The Study Of EGFR Gene Silencing By RNA Interference On Reversal Effect Of SKOV3/DDP Cell To Cisplatin-Resistance

Objective To explore the effect of vector-based small Interfering RNA (siRNA) on the expression of EGFR and its roles in reversing the cisplatin resistance on ovarian cancer cell SKOV3/DDPMethods According to EGFR mRNA sequence, we designed and synthesized shRNA targeted EGFR which were directly cloned into pSilencerTM 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/DDP by liposome. The study included the normal control cell line, the empty plasmid transfectant group, nonspecific and specific transfectant groups. RT-PCR and immunocytochemistry were performed respectively to detect the level of EGFR mRNA and its protein. The half inhibitory concentration (IC50) of cisplatin in each group was measured by methyl thiazolyl tetrazolium (MTT) assay and the chemosensitivity of the cell to cisplatin was also detectedResults DNA sequencing confirmed that the insertion sequence was completely correct. Sequence specific shRNA targeting- EGFR obviously down-regulated the expression of EGFR gene. The expression of EGFR mRNA in shRNA transfectant group was less than that in the other three groups, which was obviously down-regulated (P<0.01), and the same to the protein expression (P<0.01). The result of MTT showed that the chemosensitivity to cisplatin was increased by 2.5 times compared with the normal control group.Conclusions EGFR shRNA can effectively inhibit the expression of EGFR mRNA and its protein, and recover the chemosensitivity to cisplatin. Therefore, it could be used to reverse the drug resistance of ovarian cancer cells to chemotherapeutic agents.