肝特异性转录因子FOXA2在人结直肠癌肝转移阶梯模型中的表达变化及其意义

Expression of liver-specific transcription factors FOXA2 in human stepwise metastatic colorectal carcinoma model and its significance

目的探究人结直肠癌肝转移阶梯模型中FOXA2的表达变化及相关意义。方法采用Western blot技术和实时荧光定量PCR检测了常见结直肠癌细胞系中FOXA2的表达情况;在高表达细胞系SW-620细胞中转染FOXA2敲低质粒或阴性对照质粒,并用Western blot技术验证转染效果,以及检测上皮间质转化相关蛋白的表达变化。采用体内连续筛选法,通过在裸鼠体内进行SW-620结肠癌细胞株脾脏包膜下种植,经过连续三次肝转移筛选,构建结直肠癌肝转移阶梯模型。采用免疫组织化学染色法检测阶梯模型中肝转移瘤组织FOXA2的表达变化。采用Western blot技术检测模型中肝转移阶梯细胞的FOXA2、E-cadherin、N-cadherin、Vimentin、p-PI3K、p-Akt、PI3K、Akt相关蛋白的表达变化。采用划痕愈合实验方法分析模型中肝转移阶梯细胞迁移能力的变化。结果SW-620细胞中FOXA2的表达成功被下调后,E-cadherin表达上升,N-cadherin表达下降(P<0.05)。在结直肠癌肝转移阶梯模型中,每一代肝转移瘤的成瘤率,转移灶数量,瘤体积逐渐升高(P<0.05);每一代肝转移瘤组织FOXA2免疫组化染色的阳性率逐渐升高(P<0.05);相对于初始SW-620细胞株,经过体内连续筛选的三代肝转移瘤细胞(CRLMC)中FOXA2表达呈现阶梯状上升趋势(P<0.05),N-cadherin、Vimentin蛋白表达水平明显上升(P<0.05);E-cadherin蛋白表达水平明显下降(P<0.05),p-PI3K/PI3K、p-AKT/AKT蛋白表达之比逐渐升高(P<0.05);划痕愈合实验显示每一代肝转移瘤细胞迁移能力逐渐增强(P<0.05)。结论肝特异性转录因子FOXA2在人结直肠癌肝转移阶梯模型中的表达逐渐上升,促进了肝转移瘤细胞的上皮间质转化.

Objective To explore the expression of liver-specific transcription factors FOXA2 in human stepwise metastatic colorectal carcinoma model and its significance.Methods The expression of FOXA2 in common colorectal cancer cell lines was detected by Western blot and real-time fluorescent quantitative PCR;Transfect FOXA2 knockdown plasmid or negative control plasmid into the highly expressed SW-620 cells,and verify the transfection effect with Western blot.The expression changes of epithelial mesenchymal transformation related proteins was detected by Western blot.SW-620 ceils were transplanted into the spleen capsule of nude mice,then the liver metastatic cells were picked out and transplanted into the spleen capsule of new nude mice for two times.After three times of liver metastasis,the stepwise metastatic colorectal carcinoma model was established by in vivo selection.The Expression of FOXA2 in stepwise colorectal liver metastatic tumors were detected by Immunohistochemical staining.The expression of FOXA2、E-cadherin、N-cadherin、Vimentin、p-PI3K、p-Akt、PI3K、Akt in stepwise colorectal liver metastatic cells were detected by Western blot.The migration of stepwise colorectal liver metastatic cells were analyzed by wound healing assays.Results After down-regulating the expression of FOXA2 in SW-620 cells,the expression of EMT-related protein E-cadherin increased,while N-cadherin decreased significantly(P<0.05).The tumor formation rate,number of metastases,tumor weight and the positive rate of FOXA2 immunohistochemical staining of each generation of stepwise liver metastases gradually increased(P<0.05)in the model.After three times of liver metastasis,the expression of FOXA2,N-cadherin,Vimentin and the rate of p-PI3K/PI3K,p-AKT/AKT in colorectal liver metastatic cells screened continuously in vivo selection showed a stepwise upward trend compared with the initial SW-620 cell line(P<0.05),while the E-cadherin protein expression decreased significantly(P<0.05).Wound healing assays showed that stepwise colorectal liver metastatic cells(CRLMC)had stronger migration ability(P<0.05).Conclusion The expression of liver-specific transcription factors FOXA2 gradually increased in human stepwise metastatic colorectal carcinoma model,which promoted the epithelial mesenchymal transformation of liver metastatic cells.