探讨RNA干扰术对口腔癌细胞抗药能力之影响.docVIP

探討RNA干擾術對口腔癌細胞抗藥能力之影響 Effects of RNAi on drug resistance in oral squamous cell carcinoma Advisor : Dr. Chien Kuo, Tai Student : Ronald Lovel Date : 23 April 2010 Purpose: The project was developed to reduce the drug resistance of oral squamous cancer cell OSCC towards the chemotherapeutic drugs 5-fluorouracil 5-FU and cisplatin in order to decrease the amount of drugs injected into the cancer patients, hence, lessen the side effects. Methods: Human OSCC lines OC2 and OCSL were used as the experiment models. Experimental procedure was based on lentivirus vector-mediated delivery of siRNAs. We used this delivery system to knockdown gene expression of various multidrug resistance genes that had been reported previously. The mRNA and protein quantities were detected by real-time PCR and western blot, respectively. The drug resistance of cancer cell was quantified by its 50% drug inhibitory concentration IC50 which was detected using MTS assay. Results: We had been successful in greatly knocking down the fluorescence intensity of GFP-expressing OSCC by lentivirus vector expressing anti-GFP siRNA. This result showed that the delivery system was eligible to be applied in the OSCC gene knockdown study. In total, we had examined 11 different multidrug-resistance genes including ATP7B, AKT2, PIK3R2, IGF1R, MMP13, MRP1, MMP7, MRP2, MRP3, FRAP1 mTOR and EGFR. Knockdown of the first 6 genes resulted in no change of IC50. In contrast, mRNA expression of EGFR, an anti-apoptotic gene, had been successfully knocked down and decreased by about 80%, which resulted in 35% and 24% decrease towards 5-FU and cisplatin IC50, respectively, and also inhibit the proliferation rate of OC2. Knockdown of MMP7, a protease, resulted in 43% decrease for 5-FU IC50. Knockdown of FRAP1 mTOR , one of EGFR downstream effector gene, resulted in 28% and 36% decrease for 5-FU and cisplatin IC50, respectively. For MRP2 and MRP3, multidrug resistance-associated proteins, knockdown of these genes