ARHI基因DNA去甲基化与胰腺癌细胞凋亡相关性

DNA demethylation of ARHI and cell apoptosis in pancreatic cancer

目的探讨胰腺癌ARHI基因DNA甲基化与ARHI表达失活的关系以及其可能的作用机制。方法采用去甲基化药物5-aza—dC干预胰腺癌细胞株PANC1及P3细胞，RT—PCR方法检测ARHI mRNA表达；ATT法检测细胞增殖；流式细胞仪和TUNEL法检测细胞周期和凋亡，Western blotting法检测磷酸化star3蛋白表达。采用裸鼠胰腺癌移植瘤周围注射5-aza—dC方法，检测移植瘤细胞凋亡及ARHI蛋白表达。结果5-aza—dC干预后，PANC1、P3细胞再表达ARHI mRNA；细胞增殖有不同程度抑制；细胞凋亡率分别从（2．43±1．00）％和（1．85±0．12）％显著增加到（32．77±10．38）％和（20．56±8．29）％（P〈0．01）；P3的G1期细胞从（58．28±0．30）％显著增加到（82．58±1．00）％（P〈0．01），S期细胞从（34．27±0．83）％显著减少到（12．12±1．18）％（P〈0．05），但PANC1细胞周期元明显变化。磷酸化stat3蛋白表达均降低，但PANC1细胞磷酸化star3蛋白降低不显著；5-aza—dC治疗裸鼠胰腺癌移植瘤后3、5、7、9d抑瘤率分别达到52．7％、78．9％、78．2％和97．1％；移植瘤细胞凋亡增加；ARHI蛋白再表达。结论DNA高甲基化是ARHI基因失活的重要机制。去甲基化药物可促进胰腺癌细胞及裸鼠胰腺癌移植瘤细胞凋亡，而这与ARHI基因再表达和磷酸化stat3蛋白表达降低相关。

Objective To elucidate the relationship of demethylation with ARHI down-regulation and investigate the possible mechanism. Methods 5-aza-dC was used to interfere the pancreatic cancer cells PANC1 and P3 cells, ARHI mRNA expression was detected by RT-PCR, cell proliferation was detected by ATT method, flow cytomytry and TUNEL were performed for measurement of cell cycles and apoptosis, star3 protein expression was measured by Western blotting. 5-aza-dC was injected for xenografts in nude mice, and the apoptosis and ARHI protein expression of xenografts were determined. Results Treatment with demethylation agent 5-aza-dC resulted in the restoration of ARHI mRNA expression in P3 and PANC1 cells. 5-aza-dC suppressed the growth of pancreatic cancer cells, and the apoptosis ratios in P3 and PANC1 cells significantly increased from （2.43 ±1.00）%, （1.85 ±0.12）% to （32.77 ±10.38）% and （20.56 ±8.29）% （P〈0.01）. G1 phase ceils of P3 significantly increased from （58.28 ± 0.30） % to （ 82.58 ± 1.00） % （ P 〈 0.01 ）. S phase cells significantly decreased from （34.27 ± 0.83） % to （ 12.12 ± 1.18） % （ P 〈 0.05 ）. However, there was no change of PANC1 cell cycles. The expression of phospho-stat3 protein reduced, but the reduction in PANC1 cells was not significant. The inhibition rates of tumor xenografts in nude mice were 52.7% , 78.9% , 78.2% and 97.1% respectively after treatment with 5-aza-dC at 3, 5, 7 and 9 day. Apoptosis was increased in treatment group and ARHI protein was induced in treatment group. Conclusions DNA hypermethylation was an important mechanism of ARHI gene inactivation. Demethylation agent inhibits human pancreatic cancer cell line growth in association with ARHI re-expression and reduced p-stat3 expression.