氢醌对Jurkat细胞自噬及Akt/Foxo3a信号通路的影响

Effects of hydroquinone on autophagy and Akt/Foxo3a signal pathway in Jurkat cells

目的探究氢醌(HQ)对人T淋巴细胞白血病系Jurkat细胞自噬相关的信号通路及其作用机制。方法(1)取对数生长期的Jurkat细胞分别用终浓度为0μmol·L^(-1)(对照)、12.5μmol·L^(-1)和25μmol·L^(-1) HQ溶液的培养基培养24h后,采用蛋白质印迹试验(Western-Blotting)检测自噬关键蛋白P62、LC3B,免疫荧光法检测LC3荧光斑点聚集情况;(2)利用基因表达数据库,选取CD34+细胞样本中经HQ作用后差异表达的微小mRNA(miRNA),参考TargetScan、miRDB和miRwalk基因预测工具的结果,分析其可能调控的靶基因并进行信号通路富集分析;(3)根据分析结果,采用实时荧光定量聚合酶链式反应(RT-PCR)检测HQ染毒Jurkat细胞后蛋白激酶B/叉头框蛋白O3A(Akt/Foxo3a)信号通路中Akt、Foxo3a基因表达水平,Western-Blotting检测Akt和Foxo3a及磷酸化Akt(p-Akt)和Foxo3a(p-Foxo3a)蛋白表达水平。结果(1)与对照组相比,25μmol·L^(-1)浓度组自噬关键蛋白P62表达下降,12.5μmol·L^(-1)和25μmol·L^(-1)浓度组LC3Ⅱ/LC3Ⅰ比值升高,差异均有统计学意义(P<0.05),同时免疫荧光法结果显示LC3荧光斑点聚集随HQ浓度的升高而增多;(2)经生物信息学分析后得到AMPK、MAPK、Hedgehog和PI3K/Akt等与细胞自噬相关的信号通路;(3)RT-PCR和Western-Blotting结果显示,HQ染毒组Akt/Foxo3a通路关键基因和蛋白Akt和Foxo3a的表达水平均降低,且蛋白的磷酸化水平降低,25μmol·L^(-1)浓度组均低于12.5μmol·L^(-1)浓度组,差异均有统计学意义(P<0.05)。结论氢醌可诱导Jurkat细胞自噬水平增加,其机制可能与Akt/Foxo3a信号通路被抑制有关。

Objective To investigate the effect of HQ on autophagy in human leukemia Jurkat T-cells and its mechanism.Methods(1)Exponentially growing Jurkat cells were cultured in vitro with HQ at the concentrations of 0(control group),12.5 and 25μmol·L^(-1)for 24 hours.Western blotting was used to detect the protein levels of autophagy markers P62 and LC3B.Aggregations of LC3 fluorescent spots were determined by immunofluorescence technique.(2)Using the gene expression database,differentially expressed microRNAs(miRNAs)in CD34+cells treated with HQ were selected to predict target genes through gene prediction tools such as TargetScan,miRBD and miRwalk.Enrichment analysis was conducted on these target genes to obtain relevant signal pathways.(3)Based on the results above,RT-PCR was used to examine the expression of Akt and Foxo3a genes in Akt/Foxo3a signal pathway,while western blotting was performed to determine protein expression levels of Akt,p-Akt,Foxo3a and p-Foxo3a in Jurkat cells exposed to HQ.Results(1)Compared with the control group,the expression of autophagy key protein P62 decreased in 25μmol·L^(-1)group,and the ratio of LC3II/LC3I increased in 12.5μmol·L^(-1)and 25μmol·L^(-1)groups(P<0.05).The results of immunofluorescence showed that the aggregation of LC3 fluorescent spots increased with the increase of HQ concentration.(2)Bioinformatic analysis revealed several autophagy-related signaling pathways including AMPK,MAPK,Hedgehog and PI3K/Akt signal pathways.(3)HQ treatment decreased gene expression levels of Akt and Foxo3a in Akt/Foxo3a signal pathway,and the protein levels of Akt,p-Akt,Foxo3a and p-Foxo3a were also significantly decreased in cells treated with HQ in a concentration dependent manner(P<0.05).Conclusion HQ may promote autophagy in Jurkat cells,which may be partly mediated through the inhibition of Akt/Foxo3a signal pathway.