PTPN12甲基化调控胃癌细胞增殖的研究

Study on PTPN12 methylation regulating the proliferation of gastric cancer cells

目的探讨胃癌细胞PTPN基因启动子区甲基化及其表达与细胞生长周期的影响。方法采用甲基化特异性PCR法(MSP)检测不同分化胃癌细胞(AGS、MKN45、SGC7901、MGC803及MKN28)及正常胃黏膜细胞株(GES)中PTPN12基因甲基化状态;Western blot检测各细胞系PTPN12表达水平;流式细胞术检测甲基化抑制药物5-氮杂胞嘧啶核苷(5-azacytidine,5-Aza C)及特异性siRNA处理后胃癌细胞生长周期变化。结果各胃癌细胞株均有PTPN12基因启动子的甲基化,甲基化水平与PTPN12表达相关,且与胃癌细胞分化程度相关。5-Aza C处理后,各胃癌细胞株PTPN12蛋白表达均有所恢复。PTPN12甲基化程度最高且表达量最低的MKN45进行5-Aza C处理后,细胞生长被阻滞在G0/G1期[(56.82±6.37)%],与对照相比差异有统计学意义(P<0.05)。此外,PTPN12 siRNA转染低甲基化且高表达PTPN12的MGC803胃癌细胞,S期细胞相对增多[(51.32±7.32)%,P<0.05],细胞增殖能力增强。结论胃癌细胞PTPN12基因启动子区异常甲基化,可能是导致其表达异常的主要原因,也可能是导致胃癌发生、发展的重要机制之一。

Objective To investigate the methylation status of PTPN 12 promoter region and the effects on cell cycle of human gastric cancer cell lines .Methods The methylation status of PTPN12 promoter and expression of PTPN12 protein in different differentiated gastric cancer cell lines （AGS,MKN45, SGC7901 ,MGC803 and MKN28 ） and normal gastric mucosa cell line （ GES ） were measured by methylation specific polymerase chain reaction（MSP）and Western blot,respectively.The cell cycle distributions of gastric cancer cells treated with anti-methylation drug（5-azacytidine,5-AzaC）and specific siRNA were assessed by flow cytometry.Results The methylation of PTPN12 gene in promoter region was found in all gastric cancer cell lines.The level of PTPN12 methylation was associated with the expression of PTPN 12 protein and cell differentiation .PTPN12 expressions were recovered after treatment with 5-AzaC in gastric cancer cell lines . MKN45 cells,the highest methylation level of PTPN12 and the lowest expression of PTPN12 protein,were arrested in G0/G1 phase [（56.82 ±6.37）%]after treatment with 5-AzaC,compared with the control group had significant difference （P〈0.05）.In addition,PTPN12 siRNA was transfected into MGC803 cells,the lowest methylation level of PTPN12 and the highest expression of PTPN12 protein,and the cells in S phase was increased [ （ 51.32 ±7.32 ）%, P 〈0.05 ] , and the proliferation ability of cells was enhanced . Conclusion Hypermethylation may be the main cause that leads to the decrease of the PTPN 12 expression in gastric cancer ,and it may be the major mechanism that contributes to tumorigenesis and the progression of gastric cancer .