title: [Inhibitory effects of human papilloma virus 18 E6 gene in HeLa cell transfected with shRNA].
abstract: 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.
authors.affiliation: Department of Gynecology, Second Xiangya Hospital, Central South University, Changsha 410011, China.
authors.forename: Guang-shi
authors.initials: GS
authors.lastname: Tao
authors.forename: Ke-qiang
authors.initials: KQ
authors.lastname: Zhang
authors.forename: Yong
authors.initials: Y
authors.lastname: Chen
authors.forename: Mei-fang
authors.initials: MF
authors.lastname: Nie
authors.forename: Feng-ying
authors.initials: FY
authors.lastname: Liu
authors.forename: Yi-lin
authors.initials: YL
authors.lastname: Wu
authors.forename: Yi-zhi
authors.initials: YZ
authors.lastname: Liu
authors.forename: Xiu-shan
authors.initials: XS
authors.lastname: Wu
mesh_terms: 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
chemical_list: D004268:DNA-Binding Proteins; C052603:E6 protein, Human papillomavirus type 18; D009856:Oncogene Proteins, Viral; D012333:RNA, Messenger; D034741:RNA, Small Interfering