白头翁汤通过BUB1/STAT3信号通路抑制食管癌细胞生长

Baitouweng Tang Inhibits Growth of Esophageal Cancer Cells Through BUB1/STAT3 Signaling Pathway

目的:探讨白头翁汤通过调控苯并咪唑出芽抑制解除同源物蛋白1(BUB1)/信号转导及转录激活因子3(STAT3)信号通路抑制食管癌(EC)细胞生长的作用机制。方法:运用基因芯片技术探讨EC组织和正常组织相关基因表达,筛选出差异表达基因,运用生物信息学技术对差异表达基因进行分析。用25、50、100、200、400、800 mg·L^(-1)白头翁汤干预EC细胞,通过噻唑蓝(MTT)比色法检测EC9706细胞活力。用流式细胞术测定细胞周期和凋亡。通过实时荧光定量聚合酶链式反应(Realtime PCR)测定BUB1的表达。通过蛋白免疫印迹法(Western blot)测量BUB1、STAT3、磷酸化(p)-STAT3、细胞周期蛋白B1(CCNB1)、细胞周期蛋白依赖性激酶1(CDK1)、B细胞淋巴瘤-2(Bcl-2)、胱天蛋白酶-3(Caspase-3)和胱天蛋白酶-9(Caspase-9)蛋白水平。用划痕和Transwell侵袭实验测定细胞的迁移和侵袭能力。结果:差异表达基因参与生物学过程、信号通路和网络构建主要与细胞有丝分裂相关,BUB1是关键的核心基因。与空白组比较,白头翁汤抑制BUB1表达(P<0.05,P<0.01)。体外实验表明,与空白组比较,白头翁汤能明显抑制EC细胞的生长(P<0.05,P<0.01)、迁移和侵袭(P<0.05,P<0.01),诱导细胞凋亡(P<0.05,P<0.01)和G_(2)/M期升高(P<0.01)。与空白组比较,白头翁汤处理后,EC细胞中Caspase-3、Caspase-9表达明显升高(P<0.05,P<0.01),Bcl-2、BUB1、CCNB1、CDK1表达明显降低(P<0.05,P<0.01)。STAT3信号通路也被发现在此过程中发挥着重要作用。结论:白头翁汤可能通过下调BUB1介导STAT3信号通路抑制EC细胞的生长。

Objective:To investigate the mechanism of Baitouweng Tang in inhibiting the growth of esophageal cancer(EC)cells by regulating budding uninhibited by benzimidazoles 1(BUB1)/signal transducer and activator of transcription 3(STAT3)signaling pathway.Method:Gene chip technology was used to explore the differential gene expression between esophageal cancer tissues and normal tissues and identified differentially expressed genes.The differentially expressed genes were analyzed by bioinformatics methods.EC cells were treated with 25,50,100,200,400,800 mg·L^(-1) Baitouweng Tang.EC cell viability was detected by Thiazolyl Blue(MTT)colorimetry.Cell cycle and apoptosis were measured by flow cytometry.The expression of BUB1 was measured by real time quantitative polymerase chain reaction(Real-time PCR).The protein levels of BUB1,STAT3,phosphorylated(p)-STAT3,Cyclin B1(CCNB1),cyclin-dependent kinase 1(CDK1),B-cell lymphoma-2(Bcl-2),cysteinyl aspartate-specific proteinase(Caspase)-3,and Caspase-9 were measured by Western blot.The migration and invasion abilities of the cells were measured by wound-healing and Transwell invasion assays.Result:Differentially expressed genes were primarily involved in biological processes,signaling pathways,and network construction related to cell mitosis,with BUB1 identified as a key core gene.Compared with the control group,Baitouweng Tang inhibited BUB1 expression(P<0.05,P<0.01).In vitro experiments showed that compared with the control group,Baitouweng Tang could significantly inhibit the growth(P<0.05,P<0.01),migration and invasion(P<0.05,P<0.01)of EC cells,induce apoptosis(P<0.05,P<0.01),and cause G_(2)/M phase increase(P<0.01).After treatment with Baitouweng Tang,compared with the results in the control group,the expression of Caspase-3,and Caspase-9 in EC cells increased significantly(P<0.05,P<0.01),while the expression of Bcl-2,BUB1,CCNB1,and CDK1 decreased significantly(P<0.05,P<0.01).Moreover,the STAT3 signaling pathway was also found to play an important role in this process.Conclusion:Baitouweng Tang may inhibit the growth of EC cells by downregulating BUB1 and mediating the STAT3 signaling pathway.