双参消瘤汤调控Wnt/β-catenin信号通路防治胃癌的分子机制研究

Molecular Mechanism of Shuangshen Xiaoliu Decoction on Regulating Wnt/β-catenin Pathway in Prevention and Treatment of Gastric Cancer

目的:基于Wnt/β-catenin信号通路研究双参消瘤汤抑制胃癌细胞增殖防治胃癌的分子机制。方法:动物实验采用健康雄性BALB/c-nu小鼠依据MNNG喂养法建立胃癌小鼠模型,根据干预方法不同分为模型组、双参消瘤汤高剂量组、双参消瘤汤低剂量组、miR-338-3p agomir组、联合组(双参消瘤汤+miR-338-3p agomir);连续干预8周后采用ELISA法测定血清胃动素(MTL)、胃蛋白酶原Ⅰ(PGⅠ)、胃泌素(GAS)等水平,HE染色观察肿瘤组织病理,Real-time PCR法检测肿瘤组织miR-338-3p、SIRT2、Wnt、β-catenin mRNA表达水平,Western blot检测肿瘤组织SIRT2、Wnt、β-catenin蛋白表达水平。细胞实验根据干预方法不同分为模型组、双参消瘤汤高剂量组、双参消瘤汤低剂量组、miR-338-3p mimics组、miR-Con组;干预后采用MTT法检测细胞活性,Real-time PCR法检测细胞miR-338-3p、SIRT2、Wnt、β-catenin mRNA表达水平。结果:干预后,与模型组相比,双参消瘤汤高剂量组、双参消瘤汤低剂量组、miR-338-3p agomir组和联合组小鼠血清MTL、PGⅠ及GAS水平明显升高(P<0.05)。HE染色发现,双参消瘤汤高剂量组、双参消瘤汤低剂量组、miR-338-3p agomir组和联合组的胃组织病理学较模型组得到不同程度的改善。干预后,与模型组相比,双参消瘤汤高剂量组、双参消瘤汤低剂量组、miR-338-3p agomir组和联合组肿瘤组织miR-338-3p、SIRT2、Wnt、β-catenin mRNA表达水平明显升高(P<0.05)。干预后,与模型组相比,双参消瘤汤高剂量组、双参消瘤汤低剂量组、miR-338-3p agomir组和联合组肿瘤组织SIRT2、Wnt、β-catenin蛋白表达水平明显升高(P<0.05);干预后,与模型组相比,双参消瘤汤高剂量组、双参消瘤汤低剂量组和miR-338-3p mimics组细胞存活率均明显升高(P<0.05);与模型组相比,双参消瘤汤高剂量组、双参消瘤汤低剂量组和miR-338-3p mimics组胃癌细胞miR-338-3p、SIRT2、Wnt、β-catenin mRNA表达水平明显升高(P<0.05)。结论:双参消瘤汤抑制胃癌细胞增殖防治胃癌的分子机制与通过miR-338-3p靶向调控SIRT2促进Wnt/β-catenin信号通路活化有关。

Objective:To study the molecular mechanism of Shuangshen Xiaoliu Decoction(SXD)inhibiting cell proliferation of gastric cancer and preventing the disease based on Wnt/β-catenin signaling pathway.Methods:Animal experiments were conducted using healthy male BALB/c-nu mice to establish a gastric cancer mouse model based on MNNG feeding method.According to different intervention methods,the mice were divided into model group,SXD high dose group,SXD low dose group,miR-338-3p agomir group,and combination group(SXD+miR-338-3p agomir).After 8 weeks of continuous intervention,the levels of serum motilin(MTL),pepsinogen I(PGI)gastrin(GAS),etc.,were measured by ELISA;the pathological changes of tumor tissue were observed through HE staining;the mRNA expression levels of miR-338-3p,SIRT2,Wnt,andβ-catenin in tumor tissue were detected by Real-time PCR;and the protein expression levels of SIRT2,Wnt,andβ-catenin of tumor tissue were detected using Western blot technique.For cell experiment,according to different intervention methods,the mice were divided into model group,SXD high dose group,SXD low dose group,miR-338-3p mimics group,and miR-Con group.After the intervention,the cell activity was detected by MTT method,and the mRNA expression levels of cell miR-338-3p,SIRT2,Wnt,andβ-catenin were detected by Real-time PCR method.Results:After intervention,compared with the model group,the serum MTL,PGI and GAS levels of mice in SXD high dose group,SXD low dose group,miR-338-3p agomir group and combination group were significantly increased(P<0.05).HE staining showed that compared with the model group,the gastric histopathology in SXD high dose group,SXD low dose group,miR-338-3p agomir group and combination group were improved in varying degrees.After intervention,compared with the model group,the mRNA expression level of miR-338-3p,SIRT2,Wnt,andβ-catenin in tumor tissue of SXD high dose group,SXD low dose group,miR-338-3p agomir group,and the combination group were significantly increased(P<0.05).After the intervention,compared with the model group,the protein expression levels of SIRT2,Wnt,andβ-catenin in tumor tissue of SXD high dose group,SXD low dose group,miR-338-3p agomir group,and combination group were significantly increased(P<0.05).After the intervention,compared with the model group,the cell survival rate of SXD high dose group,SXD low dose group,and miR-338-3p agomir group were significantly increased(P<0.05).Compared with the model group,the mRNA expression levels of miR-338-3p,SIRT2,Wnt,andβ-catenin in gastric cancer cells in SXD high dose group,SXD low dose group,and miR-338-3p agomir group were significantly increased(P<0.05).Conclusion:The molecular mechanism of SXD to inhibit the proliferation of gastric cancer cells and prevent gastric cancer is related to the activation of Wnt/β-catenin signaling pathway through the targeted regulation of SIRT2 by miR-338-3p.