The Effect Of RNA Interference To EGFR Gene On Cell Cycle And Apoptosis Of Ovarian Cancer Cell Line Skov-3

Objective To construct expression vector of EGFR specific siRNA and transfect it into human ovarian cancer cell line skov3. To investigate the effect of RNA interference on the target gene expression, cell apoptosis and the cycle of the cell line.Methods According to EGFR mRNA sequence, we designed and synthesized shRNA targeted EGFR which were directly cloned into pSilencer^TM 2.1-U6 neo plasmid. DNA sequencing confirmed whether the insertion sequence was correct or not. Then transfected the recombinant plasmid into human ovarian cancer cell line skov3 with lipofectamine 2000. There were three groups: normal control; non-specific transfect and EGFR shRNA transfect. The inhibitive effect of RNAi on mRNA level and protein level were respectively detected by RT-PCR and immunocytochemistry. AnnexinV/PI double straining by flow cytometry analysis was used to evaluate apoptosis. PI straining was conducted to evaluate cell cycle arrest.Results Plasmid expressive vectors aimed directly at EGFR were successfully established. EGFR siRNA plasmid knocked down EGFR expression in Skov-3 cells obviously. Flow cytometry analysis revealed ovarian carcinoma cell line Skov-3 suffered a notable apoptosis and G₀/G₁ arrest after RNA interference mediated by siRNA targeting EGFR.Conclusion EGFR silencing by RNAi can induce cell apoptosis and arrest cell cycle at G₀/G₁ phase, which sheds light on the possible use of RNAi for further investigation of the pathogenesis and gene therapy of ovarian carcinoma.