华蟾毒精通过Wnt/β-catenin信号通路抑制人结直肠癌SW-480细胞活性及迁移能力的机制研究

Study on Mechanism of Cinobufagin Inhibiting Viability and Migration Ability of Human Colorectal Cancer SW-480 Cells through Wnt/β-catenin Signaling Pathway

目的探讨华蟾毒精抑制人结直肠癌SW-480细胞活性及迁移能力的潜在分子机制。方法使用1.0μmol/L华蟾毒精处理后,检测人结直肠癌SW-480细胞的细胞活力和迁徙能力。华蟾毒精处理后,通过高通量测序和信号通路富集分析人结直肠癌SW-480细胞中异常的信号通路。通过实时荧光定量PCR、免疫印迹和分子对接分析华蟾毒精改变人结直肠癌SW-480细胞活性及迁移能力的潜在分子机制。结果华蟾毒精处理后,人结直肠癌SW-480细胞的细胞活力在2、3、4 d时显著下降(P<0.05)。同时,人结直肠癌SW-480细胞的迁移能力显著下降。高通量和信号通路富集分析发现华蟾毒精处理人结直肠癌SW-480细胞后Wnt/β-catenin信号通路中的基因被影响最多。华蟾毒精处理人结直肠癌SW-480细胞后,发现Wnt/β-catenin信号通路下游的关键基COX2、BIRC5、CCND1、MYC、AXIN2、EPCAM、WISP1的mRNA表达水平均显著下降(P<0.05)。华蟾毒精处理人结直肠癌SW-480细胞后,分离细胞的细胞核和细胞浆发现β-catenin在细胞浆中聚集,很少入核。同时,华蟾毒精处理人结直肠癌SW-480细胞后,磷酸化的β-catenin的蛋白增加。通过分子对接发现华蟾毒精与β-catenin的第332位氨基酸T具有潜在的结合能力。通过CRISPR/CAS9技术敲除人结直肠癌SW-480细胞中的β-catenin,并在敲除了β-catenin的SW-480细胞中分别构建野生型β-catenin和突变型β-catenin T332A持续表达的稳转细胞系。华蟾毒精处理具有野生型β-catenin和突变型β-catenin的人结直肠癌SW-480细胞后,发现在野生型β-catenin细胞中Wnt/β-catenin信号通路下游的关键基COX2、BIRC5、CCND1、MYC、AXIN2、EPCAM、WISP1的mRNA表达水平均显著下降(P<0.05),而在突变型β-catenin细胞中Wnt/β-catenin信号通路下游的关键基COX2、BIRC5、CCND1、MYC、AXIN2、EPCAM、WISP1的mRNA表达水平均显著上升(P<0.05)。同时,使用华蟾毒精处理突变型β-catenin的人结直肠癌SW-480细胞后,人结直肠癌SW-480细胞的细胞活力和细胞迁移能力无显著变化。结论华蟾毒素通过结合β-catenin第332位苏氨酸,增加了β-catenin的磷酸化,抑制了β-catenin进入细胞核的量,降低了Wnt/β-catenin信号通路下游的关键基COX2、BIRC5、CCND1、MYC、AXIN2、EPCAM、WISP1的转录,最终抑制人结直肠癌SW-480细胞活性及迁移能力。

Objective To investigate the potential molecular mechanism of cinobufagin inhibiting human colorectal cancer SW-480 cell viability and migration ability.Method After treatment with 1.0μmol/L cinobufagin,the cell viability and migration ability of human colorectal cancer SW-480 cells were detected.After treatment with cinobufagin,high-throughput sequencing and signal pathway enrichment were used to analyze the abnormal signal pathways in human colorectal cancer SW-480 cells.Real-time fluorescent quantitative PCR,immunoblotting and molecular docking were used to analyze the potential molecular mechanism of cinobufagin to change the viability and migration ability of human colorectal cancer SW-480 cells.Results After cinobufagin treatment,the cell viability of human colorectal cancer SW-480 cells was decreased significantly at 2 d,3 d and 4 d(P<0.05).At the same time,the migration ability of human colorectal cancer SW-480 cells was significantly reduced.High-throughput and signal pathway enrichment analysis found that the genes in the Wnt/β-catenin signaling pathway were mostly affected after the treatment of human colorectal cancer SW-480 cells by cinobufagin.After treating human colorectal cancer SW-480 cells with cinobufagin,it was found that the mRNA expression levels of COX2,BIRC5,CCND1,MYC,AXIN2,EPCAM and WISP1 which were the key downstream of the Wnt/β-catenin signaling pathway were decreased significantly(P<0.05).After treating human colorectal cancer SW-480 cells with cinobufagin,the nucleus and cytoplasm of the cells were separated and it was found thatβ-catenin accumulated in the cytoplasm and rarely entered the nucleus.At the same time,after the treatment of human colorectal cancer SW-480 cells with cinobufagin,the phosphorylatedβ-catenin protein was increased.Through molecular docking,it is found that cinobufagin has the potential to bind to amino acid T at position 332 ofβ-catenin.CRISPR/CAS9 technology was used to knock outβ-catenin in human colorectal cancer SW-480 cells,and construct wild-typeβ-catenin and mutantβ-catenin T332 A to continuously express in SW-480 cells that knocked outβ-catenin.After treating human colorectal cancer SW-480 cells with wild-typeβ-catenin and mutantβ-catenin,it was found that the mRNA expression levels of COX2,BIRC5,CCND1,MYC,AXIN2,EPCAM and WISP1 which were the key element downstream of the Wnt/β-catenin signaling pathway were significantly decreased in wild-typeβ-catenin cells(P<0.05),while in mutantβ-catenin cells,those genes were increased significantly(P<0.05).At the same time,the cell viability and cell migration ability of human colorectal cancer SW-480 cells of human colorectal cancer SW-480 cells with mutantβ-catenin treated with cinobufagin did not change significantly.Conclusion Cinobufagin induces the phosphorylation ofβ-catenin by binding to threonine at position 332 ofβ-catenin,inhibits the amount ofβ-catenin into the nucleus and reduces COX2 which is a key downstream of the Wnt/β-catenin signaling pathway.BIRC5,CCND1,MYC,AXIN2,EPCAM and WISP1 transcription,and ultimately inhibits human colorectal cancer SW-480 cell viability and migration ability.