槲皮素调控GSTP1甲基化抑制前列腺癌LNCap细胞增殖的机制研究

Mechanism of quercetin regulating GSTP1 methylation to inhibit proliferation of LNCap cells in prostate cancer

目的探讨槲皮素通过调控谷胱甘肽S转移酶P1(GSTP1)启动子甲基化抑制前列腺癌细胞(LNCap)细胞增殖的机制。方法体外培养人前列腺癌细胞LNCap,分别使用不同浓度槲皮素(0、10、40、80μmol/L)处理,使用甲基转移酶抑制剂5′-氮杂-2′-脱氧胞苷(5′-Aza-dC)(10μmol/L)处理作为对照组。48h后,采用CCK-8法检测各组细胞增殖抑制率;流式细胞仪检测细胞凋亡水平;甲基化特异性PCR(MSP)检测GSTP1基因甲基化状态;Real-timePCR和Westernblot法分别检测各组LNCap细胞GSTP1mRNA及蛋白表达情况。结果在一定浓度范围内随着槲皮素用量增加和时间的增长,LNCap细胞增殖率下降(P<0.05);LNCap细胞中GSTP1启动子区的甲基化程度减弱(P<0.05);GSTP1的表达水平增高(P<0.05);并且GSTP1mRNA降低与GSTPl基因甲基化呈负相关(P<0.05),差异有统计学意义。结论槲皮素通过减弱LNCap启动子甲基化抑制LN-Cap细胞的生长增殖。

Objective To investigate the mechanism of quercetin inhibiting proliferation of prostate cancer cells(LNCap) by regulating promoter methylation of glutathione S transferase P1(GSTP1).Methods Human prostate cancer LNCap cells were cultured in vitro,and treated with quercetin( 0,10,40,80 μmol/L) and methyltransferase inhibitor 5’-aza-dc( 10 mol/L) as the control group. After 48 h,CCK-8 method was used to detect the inhibition rate of cell proliferation in each group. Cell apoptosis was detected by flow cytometry. Methylation status of GSTP1 gene was determined by methylation specific PCR( MSP). MRNA and protein expression of GSTP1 in LNCap cells were detected by real-time PCR and Western blot. Results The proliferation rate of LNCap cells decreased with the increase of quercetin dosage and time in a certain concentration range( P < 0. 05). The methylation of GSTP1 promoter in LNCap cells decreased( P < 0. 05). The expression level of GSTP1 increased( P <0. 05). Moreover,the decrease of GSTP1 m RNA was negatively correlated with the methylation of GSTPl gene( P< 0. 05). Conclusion Quercetin inhibited the growth and proliferation of LNCap cells by reducing the methylation of LNCap promoters.