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MiR-5000-3p confers oxaliplatin resistance by targeting ubiquitin-specific peptidase 49 in colorectal cancer.

ABSTRACT: Colorectal cancer (CRC) is the most common form of gastrointestinal malignancies. A growing number of reports focusing on oxaliplatin (OXA) resistance in CRC treatment have revealed that drug resistance is an urgent issue in clinical applications, especially for finding effective therapeutic targets. Recently, microRNAs (miRNAs) are reported to play a critical role in tumor progressions and multi-drug resistance. The main aim of this study is to establish whether miR-5000-3p is an oncogene that is resistant to OXA and further confirm its underlying regulatory role in CRC. The OXA-associated gene expression dataset in CRC cells was downloaded from Gene Expression Omnibus (GEO) database. Statistical software R was used for significance analysis of differentially expressed genes (DEGs) between OXA-resistant (OR)-CRC cells and CRC cells, and results indicated ubiquitin-specific peptidase 49 (USP49) was upregulated in OR-CRC cells. Luciferase reporter assay showed that USP49 was verified to act as a downstream target gene of miR-5000-3p. From the results of TCGA database, miR-5000-3p expression was upregulated and USP49 was downregulated in patients with CRC. The function of miR-5000-3p was detected using MTT assay, wound healing, Transwell, and flow cytometry assays. Moreover, through in vitro and in vivo experiments, miR-5000-3p expression was confirmed to be upregulated in CRC cells or OR-CRC cells comparing to normal cell lines. Molecular mechanism assays revealed that USP49 binds to the miR-5000-3p promoter to increase the expression of miR-5000-3p, resulting in cancer cells sensitized to OXA. To sum up, these results suggest that miR-5000-3p may be a novel biomarker involved in drug-resistance progression of CRC. Moreover, the drug-resistance mechanism of miR-5000-3p/USP49 axis provides new treatment strategies for CRC in clinical trials.

SUBMITTER: Zhuang YY

PROVIDER: S-EPMC8169888 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundOxaliplatin resistance is a major challenge for treatment of advanced colorectal cancer (CRC). Both acquisition of epithelial-mesenchymal transition (EMT) and suppressed drug accumulation in cancer cells contributes to development of oxaliplatin resistance. Aberrant expression of small noncoding RNA, miR-128-3p, has been shown to be a key regulator in tumorigenesis and cancer development. However, its roles in the progression of CRC and oxaliplatin-resistance are largely unknown.MethodsOxaliplatin-resistant CRC and normal intestinal FHC cells were transfected with a miR-128-3p expression lentivirus. After transfection, FHC-derived exosomes were isolated and co-cultured with CRC cells. miR-128-3p expression in resistant CRC cells, FHC cells, and exosomes was quantified by quantitative real-time PCR (RT-qPCR). The mRNA and protein levels of miR-128-3p target genes in resistant CRC cells were quantified by RT-qPCR and western blot, respectively. The effects of miR-128-3p on CRC cell viability, apoptosis, EMT, motility and drug efflux were evaluated by CCK8, flow cytometry, Transwell and wound healing assays, immunofluorescence, and atomic absorption spectrophotometry. Xenograft models were used to determine whether miR-128-3p loaded exosomes can re-sensitize CRC cells to oxaliplatin in vivo.ResultsIn our established stable oxaliplatin-resistant CRC cell lines, in vitro and vivo studies revealed miR-128-3p suppressed EMT and increased intracellular oxaliplatin accumulation. Importantly, our results indicated that lower miR-128-3p expression was associated with poor oxaliplatin response in advanced human CRC patients. Moreover, data showed that miR-128-3p-transfected FHC cells effectively packaged miR-128-3p into secreted exosomes and mediated miR-128-3p delivery to oxaliplatin-resistant cells, improving oxaliplatin response in CRC cells both in vitro and in vivo. In addition, miR-128-3p overexpression up-regulated E-cadherin levels and inhibited oxaliplatin-induced EMT by suppressing Bmi1 expression in resistant cells. Meanwhile, it also decreased oxaliplatin efflux through suppressed expression of the drug transporter MRP5.ConclusionOur results demonstrate that miR-128-3p delivery via exosomes represents a novel strategy enhancing chemosensitivity in CRC through negative regulation of Bmi1 and MRP5. Moreover, miR-128-3p may be a promising diagnostic and prognostic marker for oxaliplatin-based chemotherapy.

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MiR-5000-3p confers oxaliplatin resistance by targeting ubiquitin-specific peptidase 49 in colorectal cancer.

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