Research Of Gossypol Acetic Acid’s Influence On ER Gene Methylation And MRNA Level Of ER, PR, C-fos Gene In Human Tongue Squamous Cell Carcinoma Cal-27

Objective:To investigate the effects of GAA on proliferation in human tongue squamous cell carcinoma Cal-27 in vitro and detect the methylation level changes of the ER gene and mRNA expression level changes of the ER、PR、c-fos gene in Cal-27 cells in vitr after treated by GAA, in order to provide the experimental base and theory for prevention and therapy on carcinoma by changing methylation level of ER gene.Methods:1、Cal-27 cells were cultivated in vitro and treated by GAA,and its pre-and-post treated morphology change were observed through inverted microscope.2、CCK-8 assay were used to detect the effects on proliferation in Cal-27 cells treated by different concentration of GAA, and then IC50 of GAA was calculated.3、Methylation Specific PCR (MS-PCR) and Bisulfite Sequencing PCR (BS-PCR) were used to detect methylation level change of the ER gene in Cal-27 cells treated byGAA for 48h.4、Real-time quantitative PCR (QPCR) were used to detect mRNA expression level change of the ER、PR、c-fos gene in Cal-27 cells treated by GAA for 48h.Results:1、After adding different concentrations of GAA, cell shrinkage of the Cal-27 were observed through inverted microscope,cell bodies became smaller,round,separated from the surrounding cells, late cell death, broke into pieces and with the GAA concentration increased, extension of time, the phenomenon was more obvious.2、GAA showed proliferation inhibitary effect on Cal-27 cells treated for 24h to 72h through CCK-8 assay,and the inhibitary effects had a dose-dependent and time-dependent fashion in a certain range.IC50 of GAA for 24h、48h、72h was 12.56μmol/L、5.87μmol/L、4.00μmol/L respectively.3、 The result of MS-PCR and BS-PCR showed the hypermethylation of CpG island in ER gene were partly reversed after GAA treatment (2.5μmol/L,5.0μmol/L,10μmol/L GAA for 48h).4、Compared with the control group, the results of QPCR showed that mRNA expression of ER、PR、c-fos gene in Cal-27 cells were increased after GAA treatment (2.5μmol/L,5.0μmol/L,10.0μmol/L GAA for 48h). (P<0.05)Conclusions:1、GAA can inhibit proliferation of Human tongue squamous cell carcinoma Cal-27 cell line in a dose-dependent and time-dependent fashion.2、GAA has demethylation effect on hypermethylated region of ER gene in Human tongue squamous cell carcinoma Cal-27 cell line.3、GAA can upgrade the mRNA expression of ER、PR、c-fos gene in Human tongue squamous cell carcinoma Cal-27 cell line.