缺氧微环境下仙连解毒方通过调控HIF-1α影响结直肠癌细胞迁移的作用及机制研究

Effect and Mechanism of Xianlian Jiedu Formula on the Migration of Colorectal Cancer Cells by Regulating HIF-1α Under Hypoxic Microenvironment

目的 通过体外实验研究缺氧微环境下仙连解毒方(Xian-Lian-Jie-Du-Formula,XLJDF)抑制结直肠癌细胞迁移的有效性及其潜在机制。方法 厌氧细胞培养箱(95%N2、5%CO_(2),O_(2)浓度<1%)中培养人结直肠癌细胞HCT-116,MTT比色法检测XLJDF对细胞活力的影响;伤口愈合实验和细胞迁移实验(Transwell法)检测XLJDF对细胞迁移能力的作用;取对数生长期的细胞分组进行转染和慢病毒感染,RTPCR检测HCT-116细胞中HIF-1α mRNA的表达;蛋白免疫印迹法(Western blot)检测HCT-116细胞中HIF-1α、EMT相关蛋白的表达情况及XLJDF对TGF-β/smad信号通路的调控作用。结果 缺氧条件下,XLJDF能抑制HCT-116细胞活性(P<0.05或P<0.01)。细胞划痕实验和Transwell细胞迁移实验示,XLJDF干预后,与空白对照组相比,细胞迁移速率显著降低(P<0.05或P<0.01),传入下室的细胞数目显著减少(P<0.05或P<0.01);与空载体组对比,XLJDF能降低HIF-1α过表达细胞株的细胞迁移速率(P<0.05或P<0.01)和减少传入Transwell下室的细胞数(P<0.05或P<0.01)。Western blot法检测EMT、TGF-β/Smad通路相关蛋白表达,与常氧组相比,缺氧条件下细胞内HIF-1α蛋白表达水平显著升高,且在24 h内呈时间依赖性;XLJDF各浓度干预组,E-钙粘蛋白(E-cadherin)的表达水平显著上升,N-钙粘蛋白(N-cadherin)、波形蛋白(Vimentin)、TGF-β1、p-Smad3的表达量均降低(P<0.05或P<0.01)。结论 缺氧微环境下仙连解毒方能减弱结直肠癌细胞HCT-116迁移能力,调控HIF-1α介导的TGF-β/smad信号通路激活、逆转EMT可能是其潜在机制之一。

Objective Experiments were conducted to study the effectiveness and potential mechanism of Xianlian Jiedu Formula in inhibiting the migration of colorectal cancer cells under hypoxic microenvironment in vitro.Methods Human colorectal cancer cells HCT-116 were cultured in an anaerobic cell incubator(95% nitrogen,5% carbon dioxide,oxygen concentration <1%),and MTT colorimetry was used to detect the effect of XLJDF on cell viability;wound healing experiments and cells migration experiment(Transwell method) was used to detect the effect of XLJDF on cell migration;Cells in the logarithmic growth phase were grouped for transfection and lentivirus infection,and RT-PCR was used to detect the expression of HIF-1α mRNA in HCT-116 cells;Western blotting was used to detect the expression of HIF-1α and EMT-related proteins in HCT-116 cells and the regulation role of XLJDF on TGF-β/smad signaling pathway.Results Under hypoxic conditions,XLJDF could inhibit the activity of HCT-116 cells(P<0.05 or P<0.01).Cell scratch test and Transwell cell migration test showed that after XLJDF intervention,the cell migration rate was significantly reduced compared with the blank control group(P<0.05 or P<0.01),and the number of cells passing into the lower chamber was significantly reduced(P<0.05 or P<0.01);Compared with the empty vector group,XLJDF can reduce the cell migration rate of HIF-1α overexpressing cell lines(P<0.05 or P<0.01) and reduce the number of cells transferred into the lower chamber of Transwell(P<0.05 or P<0.01);Western blot was used to detect the expression of EMT and TGF-β/Smad pathway-related proteins;Compared with the normoxic group,the expression level of HIF-1αprotein in cells was significantly increased under hypoxic conditions,and it was time-dependent within 24 hours;After XLJDF intervention,the expression protein level of E-cadherin increased significantly,and the expression protein of Ncadherin,Vimentin,TGF-β1、and p-Smad3 were all significantly reduced(P<0.05 or P<0.01).Conclusion XLJDF can attenuate the migration ability of colorectal cancer cells HCT-116 under hypoxic microenvironment,regulating HIF-1α-mediated activation of TGF-β/smad signaling pathway and reversing EMT may be its potential mechanism.