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中华结直肠疾病电子杂志

中华结直肠疾病电子杂志 ›› 2023, Vol. 12 ›› Issue (05) : 396 -403. doi: 10.3877/cma.j.issn.2095-3224.2023.05.006

论著

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
黄怡诚, 陆晨, 孙司正, 喻春钊†()   
  1. 210011　南京医科大学第二附属医院普外科
    211112　南京医科大学附属逸夫医院普外科
    210011　南京医科大学第二附属医院普外科；211112　南京医科大学附属逸夫医院普外科
  • 收稿日期:2023-01-11 出版日期:2023-10-25
  • 通信作者: 喻春钊
  • 基金资助:
    江苏省第五期“333工程”科研项目(BRA2020091); 国家重点研发计划（政府间国际科技创新合作重点专项）(2018YFE0127300); 江苏省社会发展重点项目(BE2019759)

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

Yicheng Huang, Chen Lu, Sizheng Sun, Chunzhao Yu†()   

  1. Department of General Surgery, the Second Affiliated Hospital of Nanjing Medical University, Nanjing 210011, China
    Department of General Surgery, Sir Run Run Hospital of Nanjing Medical University, Nanjing 211112, China
    Department of General Surgery, the Second Affiliated Hospital of Nanjing Medical University, Nanjing 210011, China; Department of General Surgery, Sir Run Run Hospital of Nanjing Medical University, Nanjing 211112, China
  • Received:2023-01-11 Published:2023-10-25
  • Corresponding author: Chunzhao Yu
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黄怡诚, 陆晨, 孙司正, 喻春钊. 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[J]. 中华结直肠疾病电子杂志, 2023, 12(05): 396-403.

Yicheng Huang, Chen Lu, Sizheng Sun, Chunzhao Yu. Expression of liver-specific transcription factors FOXA2 in human stepwise metastatic colorectal carcinoma model and its significance[J]. Chinese Journal of Colorectal Diseases(Electronic Edition), 2023, 12(05): 396-403.

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。

关键词: 结直肠肿瘤, FOXA2, 阶梯转移模型, SW-620细胞株, 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.

Key words: Colorectal neoplasms, FOXA2, Stepwise metastasis model, SW-620 cell line, EMT
图1 FOXA2在SW-620中高表达。1A：通过Western blot检测正常人结肠上皮细胞系NCM-460和结直肠癌细胞系中FOXA2的蛋白质水平；1B：正常人结肠上皮细胞系NCM-460和结直肠癌细胞系中FOXA2蛋白表达水平的直方图；1C：RT-qPCR检测正常人结肠上皮细胞系NCM-460和结直肠癌细胞系中FOXA2的相对mRNA表达水平的直方图。*P＜0.05，**P＜0.01，***P＜0.001，****P＜0.0001
图2 肝转移阶梯模型小鼠肝脏转移灶肿瘤情况
表1 阶梯状肝转移模型小鼠成瘤情况和转移灶情况统计
组别 造模数量（只） 成瘤数量（只） 肝转移率（%） 转移灶数量（个/每只） 转移瘤体积（mm3/每只）
第一代 P1 10 6 60 2.83±1.17 16.67±8.42
第二代 P2 10 8 80 6.13±2.23 206.82±81.16
第三代 P3 10 9 90 7.56±3.05 774.44±151.37
检验值 7.019 105.9
P值 ＜0.05 ＜0.01
图3 肝转移阶梯细胞转移能力越来越强。3A：通过Western blot检测SW-620和肝转移阶梯细胞中EMT标记物（E-cadherin、N-cadherin和Vimentin）的蛋白质表达水平；3B：SW-620和肝转移阶梯细胞中EMT标记物（E-cadherin、N-cadherin和Vimentin）蛋白表达水平的直方图；3C：通过Western blot检测SW-620和肝转移阶梯细胞中p-PI3K、PI3K、p-AKT、AKT的蛋白质表达水平；3D：SW-620和肝转移阶梯细胞中磷酸化蛋白与总蛋白比率的直方图；3E：划痕实验检测SW-620和肝转移阶梯细胞的迁移能力；3F：划痕实验中SW-620和肝转移阶梯细胞的24小时划痕愈合率的直方图。*P＜0.05，**P＜0.01
图4 FOXA2在肝转移阶梯模型中表达逐渐升高。4A：免疫组化染色检测FOXA2在结直肠癌阶梯肝转移组织中表达的阳性率（10×/40×）；4B：结直肠癌阶梯肝转移组织中FOXA2免疫组化染色阳性率的直方图；4C：通过Western blot检测SW-620和肝转移阶梯细胞中FOXA2的蛋白质表达水平；4D：SW-620和肝转移阶梯细胞中FOXA2蛋白表达水平的直方图。*P＜0.05，**P＜0.01
图5 干扰FOXA2影响SW-620的上皮间充质转化。5A：通过Western blot检测sh-FOXA2组和sh-NC组之间FOXA2敲低效率和EMT标记物（E-cadherin、N-cadherin和Vimentin）的不同蛋白水平；5B：sh-FOXA2组和sh-NC组中FOXA2和EMT标记物（E-cadherin、N-cadherin和Vimentin）蛋白表达水平的直方图。*P＜0.05，**P＜0.01
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