基于Wnt/β-catenin信号对胡椒碱抗肿瘤作用机制的研究

Study on Anti Tumor Mechanism of Piperine Based on Wnt/β-catenin Signal

目的研究小剂量胡椒碱通过下调Wnt/β-catenin信号分子的表达调控胃癌细胞HGC-27增殖及侵袭的机制。方法体外培养人胃癌HGC-27细胞,设置梯度浓度的胡椒碱,CCK-8法检测24h细胞的活力,流式细胞术检测细胞凋亡率。将细胞分为正常组(HGC-27)细胞、对照组(Wnt/β-catenin抑制剂组)、实验组(小剂量胡椒碱)。CCK-8法检测12,24,48h细胞的活力,BrdU标记流式细胞术检测细胞增殖率、划痕实验检测细胞迁移能力,Transwell小室检测细胞侵袭能力,Western-blot法检测Wnt/β-catenin信号分子的表达以及MMP2、MMP9和N-cadherin的表达水平,RT-qPCR检测β-catenin、MMP2、MMP9、N-cadherin的mRNA表达水平。结果5μmol·L^-1的胡椒碱可以抑制细胞的增殖但无明显细胞毒性,可以显著降低HGC-27细胞的增殖能力,且具有时间依赖性,相比正常组差异具有统计学意义(P<0.05)。划痕试验和Transwell小室试验结果显示,5μmol·L^-1的胡椒碱可以降低HGC-27细胞的迁移能力和侵袭能力,相比正常组差异具有统计学意义(P<0.05)。胡椒碱可以显著下调Wnt/β-catenin信号分子的表达,以及关键蛋白MMP2、MMP9、N-cadherin的表达水平,相比正常组差异具有统计学意义(P<0.05)。RT-qPCR结果显示,β-catenin、MMP2、MMP9、N-cadherin的mRNA表达水平相比正常组显著下调(P<0.05)。结论小剂量的胡椒碱可以通过下调Wnt/β-catenin信号分子的表达调控胃癌细胞HGC-27的增殖及侵袭,可能是胡椒碱抗肿瘤的作用机制之一。

OBJECTIVE To study the mechanism of piperine at low dose regulating the proliferation and invasion abilities of gastric cancer HGC-27 cells by down-regulating the expression of Wnt/β-catenin signaling molecules.METHODS HGC-27 cells were cultured in vitro and exposed to gradient concentration of piperine.The cell viability after 24 h was measured by CCK-8 assay,and apoptotic rate was analyzed by flow cytometry.The cells were divided into normal group(HGC-27 cell), control group(Wnt/β-catenin inhibitor group) and experimental group(low dose piperine).CCK-8 assay was used to detect cell viability after 12,24,48 h.BrdU labeled flow cytometry was used to detect cell proliferation rate,the cell migration ability was detected by scratch test,the cell invasion ability was detected by Transwell assay.The protein expression of Wnt/β-catenin and the matrix metalloproteinases MMP2,MMP9 and cadherin N-cadherin were determined by Western blot.The mRNA expression of β-catenin,MMP2,MMP9 and N-cadherin was detected by RT-qPCR.RESULTS Compared with normal group,piperine at 5 μmol·L^-1 inhibited cell proliferation without obvious cytotoxicity,time-dependently reduced the proliferation of HGC-27 cells with statistical significant(P<0.05).The results of scratch test and Transwell experiment showed that piperine at 5 μmol·L^-1 reduced the migration and invasion abilities of the HGC-27 cells as compared with normal group(P<0.05).Piperine significantly reduced the expression of Wnt/β-catenin signaling molecules,as well as the protein levels of MMP2,MMP9 and N-cadherin as compared with normal group(P<0.05).RT-qPCR results showed that the mRNA expression levels of β-catenin,MMP2,MMP9 and N-cadherin were significantly lower than those in normal group(P<0.05).CONCLUSION Piperine at low dose can regulate the proliferation and invasion of gastric cancer HGC-27 cells by down-regulating the expression of Wnt/β-catenin signaling molecules,which may be one of the anti-tumor mechanisms of piperine.