Disruption of the c-Myc/miR-200b-3p/PRDX2 regulatory loop enhances tumor metastasis and chemotherapeutic resistance in colorectal cancer

Zhenbing Lv, Jinlai Wei, Wenxian You, Rong Wang, Jingkun Shang, Yongfu Xiong, Hua Yang, Xuanhua Yang, Zhongxue Fu

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.