姜黄素通过上调miR-34a抑制人神经母细胞瘤SH-SY5Y细胞的恶性生物学行为

Curcumin inhibiting malignant biological behaviors of human neuroblastoma SH-SY5Y cells by up-regulating miR-34a

目的探讨姜黄素对人神经母细胞瘤SH-SY5Y细胞恶性生物行为的影响及其可能作用机制。方法CCK8法检测姜黄素(0、6.25、12.5、25、50和100μmol·L^(-1))作用下细胞活力并筛选出后续姜黄素处理浓度(0、6.25、12.5、25μmol·L^(-1)),流式细胞法、划痕实验、Transwell实验和Western blot分别检测细胞凋亡、迁移、侵袭及相关蛋白水平。使用qRT-PCR法检测SH-SY5Y细胞miR-34a表达水平,构架miR-34a低表达SH-SY5Y细胞系(miR-34a inhibitor组)及其阴性对照(NC组)、对照组(Control组),三组细胞系均给予姜黄素(25μmol·L^(-1))处理分别记为黄素+miR-34a inhibitor组、姜黄素+NC组、姜黄素组,另设置空白对照组,采用上述相同方法检测各组细胞凋亡、迁移、侵袭及相关蛋白水平和p-p65、p-IκBα蛋白水平。结果当姜黄素干预浓度达到12.5μmol·L^(-1)时,SH-SY5Y细胞存活率显著低于无姜黄素处理的细胞(P<0.05);姜黄素(12.5、25μmol·L^(-1))组细胞凋亡率、Cleaved cas3/cas3、Cleaved cas9/cas9、E-cadherin蛋白表达和miR-34a表达水平均显著高于姜黄素(0μmol·L^(-1))组(P<0.01),划痕细胞闭合率、侵袭细胞数目和N-cadherin、Snail蛋白表达均显著低于黄素(0μmol·L^(-1))组(P<0.05);姜黄素组细胞凋亡率和Cleaved cas3/cas3、Cleaved cas9/cas9、E-cadherin蛋白表达均显著高于空白对照组(P<0.01),而划痕细胞闭合率、侵袭细胞数目和N-cadherin、Snail、p-p65/p65、p-IκBα/IκBα蛋白表达均显著低于空白对照组(P<0.01);姜黄素+miR-34a inhibitor组细胞凋亡率和Cleaved cas3/cas3、Cleaved cas9/cas9、E-cadherin蛋白表达均显著低于姜黄素+NC组(P<0.01),而划痕细胞闭合率、侵袭细胞数目和N-cadherin、Snail、p-p65/p65、p-IκBα/IκBα蛋白表达均显著高于姜黄素+NC组(P<0.01)。结论姜黄素可以促进SH-SY5Y细胞凋亡、抑制细胞迁移和侵袭,可能与上调miR-34a表达抑制NF-κB信号通路有关。

Objective To explore the effects and possible action mechanism of curcumin on malignant biological behaviors of human neuroblastoma SH-SY5Y cells.Methods The cells viability treated with curcumin(0,6.25,12.5,25,50,100μmol·L~(-1))was detected by CCK8,and the subsequent treatment concentration of curcumin(0,6.25,12.5,25μmol·L~(-1))were screened out.The cells apoptosis,migration,invasion and levels of related proteins were detected by flow cytometry,scratch assay,Transwell assay and Western blot.The expression level of miR-34a in SH-SY5Y cells was detected by qRT-PCR.The SH-SY5Y cells line with low-expression miR-34a(miR-34a inhibitor group),its negative control(NC group)and control group(Control group)were set up.The cell lines in the three groups were treated with curcumin(25μmol·L~(-1))and included into curcumin+miR-34a inhibitor group,curcumin+NC group and and curcumin group,respectively.And blank control group was set up.The cells apoptosis,migration and invasion,and levels of related proteins,p-p65 and p-IκBαin each group were detected by the above-mentioned method.Results The survival rate of SH-SY5Y cells treated with curcumin(12.5μmol·L~(-1))was significantly lower than that without curcumin treatment(P<0.05).The apoptosis rate,expressions of Cleaved cas3/cas3,Cleaved cas9/cas9 and E-cadherin,and expression level of miR-34a in curcumin(12.5,25μmol·L~(-1))groups were significantly higher than those in curcumin(0μmol·L~(-1))group(P<0.01),while closure rate of scratched cells,number of invasion cells,expressions of N-cadherin and Snail were significantly lower than those in curcumin(0μmol·L~(-1))group(P<0.05).The apoptosis rate,expressions of Cleaved cas3/cas3,Cleaved cas9/cas9 and E-cadherin in curcumin group were significantly higher than those in blank control group(P<0.01),while closure rate of scratched cells,number of invasion cells,expressions of N-cadherin,Snail,p-p65/p65 and p-IκBα/IκBαwere significantly lower than those in blank control group(P<0.01).The apoptosis rate,expressions of Cleaved cas3/cas3,Cleaved cas9/cas9 and E-cadherin in curcumin+miR-34a inhibitor group were significantly lower than those in curcumin+NC group(P<0.01),while closure rate of scratched cells,number of invasion cells,expressions of N-cadherin,Snail,p-p65/p65 and p-IκBα/IκBαwere significantly higher than those in curcumin+NC group(P<0.01).Conclusion Curcumin can promote apoptosis of SH-SY5Y cells,inhibit cells migration and invasion,which may be related to up-regulating miR-34a expression and inhibiting NF-κB signaling pathway.