| Objective:To investigate the methylation status of hepatoma cell line miRNA-214 promoter region. At different concentrations, methyl transferase enzyme inhibitor promoter methylation and miRNA-214 impact, further analysis of hepatoma cell line proliferation inhibition of the RNA methylation.Methods:Using bioinformatics software online prediction miRNA-214 and the promoter region of CpG sites; extract LO-2, Huh7, HepG2 and QSG-7701 four kinds of liver cell line DNA by bisulfite sequencing method (bisulfite-sequencing PCR, BSP) analysis of the mi-RNA CpG sites methylation; use 0 umol/L,5umol/L and lOumol/L three different concentrations of methyl transferase inhibitor (5-Aza-CdR) interference liver cell line promoter methylation and miRNA-214 to detect the expression of different concentrations treated by RT-PCR method; by the following three concentrations of cell proliferation assay above inhibitor on liver cell proliferation.Results:(1)BSP calculated, miRNA-214 promoter methylation frequency, the untreated group showed, LO-2, Huh7, HepG2, and the degree of methylation QSG-7701 four kinds of liver cells in the promoter region were 51.42%, 80.00%,60.00% and 68.57%. And normal liver cells (LO-2) compared hepatoma cells (Huh7, HepG2 and QSG-7701) of miRNA-214 starts methylation of the promoter region were significantly increased, there are significant differences (F=25.9, p<0.001 difference).(2)By PCR, the expression of miRNA-214 four kinds of untreated hepatocytes (LO-2, Huh7, HepG2 and QSG-7701) were 2.84±0.52,1.00± 0.08,1.00±0.06 and 1.00±0.12. Compared with normal liver cells, low expression levels of miRNA-214 three kinds of liver cancer cells, the results were statistically significant (F=35.39, p<0.001). In 5umol/L and 10umol/L two concentrations, treated miRNA-214 expression level of 4 kinds of liver cells were 1.66±0.06,0.58±0.12,0.73±0.13 and 0.83±0.04; 1.00±0.29,0.34± 0.07,0.10±0.01 and 0.30±0.06. With the concentration of interferon, miRNA-214 expression level of 4 kinds of liver cells is reduced. Increasing concentrations of three treatment groups, miRNA-214 expression was statistically significant (F=21.9, p<0.001; F=40.5, p<0.001; F=98.1, p <0.001;F=61.6,p<0.001).(3) Cell proliferation assay, the untreated group four kinds of liver cell line (LO-2, Huh7, HepG2 and QSG-7701) proliferation levels were 0.18±0.03,0.81 ±0.61,0.68±0.28 and 3.79±0.16; in 5μmol/L and under 10μmol/L concentration, treated hepatocytes (LO-2, Huh7, HepG2 and QSG-7701) proliferation level of 0.17±0.07,1.14±0.76,0.42±0.18 and 2.70±0.107; 0.16 ±0.04,1.55±0.87,0.37±0.19 and 0.45±0.37. Compared with normal liver cells, Huh7 cells by demethylation activity increased cell proliferation after treatment (F=72.89, p<0.05), HepG2 cells and QSG-7701 by demethylation treatment of cell proliferation activity decreased slightly (F=95.36, p<0.05; F =381.46, p<0.05), a statistically significant difference.Conclusions:The BSP calculations, we found that three kinds of liver cancer cells showed miRNA-214 promoter hypermethylation. Interference treated with different concentrations, the cancer miRNA-214 promoter methylation decreased significantly reduce the expression levels of miRNA-214, but promoted the proliferation of hepatoma cells. Our results confirm, DNA methylation can regulate the expression of miRNA-214 in liver cancer cells, and further affect the progression of liver cancer. |
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