[Inhibitory effects of human papilloma virus 18 E6 gene in HeLa cell transfected with shRNA].
OBJECTIVE
To investigate the inhibition of HPV18E6 gene in HeLa cell transfected with plasmid expressing human papilloma virus 18 E6 (HPV18E6) short hairpin RNA (shRNA).


METHODS
We synthesized two HPV18E6 shRNA frames and sub-cloned them into pSUPER which can express shRNA in mammalian cells to construct pE6-1shRNA and pE6-2shRNA which were mutant in E6 shRNA frame. The pE6-1shRNA, pE6-2shRNA and pcDNA3.1 were co-transfected into HeLa cells by cationic liposome respectively and the positive transfectants were selected by G418. The HPV18E6 mRNA and protein expression level was detected by semi-quantitative RT-PCR and streptavidin-peroxidase conjugated method (SP) to assay the inhibitory effects of pE6shRNA.


RESULTS
We successfully constructed several new HeLa cell lines transfected with pE6-1shRNA and pE6-2shRNA. In the HeLa cells without transfection and the HeLa cells transfected with pE6-1shRNA plasmid, the HPV18E6 mRNA levels were 1.14 +/- 0.45, 0.76 +/- 0.28 respectively, and the difference of HPV18E6 mRNA levels was significant (P < 0.05). The inhibition efficiency of HPV18E6 gene mRNA was 33.3% and the HPV18E6 protein levels were declined after transfection with pE6-1shRNA. In the HeLa cells transfected with pE6-2shRNA and pSUPER plasmids, HPV18E6 mRNA and protein expression levels were not different from those in wild HeLa cells.


CONCLUSIONS
The pE6-1shRNA plasmid can inhibit HPV18E6 expression in HeLa cells, which is persistent, specific and heritable.
Tao
Guang-shi
GS
Department of Gynecology, Second Xiangya Hospital, Central South University, Changsha 410011, China.
Zhang
Ke-qiang
KQ
Chen
Yong
Y
Nie
Mei-fang
MF
Liu
Feng-ying
FY
Wu
Yi-lin
YL
Liu
Yi-zhi
YZ
Wu
Xiu-shan
XS
D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D005260:Female; D006367:HeLa Cells; D006801:Humans; D007150:Immunohistochemistry; D009856:Oncogene Proteins, Viral / Q000235:genetics* / Q000378:metabolism; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D002583:Uterine Cervical Neoplasms / Q000473:pathology / Q000821:virology
D004268:DNA-Binding Proteins; C052603:E6 protein, Human papillomavirus type 18; D009856:Oncogene Proteins, Viral; D012333:RNA, Messenger; D034741:RNA, Small Interfering