studies have demonstrated that E6 and E7 oncoproteins of HPV-16 could induce cell proliferation and immortalization. Repression of E6 and/or E7 oncogenes may induce cervical cancer cells to undergo apoptosis or senescence. The purpose of this study was to determine whether activation of the p53 and retinoblastoma (Rb) pathway by HPV-16 E6 and E7 repression was responsible for apoptosis and senescence of cervical cancer cells and to explore the potential of an antisense RNA (AS) transcript for gene therapy of cervical cancer.


METHODS
The antisense RNA directed against HPV-16 E6 and E7 (16AS) was constructed, and its effects on cell apoptosis and senescence of SiHa cervical carcinoma cells harboring HPV-16 were analyzed. The efficiency of 16AS was evaluated with RT-PCR, Western blotting, flow cytometry analysis, Hoechst 33258 staining, senescent cell morphology observation and senescence-associated beta-galactosidase staining.


RESULTS
The sufficient repression of HPV-16 E6 and E7 oncogenes were achieved in 16AS-transfected SiHa cells, which led to obvious apoptosis and replicative senescence of tumor cells. Furthermore, the downregulation of HPV-16 E6 and E7 by 16AS transfection resulted in remarkable increase of both p53 expression and hypophosphorylated p105Rb level in SiHa cells.


CONCLUSIONS
These results demonstrate that reduction of E6 and E7 expression is sufficient to induce SiHa cells to undergo apoptosis and senescence and suggest that transfection of cervical cancer cells with HPV-16 E6 and E7 antisense RNA is a potential approach to treat HPV-16-positive cervical cancers.
Sima
Ni
N
Cancer Biology Research Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1095 Jiefang Ave., Wuhan, Hubei 430030, PR China.
Wang
Shixuan
S
Wang
Wei
W
Kong
Debo
D
Xu
Qian
Q
Tian
Xu
X
Luo
Aiyue
A
Zhou
Jianfeng
J
Xu
Gang
G
Meng
Li
L
Lu
Yunping
Y
Ma
Ding
D
D017209:Apoptosis / Q000235:genetics*; D045744:Cell Line, Tumor; D016922:Cellular Senescence / Q000235:genetics; D005260:Female; D015316:Genetic Therapy / Q000379:methods; D049452:Green Fluorescent Proteins / Q000235:genetics; D052162:Human papillomavirus 16 / Q000235:genetics; D006801:Humans; D009856:Oncogene Proteins, Viral / Q000235:genetics*; D050725:Papillomavirus E7 Proteins; D030361:Papillomavirus Infections / Q000235:genetics / Q000473:pathology / Q000821:virology; D016372:RNA, Antisense / Q000235:genetics*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D012097:Repressor Proteins / Q000235:genetics*; D014162:Transfection; D002583:Uterine Cervical Neoplasms / Q000235:genetics* / Q000473:pathology / Q000628:therapy / Q000821:virology
C068651:E6 protein, Human papillomavirus type 16; D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins; D016372:RNA, Antisense; D012333:RNA, Messenger; D012097:Repressor Proteins; C485184:enhanced green fluorescent protein; C059731:oncogene protein E7, Human papillomavirus type 16; D049452:Green Fluorescent Proteins