从Wnt通路抑制因子表观遗传修饰调控研究重楼皂苷Ⅶ抗结直肠癌机制

Epigenetic modification regulation of Wnt pathway inhibitors to study the mechanism of polyphyllin Ⅶ against colorectal cancer

目的从Wnt通路抑制因子表观遗传修饰调控研究重楼皂苷Ⅶ(PPLVⅡ)抗结直肠癌机制。方法实验分三组,空白对照组、结直肠癌模型组和PPLVII治疗组。采用TUNEL法检测小鼠的细胞凋亡情况,TOPglow/FOPglow TCF试剂盒用于检测Wnt信号传导途径活性,应用双抗体夹心法ELISA(sandwich ELISA)检测cleaved caspase-3、cleaved caspase-8、cleaved caspase-9、Bax、Bcl-2以及Cdk-4、Cdk-6、Cyclin D1和p21的表达,用免疫蛋白印迹检测细胞中的β-连环蛋白,实时荧光定量PCR检测干细胞相关基因OCT4、SOX2、c-Myc和TCF4的表达,检测lgG、β-catenin和H3k9Ac与MMP-9启动子的结合。结果 TUNEL免疫组织化学分析与对照组相比,PPLVII诱导肿瘤细胞凋亡;PPLVII治疗后cleaved-caspase-3、cleaved-caspase-8、cleaved-caspase-9和Bax表达增加,而Bcl-2则降低,差异有统计学意义(P <0.05);Cyclin D1、CDK-4、CDK-6表达下降,而p21表达上调,差异有统计学意义(P <0.01);PPLVII干预治疗后检测治疗组的TOP/FOP比率降低,差异有统计学意义(P <0.05);在PPLVII进行干预治疗后β-连环蛋白灰度值下降,差异有统计学意义(P <0.05);PPLVII进行干预治疗后OCT4、SOX2、c-Myc和TCF4相关基因的表达下调,差异有统计学意义(P <0.05)。结论重楼皂苷Ⅶ干预治疗通过降低Wnt通路抑制因子的表达对结直肠癌有一定的改善和治疗作用。

Objective Inquiry PolyphyllinⅦresistance mechanism of colorectal cancer from Wnt pathways inhibitory factor the epigeNETsic modifications regulation.Methods The experiment is divided into three groups:blank control group,model group and colorectal cancer polyphyllinⅦtreatment group.TUNEL assay was used to detect apoptosis in mice.TOPglow/FOPglow TCF kit was used to detect activity of Wnt signal transduction pathway.Sandwich ELISA was used to detect expression of caspase-3,caspase-8,caspase-9,Bax,bcl-2,cdk-4,cdk-6,Cyclin D1 and p21.The expression of stem cell related genes OCT4,SOX2,c-myc and TCF4 was detected by real-time fluorescence quantitative PCR,and the binding of lgG,beta-catenin and H3 k9 Ac to mmp-9 promoter was detected.Results Compared with the control group,TUNEL immunohistochemical analysis showed that PPLVII significantly induced apoptosis of tumor cells.After PPLVII treatment,cleaved caspase-3,cleaved caspase-8,cleaved caspase-9 and Bax expression were increased,while Bcl-2 expression was decreased,the difference was significant(P<0.05).The expression of Cyclin D1,CDK-4 and CDK-6 decreased,while the expression of p21 was up-regulated,the difference was significant(P<0.01).The TOP/FOP ratio in the treatment group was decreased after PPLVII intervention.Betacatenin was detected by western blotting,The results showed that the gray level of PPLVII intervention decreased,the difference was significant(P<0.05).The expressions of OCT4,SOX2,c-Myc and TCF4 related genes were down-regulated after PPLVII intervention,the difference was significant(P<0.05).Conclusion Treatment by reducing PPLVII Wnt pathways of inhibiting factor expression in colorectal cancer have certain improvement and treatment effect.