Effect Of Arsenic Trioxide On Demethylation Of SHP-1 Gene In Cutaneous T Cell Lymphoma Cells And Its Relative Mechanisms

Objective:1.To investigate the methylation status of SHP-1 promoter in Hut102 cells,another cell line of cutaneous T cell lymphoma.2.If the SHP-1 promoter is hypermethylated,we treated Hut102 cells with As₂O₃ and analysed the changes of the methylation status of SHP-1 promoter,SHP-1 mRNA and protein levels and the proliferation and apoptosis of Hut102 cells before and after the treatment.3.To investigate the changes of STAT3,p-STAT3 and its downstream genes mRNA and protein levels along with SHP-1 changes in Hut 102 and Hut78 cells.We investigated the effects of As₂O₃ on demethylation of SHP-1 and the effect of As₂O₃ demethylation on Hut102,Hut78 cell biology and these results may provide helpful information for As₂O₃ used to the treatment of advanced-stage MF/SS.Methods:1.The methylation status of SHP-1 promoter in Hut102 cells was detected by methylation specific polymerase chain reaction（MSP）.2.The changes of methylation status of SHP-1 promoter in Hut102 cells before and after As₂O₃ treatment were detected by MSP.3.The expression levels of SHP-1 mRNA in Hut102 cells before and after AS2O3 treatment were analysed by Real-time polymerase chain reaction（Real-time PCR）.4.The expression levels of SHP-1 protein in Hut102 cells before and after As₂O₃ treatment were analysed by Western Blot.5.The changes of Hut102 cells proliferation induced by As₂O₃ were detected by MTT method.6.The changes of Hut102 cells apoptosis induced by As₂O₃ were detected by flow cytometry with Annexin V/PI staining.7.The expression levels of STAT3,p-STAT3,Bcl-2,VEGF,etc protein in Hut102 and Hut78 cells before and after As₂O₃ and SSG treatment were analysed by Western Blot.8.The expression levels of Bcl-2,VEGF,etc mRNA in Hut102 and Hut78 cells before and after As₂O₃ and SSG treatment were analysed by Real-time polymerase chain reaction（Real-time PCR）.9.The changes of Hut102 and Hut78 cells proliferation after SSG treatment were detected by MTT method.10.The changes of Hut102 cells apoptosis after SSG treatment were detected by flow cytometry with Annexin V/PI staining.11.All data were processed with SPSS for windows software version 19.0.The means of different samples were compared by using one-way ANOVA.If the difference was statistically significant,further LSD-t tests were used to analyzed the means.The level of significance was set at 0.05.Results:1.The MSP results indicated that the SHP-1 promoter in Hutl02 cells was completely methylated and there was no unmethylation strap.2.The methylation straps of SHP-1 promoter in Hut 102 cells treated with 5.0μmol/L As₂O₃ were weakened along with the prolonging of treatment time,meanwhile the unmethylation straps were enhanced.3.The expression levels of SHP-1 mRNA in Hutl02 cells increased along with the increase of As₂O₃ concentration and treatment time（P<0.05）.4.The SHP-1 protein was not detectable in untreated Hut 102 cells.After treating Hut 102 cells with 5.0μmol/L As₂O₃,the expression of SHP-1 protein increased along with the prolonging of treatment time（P<0.05）.5.As₂O₃ elicited significant inhibition of Hut102 cells proliferation and its inhibitory effects were enhanced along with the increase of concentration and time（P<0.05）.6.As₂O₃ could induce apoptosis of Hut102 cells and the apoptosis rates were significantly higher along with the increase of As₂O₃ concentration and treatment time（P<0.05）.7.The expression of VEGF protein,p-STAT3 protein and its downstream proteins decreased along with the increasing SHP-1 protein（P<0.05）.However,the expression of SHP-1 protein decreased,and the VEGF protein,p-STAT3 protein and its downstream proteins increased when SSG was used（P<0.05）.8.The mRNA level of VEGF and the downstream gene of STAT3 decreased along with the increasing SHP-1 mRNA（P<0.05）.Nevertheless,the expression of SHP-1 mRNA decreased,and the expression of VEGF and the downstream gene of STAT3 mRNA increased when SSG was used（P<0.05）.9.The effect of As₂O₃ on the proliferation inhibition of Hut102 and Hut78 cells was impaired by SSG（-P<0.05）.10.The ability of As₂O₃ on the apoptosis of Hut102 and Hut78 cells was also attenuated by SSG（P<0.05）.Conclusion:1.The SHP-1 promoter in Hut102 cells is completely methylated resulting in the transcriptional silencing of SHP-1.2.As₂O₃ can cause re-expression of SHP-1 mRNA and protein in Hut102 cells via the demethylation effect,and inhibit proliferation and induce apoptosis of Hut102 cells.Probably,the influence on the proliferation and apoptosis was implemented by accommodating VEGF and anti-apoptosis gene Bcl-2,etc.,through STAT3 protein.As₂O₃ may be active in treatment of advanced-stage MF/SS.