Notch通路上调miR-223抑制FBXW7表达对结肠癌 HCT116细胞生物学的影响

摘要：目的 探讨microRNAs（miRNAs）对结肠癌HCT116细胞增殖和凋亡的影响及其作用机制。方法 经实时 定量 PCR 和 Western blot 检测 20 例结肠癌组织及癌旁正常组织标本中 FBXW7 mRNA 和其蛋白水平。通过 TargetScan 工具预测、定量 PCR 和荧光素酶活性实验鉴定与 FBXW7 结合的 miRNAs，瞬时转染 miR-223 和其对照 miCtr、抑制剂（Inhibitor）入HCT116细胞后检测FBXW7 mRNA和蛋白水平，CCK-8和流式细胞术分别检测细胞活性 和凋亡率。同时通过 siRNA 下调 Notch3 表达后，检测 miR-223 水平以及细胞凋亡率。结果 结肠肿瘤组织中 FBXW7 mRNA与蛋白含量低于癌旁组织（P＜0.05），预测并证实miR-223与miR-25能够与FBXW7基因特异结合。 HCT116细胞中瞬时转染miR-223后，FBXW7 mRNA水平和蛋白表达量下调（P＜0.01），细胞活性增加（P＜0.05），而 细胞凋亡率降低（P＜0.01）。下调Notch3通路后，miR-223表达水平下降（P＜0.001），FBXW7 mRNA水平上升（P＜ 0.01），细胞凋亡率下降（P＜0.05）。结论 Notch通路上调miR-223水平，进而抑制FBXW7基因的表达，最终促进结 肠癌细胞HCT116的增殖并抑制其凋亡。

The effect of upregulating miR-223 by Notch pathway to inhibit the expression of FBXW7 on the biology of colorectal cancer HCT116 cells

Abstract: Objective To investigate the effects of microRNAs (miRNAs) on the proliferation and apoptosis of colorectal cancer HCT116 cells and its mechanism. Methods The FBXW7 mRNA and protein levels were detected by realtime quantitative PCR and Western blot assay in 20 clinical specimens. After predicted by TargetScan tool, verified by quantitative PCR and luciferase activity assay, the miRNAs combined with FBXW7 were identified. The FBXW7 mRNA and protein levels were detected after transient transfection of miR-223 and its control miCtr and inhibitor (inhibitor) into HCT116 cells. CCK-8 and flow cytometry were used respectively to detect the cell viability and apoptosis rate. In addition, the level of miR-223 and the number of apoptosis were detected after down-regulating the expression of Notch3 by siRNA. Results The FBXW7 mRNA and protein levels were lower in colorectal tumor tissues than those in adjacent tissues (P＜ 0.05). It was predicted and confirmed that miR-223 and miR-25 could specifically bind to FBXW7 gene. After transfection of miR-223 in HCT116 cells, the FBXW7 mRNA and protein levels were down-regulated (P＜0.01), the cell viability was increased (P＜0.05) and the number of apoptotic cells was decreased (P＜0.01). After down-regulation of Notch3 pathway, miR-223 mRNA levels decreased (P＜0.001), FBXW7 mRNA levels increased (P＜0.01) and apoptotic cells decreased (P＜ 0.05). Conclusion Notch pathway upregulates the miR-223 level, which inhibits the expression of FBXW7 and eventually promotes the proliferation and inhibits its apoptosis of colon cancer cell line HCT116.