miR-92a/Dickkopf相关蛋白1介导丁酸钠调控Wnt/β-catenin信号通路抑制结肠癌细胞增殖

Sodium Butyrate Inhibits Colon Cancer Proliferation Through miR-92a/DKK1-Mediated Inhibition of the Wnt/β-catenin Pathway

近年来已有研究表明,组蛋白去乙酰化酶抑制剂(HDACs)丁酸钠可以抑制结肠癌细胞增殖,促进结肠癌细胞凋亡,但其分子调控机制仍有待于深入研究。在本研究中,CCK-8检测、5-乙炔基-2-脱氧尿嘧啶核苷(Edu)增殖实验和流式细胞术分析证实,丁酸钠处理结肠癌细胞24 h后结肠癌细胞增殖能力明显减弱,凋亡率明显增加(P<0.01)。进一步转录物组测序结果表明,丁酸钠处理结肠癌细胞后,Wnt/β-catenin信号通路的拮抗剂Dickkopf相关蛋白1(DKK1)水平的表达显著高于未处理组(P<0.01);荧光定量逆转录-聚合酶链反应(qRT-PCR)和Western印迹分析进一步验证,丁酸钠可以显著诱导DKK 1的表达水平,同时抑制Wnt/β-catenin信号通路下游靶基因c-Myc的表达水平(P<0.01)。沉默结肠癌细胞的DKK 1后进一步使用丁酸钠进行处理,Edu增殖实验和流式细胞术结果显示,抑制结肠癌细胞中DKK 1后可以逆转丁酸钠对结肠癌细胞的作用(P<0.01)。生物信息学预测数据库分析显示,DKK 1可能与miR-92靶向结合;qRT-PCR结果显示,丁酸钠可以明显降低结肠癌细胞中miR-92a的表达(P<0.01);Western印迹技术和双萤光素酶报告基因检测证实,miR-92a与DKK 1 mRNA的3′-UTR存在靶向结合关系(P<0.01)。过表达结肠癌细胞中的miR-92a后进一步使用丁酸钠进行处理也能逆转丁酸钠对结肠癌细胞的作用(P<0.01)。总之本研究明确了,丁酸钠可以通过miR-92a/DKK1调控Wnt/β-catenin信号通路进而抑制结肠癌细胞增殖,促进结肠癌细胞凋亡。

Recent studies have shown that the histone deacetylase(HDAC)inhibitor sodium butyrate can inhibit the proliferation and promote apoptosis of colon cancer cells,but its molecular mechanism remains unclear.We used the CCK-8 assay,5-acetyl-2-deoxyuridine(EdU)proliferation assay and flow cytometry analysis to show that sodium butyrate inhibited the proliferation of colon cancer cells,and their apoptosis rate was significantly increased(P<0.01).Further RNA-seq analysis showed that the level of Dickkopf-related protein 1(DKK1),an antagonist of the Wnt/β-catenin signaling pathway,was significantly increased in sodium butyrate-treated colon cancer cells(P<0.01).Fluorescence quantitative reverse transcription-polymerase chain reaction(fluorescence quantitative reverse transcription-polymerase chain reaction,qRT-PCR)analysis and Western blot further verified that sodium butyrate could significantly promote the expression of DKK 1 while inhibiting the expression of c-Myc,a downstream target gene of the Wnt/β-catenin signaling pathway(P<0.01).The EdU assay and flow cytometry results showed that inhibition of DKK1 in colon carcinoma cells could reverse the regulation of sodium butyrate on colon carcinoma cells(P<0.01).Bioinformatics prediction showed that DKK1 may bind to miRNA-92a,and qRT-PCR showed that sodium butyrate could significantly downregulate miRNA-92a expression in colon carcinoma cells.The Western blot and dual luciferase reporter gene assays further confirm that the binding relationship between miRNA-92a and the 3′-UTR of DKK 1 mRNA(P<0.01).Finally,overexpression of miRNA-92a in colon carcinoma cells could also reverse the regulation of sodium butyrate on proliferation and apoptosis in colon carcinoma cells(P<0.01).In conclusion,this study clarified that sodium butyrate can regulate the Wnt/β-catenin signaling pathway through miR-92a/DKK1,and then inhibit cell proliferation and promote apoptosis of colon carcinoma cells.