title: Silencing of HPV 18 oncoproteins With RNA interference causes growth inhibition of cervical cancer cells.
abstract: Silencing the expression of human papillomavirus (HPV) oncoproteins should have therapeutic benefits for cervical cancer. The authors' objective was to study RNA interference of the HPV 18 E6/E7 bicistronic mRNA with E6 small interfering RNA (siRNA) and E7 siRNA and determine the effect of each siRNA on oncoprotein expression, resultant cell growth, and downstream molecular effects. RNA interference was used to knockdown HPV 18 E6 and E7 oncoproteins on the HPV 18 positive cervical cancer cell lines HeLa and C4I. Western blotting was used to assay for each oncoprotein expression and select downstream molecular targets. Cell cycle analyses, cell viability assays, and colony formation assays were performed to determine the effect of treatment by both HPV 18 E6 siRNA and E7 siRNA. The transfection reagent oligofectamine and Tax siRNA were used as negative controls. Transfection with E6 siRNA caused complete loss of E6 but not E7 oncoprotein. However, E7 siRNA induced complete loss of both E6 and E7 oncoproteins. E6 siRNA mediated the reexpression of p53 protein and a moderate decrease in phosphorylated retinoblastoma protein expression (pRb), resulting in decreased colony formation. Transfection with E7 siRNA mediated a robust increase in p53 expression and complete loss of pRb, resulting in a marked decrease in colony formation compared to the E6 siRNA (P =.001). Flow cytometry revealed significantly increased apoptotic cells with E7 siRNA compared to E6 siRNA and control. RNA interference targeting the E7 portion of the bicistronic HPV 18 mRNA can silence both E6 and E7 oncoproteins and is most effective in cervical cancer growth inhibition.
authors.affiliation: Division ofGynecologic Oncology, Hamon Center of Therapeutic Oncology Research, University ofTexas Southwestern Medical Center, Dallas, TX75390-9032, USA. jayanthi.lea@utsouthwestern.edu
authors.forename: Jayanthi S
authors.initials: JS
authors.lastname: Lea
authors.forename: Noriaki
authors.initials: N
authors.lastname: Sunaga
authors.forename: Mitsuo
authors.initials: M
authors.lastname: Sato
authors.forename: Geetha
authors.initials: G
authors.lastname: Kalahasti
authors.forename: David S
authors.initials: DS
authors.lastname: Miller
authors.forename: John D
authors.initials: JD
authors.lastname: Minna
authors.forename: Carolyn Y
authors.initials: CY
authors.lastname: Muller
mesh_terms: D002453:Cell Cycle; D002455:Cell Division; D003114:Colony-Forming Units Assay; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000235:genetics; D005260:Female; D015967:Gene Expression Regulation, Viral; D006367:HeLa Cells; D052161:Human papillomavirus 18 / Q000235:genetics*; D006801:Humans; D007700:Kinetics; D008175:Lung Neoplasms; D009856:Oncogene Proteins, Viral / Q000235:genetics; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics; D014162:Transfection; D002583:Uterine Cervical Neoplasms / Q000517:prevention & control / Q000821:virology*; D014764:Viral Proteins / Q000235:genetics
chemical_list: D017931:DNA Primers; D004268:DNA-Binding Proteins; C052603:E6 protein, Human papillomavirus type 18; C052633:E7 protein, Human papillomavirus type 18; D009856:Oncogene Proteins, Viral; D034741:RNA, Small Interfering; D012367:RNA, Viral; D014764:Viral Proteins