Effect of HPV-16 LCR internal meCpG site demethylation on HPV positive head and neck tumor cells / 中国耳鼻咽喉头颈外科

ABSTRACT

OBJECTIVE To investigate the effect of HPV-16 LCR internal meCpG site demethylation on HPV positive head and neck tumor cells. METHODS After 5-azoxy-2-deoxycytidine(5-Aza-2'-dc) demethylation treatment, the methylation status of CpG sites in the long regulatory region of UM-SCC47, CaSki and SiHa tumor cells was detected by BSP assay. The expression of HPV 16 E6 and E7 mRNA was detected by real-time PCR. The effects of 5-Aza-2'-dc on cell growth and apoptosis were detected. Using Lipolnfectamine RNAiMAX to transfect siRNA, the expression of E6 and E7 was silenced, and then the changes of biological state of cell proliferate inhibition, apoptosis and cycle were detected. RESULTS Cells treated with 5-Aza-2, -dc at a concentration of 0.5 μmol/L for 96 h had the best demethylation. After demethylation of 5-Aza-2'-dc, the expression levels of E6 and E7 mRNA in UM-SCC47 cells and CaSki cells were significantly decreased compared with the control group(t=1.356, 2.623, P=0.031, 0.005; t=1.798, 2.015, P=0.011, 0.009). The expression levels of E6 and E7 mRNA in SiHa cells were not significantly differences compared with the control group(t=1.591, 1.153, P=1.105, 0.753); After treated with 5-Aza-2'-dc, cells proliferation were significantly inhibited. After E6 and E7 expression was silenced, the growth of all three kinds of cells was inhibited. The apoptosis of all three kinds of cells were significantly increased, cells arrested in S phase and G2/M phase were significantly increased compared with the control groups, the difference were statistically significant. CONCLUSION After demethylation, the response of HPV-16 positive tumor cells with different methylation status in the long regulatory region is completely different, indicating that in the HPV-16 positive tumors, there may be some information about the methylation status in the long regulatory region Different tumor characteristics and mechanism of cancer.