Department of Colorectal Surgery, Harbin Medical University Cancer Hospital, Harbin, China.
J Cell Mol Med. 2019 Jan;23(1):630-640. doi: 10.1111/jcmm.13968. Epub 2018 Oct 19.
Colorectal cancer (CRC) remains both common and fatal, and its successful treatment is greatly limited by the development of stem cell-like characteristics (stemness) and chemoresistance. MiR-30-5p has been shown to function as a tumor suppressor by targeting the Wnt/β-catenin signaling pathway, but its activity in CRC has never been assessed. We hypothesized that miR-30-5p exerts anti-oncogenic effects in CRC by regulating the USP22/Wnt/β-catenin signaling axis. In the present study, we demonstrate that tissues from CRC patients and human CRC cell lines show significantly decreased miR-30-5p family expression. After identifying the 3'UTR of USP22 as a potential binding site of miR-30-5p, we constructed a luciferase reporter containing the potential miR-30-5p binding site and measured the effects on USP22 expression. Western blot assays showed that miR-30-5p decreased USP22 protein expression in HEK293 and Caco2 CRC cells. To evaluate the effects of miR-30-5p on CRC cell stemness, we isolated CD133 + CRC cells (Caco2 and HCT15). We then determined that, while miR-30-5p is normally decreased in CD133 + CRC cells, miR-30-5p overexpression significantly reduces expression of stem cell markers CD133 and Sox2, sphere formation, and cell proliferation. Similarly, we found that miR-30-5p expression is normally reduced in 5-fluorouracil (5-FU) resistant CRC cells, whereas miR-30-5p overexpression in 5-FU resistant cells reduces sphere formation and cell viability. Inhibition of miR-30-5p reversed the process. Finally, we determined that miR-30-5p attenuates the expression of Wnt/β-catenin signaling target genes (Axin2 and MYC), Wnt luciferase activity, and β-catenin protein levels in CRC stem cells.
结直肠癌（CRC）仍然很常见且致命，其成功治疗受到干细胞样特征（干性）和化疗耐药性发展的极大限制。miR-30-5p 通过靶向 Wnt/β-catenin 信号通路已被证明具有肿瘤抑制作用，但它在 CRC 中的活性从未被评估过。我们假设 miR-30-5p 通过调节 USP22/Wnt/β-catenin 信号轴在 CRC 中发挥抗癌作用。在本研究中，我们证明 CRC 患者组织和人 CRC 细胞系中 miR-30-5p 家族表达明显降低。在确定 USP22 的 3'UTR 是 miR-30-5p 的潜在结合位点后，我们构建了包含潜在 miR-30-5p 结合位点的荧光素酶报告基因，并测量了对 USP22 表达的影响。Western blot 检测表明，miR-30-5p 降低了 HEK293 和 Caco2 CRC 细胞中的 USP22 蛋白表达。为了评估 miR-30-5p 对 CRC 细胞干性的影响，我们分离了 CD133+CRC 细胞（Caco2 和 HCT15）。然后，我们确定，虽然 miR-30-5p 在 CD133+CRC 细胞中通常降低，但 miR-30-5p 过表达显著降低了干细胞标记物 CD133 和 Sox2 的表达、球体形成和细胞增殖。同样，我们发现 miR-30-5p 在 5-氟尿嘧啶（5-FU）耐药 CRC 细胞中的表达通常降低，而 miR-30-5p 在 5-FU 耐药细胞中的过表达降低了球体形成和细胞活力。miR-30-5p 的抑制作用逆转了这一过程。最后，我们确定 miR-30-5p 减弱了 CRC 干细胞中 Wnt/β-catenin 信号靶基因（Axin2 和 MYC）、Wnt 荧光素酶活性和 β-catenin 蛋白水平的表达。
