DKK4增强结肠癌细胞5-FU耐药的作用和意义

Effect of DKK4 Enhancing 5-FU Resistance in Colon Cancer

摘要: 目的：观察DKK4在结肠癌肝转移组织中的表达差异和DKK4基因对结肠癌细胞耐受5-FU的作用。方法：收集17例同时性结肠癌肝转移肿瘤组织，通过免疫组化分析原发灶和转移灶DKK4蛋白的表达差异。利用CRISPR-Cas9技术敲除人结肠癌SW480细胞DKK4基因，使用蛋白电泳和RT-PCR评估基因编辑效果，并分析DKK4表达对结肠癌细胞耐受5-FU杀伤的影响。结果：在17例结肠癌肝转移组织中，原发灶DKK4染色阳性率为20% (2/10)，肝转移灶染色阳性率70% (7/10)，提示肝转移灶DKK4蛋白表达明显高于结肠原发灶。利用CRISPR/Cas9技术成功敲除人结肠癌SW480细胞DKK4基因，在含有5%、10% 5-FU的培养基中，WT-DKK4 SW480细胞可继续增殖，但DKK4-KO SW480细胞则随5-FU浓度增加而加速死亡。结论：结肠癌肝转移灶组织中DKK4表达更高，且DKK4表达水平对结肠癌细胞耐受5-FU杀伤有极为重要的影响，提示DKK4可能是影响肿瘤耐受5-FU化疗的重要因素和潜在治疗靶点。

Abstract: Aims: To investigate the expression difference of DKK4 protein between primary colon cancer and liver metastases, and fluorouracil-tolerated effect of DKK4 gene in human colon cancer cells. Methods: Synchronous colon cancer liver metastasis cases and tissue samples were collected, ex-pression of DKK4 between colon cancer and liver metastasis were analyzed via immunohistochem-istry (IHC). DKK4 gene in human SW480 colon cancer cells was knocked out via CRISPR/Cas9 tech-nique and gene editing efficiency was analyzed by Western Blotting and Real-time PCR. Also, 5-FU resistance was tested for investigating the importance of DKK4 in colon cancer. Results: In 17 colon cancer liver metastasis cases, positive rate of DKK4 expressions were 35% in primary site and 75% in liver metastasis, which showed that liver metastases had higher DKK4 expression than colon primary sites. We successfully knocked out DKK4 gene in human SW480 colon cancer cells by using CRISPR/Cas9. Wild-type SW480 cells grew normally in 5%/10% 5-FU culture medium, while DKK4-KO SW480 cells accelerated death with increasing 5-fu concentration. Conclusion: DKK4 ex-pression was higher in liver metastasis than in colon primary site, and was critical to 5-FU tolerance in colon cancer. This result indicated that DKK4 was an important factor in 5-FU resistance and a potential therapeutics target.