Effects of lentivirus-mediated c-met RNAi on biological behaviors of human papillary thyroid cancer K1 cells

AIM: To evaluate the expression of c-Met in papillary thyroid cancer (PTC) by constructing lentiviral vectors for RNA interference (RNAi) of c-met gene and detecting its silencing effect on the biological behaviors of human papillary thyroid cancer cell line K1 cells. METHODS: Immunohistochemical assay was performed to detect the expression of c-Met protein in 35 cases of PTC and 25 cases of benign thyroid disease. Lentiviral vector for RNA of c-met gene was constructed and the silencing effect was detected by RT-PCR and Western blotting. The colony-forming ability, cell cycle, migration and invasion of K1 cells were measured by colony-forming assay, flow cytometry, wound-healing observation and Transwell experiment, respectively. In vivo tumorigenicity assay was performed to analyze in vivo proliferation of K1 cells in a xenograft model. RESULTS: The expression of c-Met in PTC was significantly higher than that in benign thyroid tissues. Lentiviral RNAi vectors targeting c-met gene were successfully constructed, and they efficiently inhibited the expression of c-met at mRNA and protein levels. Transfection of c-met lentiviral RNAi vectors inhibited the colony formation, cell cycle progression, migration, invasion and tumorigenicity of K1 cells. CONCLUSION: Lentivirus-mediated c-met RNAi efficiently inhibits colony formation, cell cycle progression, migration, invasion and tumorigenicity of K1 cells.     

Imperatorin increased sensitivity of lung cancer CD133+ cell subsets to gefitinib by down-regulating c-met expression

AIM: To investigate the role of imperatorin in reversing the resistance of the PC9 CD133⁺ cell subsets to gefitinib. METHODS: MTT assay was performed to evaluate the viability of PC9 cells treated with imperatorin and gefitinib. The expression of c-met, activation of caspases and phosphorylation of epidermal growth factor receptor (EGFR), PI3K and AKT in the PC9 cells treated with imperatorin and gefitinib were determined by Western blot. The percentage of CD133⁺ cell subsets population and the apoptotic rate of the PC9 cells treated with imperatorin and gefitinib were analyzed by flow cytometry. RESULTS: The sensitivity of the PC9 CD133⁺ cell subsets to gefitinib was significantly lower than that of the PC9 CD133^- cell subsets. Treatment with gefitinib alone significantly inhibited the protein levels of EGFR/PI3K/AKT in the PC9 CD133^- cell subsets but not the PC9 CD133⁺ cell subsets. Treatment with gefitinib alone increased the percentage of CD133⁺ cell subsets population in the PC9 cells. However, combination of gefitinib with imperatorin significantly inhibited the enrichment of CD133⁺ cell subsets population. Imperatorin down-regulated c-met expression, suggesting the c-met was the target of imperatorin in the PC9 CD133⁺ cell subsets. The results of MTT assay, Western blot analysis and flow cytometry indicated that imperatorin increased the gefitinib induced inhibition of PI3K/AKT protein levels by down-regulating the expression of c-met, which subsequently induced the cleavage of caspases and apoptosis in the PC9 CD133⁺ cell subsets.CONCLUSION: Imperatorin increases the sensitivity of lung cancer CD133⁺ cell subsets to gefitinib by down-regulating the expression of c-met, and the synergistic anti-tumor effect exists between imperatorin and gefitinib.