Abstract: Demethylation Effects of TanshinoneⅡA on SMMC-7721 CellsYe ZHANG, Bin YANG, Tong BAI, Yingtang GAO, Hui LIU, Ying LUO, PengWANG, Zhi DUCorrespondence to: Zhi DU, E-mail: zhi-du@163.comThe Third Central Hospital of Tianjin, Tianjin Key Laboratory of Artificial Cell, Tianjin 300170, ChinaThis study was supported by Tianjin Health Bureau Funded Project (No. 2010KZ17 and 09KY04)Abstract Objective: To investigate the demethylation effects of tanshinoneⅡA on tumor suppressor genes of hepatocellularcarcinoma cells. Methods: SMMC-7721 cells were devided into three groups during the culture course in vitro. In tanshinone ⅡAgroup, cells were treated with tanshinoneⅡA at different doses in the culture media. Cells in the positive control group were cultured inthe media with 5-Aza-2'-deoxycytidine at 10 μmol/L. Cells in the negative group were cultured with blank media. The methylationstatus of tumor suppressor gene profile including TFPI2, SPARC, DKK3, P16, APC and MGMT was analyzed in different groups bymethylation-specific polymerase chain reaction ( PCR ). The mRNA expression of genes was examined by reverse transcription PCRand the protein expression was tested by immunocytochemistry staining. By bisulfite sequencing, methylation status of CpG sites inSPARC promoter region was estimated in different groups. Results: Both methylated and unmethylated status of TFPI2, P16 and APCcould be detected in the three groups by methylation-specific PCR. In the negative control group, methylated segments of SPARC,DKK3 and MGMT were only detected, however, both methylated and unmethylated segments of these genes were found in tanshinoneⅡA group and 5-Aza-2'-deoxycytidine group. SPARC, DKK3 and MGMT were totally methylated while TFPI2, P16 and APC werepartially methylated in SMMC-7721 cells. The mRNA expression of SPARC, DKK3 and MGMT could be restored in tanshinoneⅡAgroup compared with negative control group. Immunocytochemistry showed positive expression of SPARC in tanshinoneⅡA group.Methylation frequencies of CpG sites in SPARC promoter region were 96.53% (139/144) in the negative control group, 18.75% ( 27/144 ) in 5-Aza-2'-deoxycytidine group and 45.14% ( 65/144 ) in tanshinoneⅡA group, respectively. There was a significant differencein methylation frequencies of CpG sites among the three groups ( P < 0.001 ). Parts of methylated CpGs were converted tounmethylation status in both tanshinoneⅡA group and 5-Aza-2'-deoxycytidine group. Compared with that in the negative controlgroup, the mRNA expression of DNA methytransferaseⅠwas downregulated by 0.36-0.78 folds in tanshinoneⅡA group. Conclusion:TanshinoneⅡA might convert the methylation status of some tumor suppressor genes in SMMC-7721 cells by downregulating DNAmethytransferaseⅠ.Keywords Hepatocellular carcinoma; Tumor suppressor gene; Methylation; TanshinoneⅡA