Abstract

SALL4 is a transcription factor highly expressed in diverse cancers and is implicated in the development of cancer. SALL4 has been implied to play a cancer-promoting role in colon cancer (CC), but the molecular mechanism remains unclear. Chromatin immunoprecipitation assay and dual-luciferase assay were conducted to verify the binding relationship of SALL4 and ROBO2. qRT-PCR detected the mRNA expression levels of SALL4 and ROBO2, and the flow cytometry analyzed the cell cycle distribution. Western blot examined SALL4 expression, and cell cycle/cell stemness-related proteins. The impact of SALL4 and ROBO2 on the proliferation capacity of cells and tumor cell stemness was elucidated by MTT, colony formation, and sphere-forming assays. SALL4 and ROBO2 were up-regulated in CC, and SALL4 could activate the transcription of ROBO2. Down-regulated SALL4 was able to significantly restrain the proliferation capacity of CC cells and arrest the cell cycle in G0/G1 phase by repressing the expression of cyclin B, cyclin E, and cyclin D1. Besides, the rescue assay results indicated that up-regulated ROBO2 could reverse the repressive impact of down-regulated SALL4 on the proliferation of CC cells and accelerate the progression of the cell cycle, thus promoting the sphere-forming of tumor stem cells. SALL4 advanced the proliferation of CC and cell stemness through direct activation of ROBO2 expression, implied the novel mechanism of SALL4 in CC, and pointed out that SALL4/ROBO2 axis was likely to be a potential target for clinical treatment of CC.

摘要

SALL4 是一种在多种癌症中高表达的转录因子，与癌症的发展有关。SALL4 已被暗示在结肠癌 (CC) 中发挥促癌作用，但其分子机制仍不清楚。通过染色质免疫沉淀实验和双荧光素酶实验验证SALL4和ROBO2的结合关系。qRT-PCR检测SALL4和ROBO2 mRNA表达水平，流式细胞仪分析细胞周期分布。Western blot 检测了 SALL4 表达和细胞周期/细胞干性相关蛋白。通过MTT、集落形成和球体形成测定阐明了SALL4和ROBO2对细胞增殖能力和肿瘤细胞干性的影响。SALL4和ROBO2在CC中表达上调，并且SALL4可以激活ROBO2的转录。下调的SALL4能够通过抑制cyclin B、cyclin E和cyclin D1的表达，显着抑制CC细胞的增殖能力，使细胞周期停滞在G0/G1期。此外，救援实验结果表明，上调的ROBO2可以逆转下调的SALL4对CC细胞增殖的抑制作用，加速细胞周期的进程，从而促进肿瘤干细胞的球形形成。SALL4通过直接激活ROBO2表达促进CC增殖和细胞干性，暗示SALL4在CC中的新机制，并指出SALL4/ROBO2轴可能成为临床治疗CC的潜在靶点。