Department of Gastrointestinal Surgery, The First Affiliated Hospital, Chongqing Medical University, Chongqing, 400016, China.
Department of Gastrointestinal Surgery, Nanchong Central Hospital, Nanchong, 637000, Sichuan, China.
J Transl Med. 2017 Dec 19;15(1):257. doi: 10.1186/s12967-017-1357-7.
Metastasis is a major threat to colorectal cancer (CRC) patients. We have reported that peroxiredoxin-2 (PRDX2) is associated with CRC invasion and metastasis. However, the mechanisms regulating PRDX2 expression remain unclear. We investigate whether microRNAs (miRNAs) regulate PRDX2 expression in CRC progression.
Quantitative real-time polymerase chain reaction (qPCR) was used to measure microRNA-200b-3p (miR-200b-3p) expression. Immunohistochemistry (IHC) was performed to detect c-Myc and PRDX2 protein levels in CRC tissue samples (n = 97). Western blot was used to quantify PRDX2, c-Myc, AKT2/GSK3β pathway-associated proteins and epithelial-mesenchymal transition (EMT)-related proteins in CRC cells. Luciferase reporter assays were used to analyze the interaction between miR-200b-3p and 3'untranslated region (3'UTR) of PRDX2 mRNA and AKT2 mRNA as well as c-Myc and the miR-200b-3p promoter. Chromatin immunoprecipitation (ChIP) assay was used to evaluate binding of c-Myc to the miR-200b-3p promoter. Invasive assay and metastatic model were used to assess invasive and metastatic capacities of CRC cells in vitro and in vivo. Moreover, drug-induced apoptosis was measured by flow cytometry.
We found that miR-200b-3p was significantly downregulated, whereas c-Myc and PRDX2 were upregulated in metastatic CRC cells and CRC tissues compared to their counterparts. An inverse correlation existed between c-Myc and miR-200b-3p, and between miR-200b-3p and PRDX2. We also found that PRDX2 was a target of miR-200b-3p. Importantly, overexpression of nontargetable PRDX2 eliminated the suppressive effects of miR-200b-3p on proliferation, invasion, EMT, chemotherapeutic resistance and metastasis of CRC cells. Moreover, c-Myc bound to the promoter of miR-200b-3p and repressed its transcription. In turn, miR-200b-3p disrupted the stability of c-Myc protein by inducing c-Myc protein threonine 58 (T58) phosphorylation and serine 62 (S62) dephosphorylation via AKT2/GSK3β pathway.
Our findings reveal that the c-Myc/miR-200b/PRDX2 loop regulates CRC progression and its disruption enhances tumor metastasis and chemotherapeutic resistance in CRC.
转移是结直肠癌（CRC）患者的主要威胁。我们已经报告过过氧化物酶 2（PRDX2）与 CRC 侵袭和转移有关。然而，调节 PRDX2 表达的机制仍不清楚。我们研究了 microRNAs（miRNAs）是否调节 CRC 进展过程中的 PRDX2 表达。
采用实时定量聚合酶链反应（qPCR）测量微小 RNA-200b-3p（miR-200b-3p）的表达。免疫组织化学（IHC）检测 97 例 CRC 组织样本中 c-Myc 和 PRDX2 蛋白水平。Western blot 用于定量 CRC 细胞中 PRDX2、c-Myc、AKT2/GSK3β 通路相关蛋白和上皮-间充质转化（EMT）相关蛋白。荧光素酶报告分析用于分析 miR-200b-3p 与 PRDX2 mRNA 和 AKT2 mRNA 的 3'非翻译区（3'UTR）以及 c-Myc 和 miR-200b-3p 启动子之间的相互作用。染色质免疫沉淀（ChIP）实验用于评估 c-Myc 与 miR-200b-3p 启动子的结合。体外侵袭和转移模型用于评估 CRC 细胞的侵袭和转移能力。此外，通过流式细胞术测量药物诱导的细胞凋亡。
与相应的对照相比，我们发现转移性 CRC 细胞和 CRC 组织中 miR-200b-3p 明显下调，而 c-Myc 和 PRDX2 则上调。c-Myc 与 miR-200b-3p 呈负相关，miR-200b-3p 与 PRDX2 呈负相关。我们还发现 PRDX2 是 miR-200b-3p 的靶标。重要的是，非靶向 PRDX2 的过表达消除了 miR-200b-3p 对 CRC 细胞增殖、侵袭、EMT、化疗耐药和转移的抑制作用。此外，c-Myc 与 miR-200b-3p 启动子结合并抑制其转录。反过来，miR-200b-3p 通过 AKT2/GSK3β 通路诱导 c-Myc 蛋白 threonine 58（T58）磷酸化和 serine 62（S62）去磷酸化，破坏 c-Myc 蛋白的稳定性。
我们的研究结果表明，c-Myc/miR-200b/PRDX2 环调节 CRC 的进展，其破坏增强了 CRC 的肿瘤转移和化疗耐药性。
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