Research On The Function Of5-Aza-cytidine To The Methylation Related Genes Of Canine Breast Cancer

The experiment aims to explore the relation between occurrence and methylation of caninebreast tumor by histopathological analysis and methylation detection of clinical tissue samples andblood samples. Meanwhile, explore the effective drug concentration and time of5-Azacytidine onCHMm and the enfluence of its methylation and gene expression.24cases of collected clinical tissue samples were analyzed by making paraffin section withHE staining. After extraction of the genome DNA of canine blood (24cases) and CHMm, bysodium bisulfite modification and purification, methylation specific PCR (MSP) was applicated fordetecting the states of DAPK1and MGMT in blood and cells.CHMm was trained in vitro, the inhibition rate of cells was detected by MTT method withdifferent drug concentrations (0.25μmol/L、0.5μmol/L、1μmol/L、2.5μmol/L、5μmol/L、10μmol/L、25μmol/L、50μmol/L、100μmol/L) after24h,48h,72h on cell proliferation.According the cellgrowth inhibition rate calculation, determined the effective drug concentration and time. Thencanine breast tumor cells were processed with different concentrations of5-Azacytidine after sometime before real-time PCR detection for the mRNA expression of genes DAPK1, MGMT, BRCA2and P27and CyclinD1.Results:1. Through pathology classification,24cases of samples included11benign tumors(45.83%) and13malignant tumors (54.17%); DAPK1and MGMT genes appeared with genemethylation in cells and blood samples, in which cells and9cases of malignant tumor bloodsamples fully methylated (37.50%),4cases of malignant tumor blood samples and9cases ofbenign tumor blood samples partly methylated (54.17%), there was no methylation in2cases ofbenign tumor blood samples (8.33%), the results show that the DAPK1and MGMT methylationphenomenon was obvious in canine breast tumor.2. Detection of cell growth inhibition rate showed every drug concentration and time cancause different restrain effects on the growth of cells. Intensity of inhibition increased with theincrease of drug concentration and the extension of time, and1μmol/L、2.5μmol/L、5μmol/L、10μmol/L、25μmol/L and50μmol/L were choosed as experimental drug concentrations with timeof72h for subsequent experiment.3. With different concentrations of5-Azacytidine processed in canine breast tumor cells after72h, detection of real-time PCR showed DAPK1, MGMT, BRCA2, P27mRNA expressiongradually increased along with the increase of drug concentration, CyclinD1mRNA expressiongradually decrease along with the increase of drug concentration, difference of high dose groups were very significant compared with control group.