| Summary: | 碩士 === 國立中正大學 === 分子生物研究所 === 96 === 5-Fluorouracil (5-FU) is one of the widely used anticancer chemotherapeutic agents for treatment of hepatocellular carcinoma (HCC) and oral squamous cell carcinoma (OSCC). The cytotoxic effects of 5-FU occur following its conversion to 5-fluoro-deoxyuridine monophosphate (5-FdUMP), an irreversible inhibitor of thymidylate synthase (TS) to inhibit synthesis of dTMP and dTTP, a precursor nucleotide for DNA molecules. The cytosolic enzyme dihydropyrimidine dehydrogenase (DPD) plays an important role in 5-FU degradation via catalyzing 5-FU to fluoro-β-alanine and fluoroacetate. In addition, several studies have demonstrated that a long term exposure of 5-FU could induce drug resistance to 5-FU in many cancer cells. Therefore, we intend to use a lentiviral vector-mediated RNAi expression system to achieve a long-term, efficient knockdown of DPD in cancer cell for the enhancement of sensitivity to 5-FU. In addition, we proposed to knock down TS by RNAi for inhibiting cancer cell proliferation. In this study, we reported that the knockdown of DPD could not sufficiently enhance HepG2`s sensitivity to 5-FU even though the expression of DPD mRNA was sufficiently down-regulated ranged from 43% at MOI 1 to 86% at MOI 10 by Lenti-shDPD468. However, we also reported that the Lenti-shTS219 could sufficiently inhibit the cells growth by 53% and the expression of TS mRNA by 82% at MOI 1 in HepG2 cells. Furthermore, in OSCC cell line TW206, Lenti-shTS734 could inhibt the cells growth ranged from 35% at MOI 1 to 78% at MOI 5 and the expression of TS mRNA ranged from 61% at MOI 1 to 92% at MOI 5. These observation suggest that the lentiviral vector-mediated RNAi expression system could sufficiently inhibit gene expression in cancer cells. Particularly, the knockdown of TS could inhibit the cells growth. Although the knockdown of DPD could not enhance the sensitivity to 5-FU, the RNAi expression system would be used to knock down other drug resistant gene.
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