miR-448/RTN4轴调控结肠癌细胞对5-氟尿嘧啶耐药性的机制研究

Mechanism of Mir-448/RTN4 Axis Regulating 5-fluorouracil Resistance in Colon Cancer Cells

目的探讨结肠癌细胞对5-氟尿嘧啶(5-Fu)的抵抗及潜在机制。方法使用5-Fu筛选出HCT116和HT295-Fu抗性细胞系(HCT116/FR和HT29/FR细胞)。通过高通量测序和siRNA功能筛选鉴定5-Fu处理后HCT116/FR细胞中影响细胞凋亡水平的关键基因。过表达该基因(RTN4)后检测HCT116/FR和HT29/FR细胞的凋亡水平和细胞活力水平。过表达或敲低此miRNA(miR-448)后检测HCT116、HT29、HCT116/FR和HT29/FR细胞的细胞活力、细胞凋亡水平和RTN4的表达水平。26例结肠癌患者,按照化疗效果分为对化疗敏感型结肠癌患者(Sensitive)与化疗抵抗型结肠癌患者(Resistant),并采用免疫组织化学染色方法检测RTN4在患者中的表达水平。结果当敲低RTN4时,HCT116/FR细胞的凋亡水平下降(P<0.05)。5-Fu处理HCT116/FR细胞和HT29/FR细胞后,RTN4的mRNA水平均下调(P<0.05)。敲低RTN4后并用5-Fu处理,HCT116和HT29显示出对5-Fu抑制增殖的抵抗(P<0.05)。过表达RTN4后,HCT116/FR和HT29/FR细胞的凋亡水平上升(P<0.05)。5-Fu处理后,HT29/FR和HCT116/FR细胞中miR-448的表达水平上升(P<0.05)。5-Fu处理后并过表达miR-448后,HCT116和HT29细胞的凋亡水平下降(P<0.05)。在HCT116/FR细胞中,过表达miR-448后,RTN4的表达水平下降;敲低miR-448后,RTN4的表达水平上升(P<0.05)。在HT29/FR细胞中,过表达miR-448后,RTN4的表达水平下降;敲低miR-448后,RTN4的表达水平上升(P<0.05)。免疫组织化学分析发现对化疗敏感的结肠癌患者癌组织中RTN4表达水平较高(P<0.05)。结论当结肠癌细胞HCT116和HT29遇到5-Fu后,细胞中miR-448的表达水平上升,miR-448靶向RTN4 mRNA的3端非编码区并降解了RTN4 mRNA,减少了RTN4的蛋白表达,最终提升了结肠癌细胞HCT116和HT29对5-Fu的抗性。

Objective To investigate the resistance and potential mechanism of colon cancer cells to 5-fluorouracil(5-Fu).Methods HCT116 and HT295-Fu resistant cell lines(HCT116/FR and HT29/FR cells)were screened using 5-Fu.High-throughput sequencing and siRNA functional screening identified key genes affecting apoptosis level in HCT116/FR cells treated with 5-Fu.The apoptosis level and cell viability of HCT116/FR and HT29/FR cells were detected after overexpression of RTN4.The cell viability,apoptosis and RTN4 expression levels of HCT116,HT29,HCT116/FR and HT29/FR cells were detected after overexpression or knockdown of this miRNA(miR-448).Meanwhile,26 patients with colon cancer were included.According to the effect of chemotherapy,they were divided into chemotherapy Sensitive colon cancer patients and chemotherapy Resistant colon cancer patients.Immunohistochemical staining was used to detect the expression level of RTN4 in patients.Results When RTN4 was knocked down,the apoptosis level of HCT116/FR cells was decreased(P<0.05).The mRNA levels of RTN4 were down-regulated in both HCT116/FR cells and HT29/FR cells treated with 5-Fu(P<0.05).After RTN4 was knocked down and treated with 5-Fu,HCT116 and HT29 showed resistance to 5-Fu inhibition of proliferation(P<0.05).After overexpression of RTN4,the apoptosis level of HCT116/FR and HT29/FR cells was increased(P<0.05).After 5-Fu treatment,the expression level of miR-448 in HT29/FR and HCT116/FR cells increased(P<0.05).After 5-Fu treatment and overexpression of miR-448,the apoptosis level of HCT116 and HT29 cells was decreased(P<0.05).In HCT116/FR cells,the expression level of RTN4 decreased after overexpression of miR-448.After miR-448 knockdown,the expression level of RTN4 was increased(P<0.05).In HT29/FR cells,the expression level of RTN4 decreased after overexpression of miR-448.After miR-448 knockdown,the expression level of RTN4 was increased(P<0.05).Immunohistochemical analysis showed that RTN4 expression level was higher in colon cancer tissues sensitive to chemotherapy(P<0.05).Conclusion When colon cancer cells HCT116 and HT29 encounter 5-Fu,the expression level of miR-448 in the cells increases.miR-448 targets the 3-terminal non-coding region of RTN4 mRNA and degrades RTN4 mRNA and reduces RTN4 protein.expression,and finally increased the resistance of colon cancer cells HCT116 and HT29 to 5-Fu.