NKD1调控Wnt/β-catenin信号通路影响结直肠肿瘤细胞周期的机制研究

Study on the mechanism of NKD1 regulating Wnt/β-catenin signaling pathway affecting colorectal cancer cell cycle

目的探讨NKD1调控Wnt/β-catenin信号通路影响结直肠肿瘤细胞周期的作用机制。方法利用免疫组化和RT-PCR方法,检测NKD1在结直肠肿瘤癌组织和癌旁组织中的表达。选择人结直肠癌细胞SW620和HT29,分别转染NKD1干扰(NKD1 siRNA)慢病毒载体,RT-PCR和Western blot检测转染效果;定量蛋白质组学联合生信分析筛选NKD1富集的生物学功能及信号通路;CCK8、平板克隆实验检测NKD1对细胞增殖能力的影响;RT-PCR和Western blot检测NKD1对细胞周期标志物表达的影响;Western blot检测Wnt/β-catenin信号通路核心分子的蛋白表达。结果NKD1在结直肠癌组织中表达高于癌旁组织(P<0.05);下调NKD1导致SW620和HT29细胞中NKD1 mRNA和蛋白表达水平均降低(P<0.05);定量蛋白质组学联合生信分析显示,下调NKD1差异蛋白主要富集细胞增殖和细胞周期生物过程,且显著富集细胞周期信号通路;相比对照组(Control组和NC组),siNKD1组SW620和HT29细胞增殖能力明显下降(P<0.05),细胞周期标志物CyclinD1、CDK2、CDK4、CDK6 mRNA和蛋白表达均降低(P<0.05)。下调NKD1促进Wnt/β-catenin信号通路核心分子β-catenin、GSK3β、Axin2、c-Jun蛋白表达升高(P<0.05)。结论NKD1异常表达与结直肠癌发病机制相关,NKD1通过负向调控Wnt/β-catenin信号通路促进结直肠癌细胞增殖和细胞周期。

Objective To investigate the effect of NKD1 on colorectal cancer cell cycle by regulating Wnt/β-catenin signaling pathway.Methods Immunohistochemistry and RT-PCR were used to detect the expression of NKD1 in colorectal cancer tissues and adjacent tissues.Human colorectal cancer cells SW620 and HT29 were transfected with NKD1 interference(NKD1 siRNA)lentiviral vector,and the transfection efficiency was detected by RT-PCR and Western blot;the biological functions and signaling pathways of NKD1 enrichment were screened by quantitative proteomics combined with bioinformatics analysis;CCK8 and clone formation assay were used to detect the proliferation ability of NKD1 on SW620 and HT29 cells;the effects of NKD1 on the expression of cell cycle markers were detected by RT-PCR and Western blot;the protein expression of Wnt/β-catenin signaling pathway was detected by Western blot.Results The expression of NKD1 in colorectal cancer tissues was higher than that in adjacent tissues(P<0.05);the mRNA and protein expression levels of NKD1 in SW620 and HT29 cells were significantly decreased after NKD1 silencing(P<0.05);quantitative proteomics analysis showed that down-regulation of NKD1 differential proteins mainly enriched cell proliferation and cell cycle biological processes;Compared with the control group and NC group,the proliferation ability of SW620 and HT29 cells in siNKD1 group was significantly decreased(P<0.05),and the mRNA and protein expression of cell cycle markers,including CyclinD1,CDK2,CDK4 and CDK6 were decreased(P<0.05).The expression ofβ-catenin,GSK3β,Axin2 and c-Jun were promoted by NKD1 silencing(P<0.05).Conclusion The abnormal expression of NKD1 is related to the pathogenesis of colorectal cancer.NKD1 promotes the proliferation and cell cycle of colorectal cancer cells by negatively regulating Wnt/β-catenin signaling pathway.