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MiR-188-5p promotes oxaliplatin resistance by targeting RASA1 in colon cancer cells.

ABSTRACT: The efficacy of chemotherapy for colon cancer is limited due to the development of chemoresistance. MicroRNA (miR)-188-5p is downregulated in various types of cancer. The aim of the present study was to explore the molecular role of miR-188 in oxaliplatin (OXA) resistance. An OXA-resistant colon cancer cell line, SW480/OXA, was used to examine the effects of miR-188-5p on the sensitivity of colon cancer cells to OXA. The target of miR-188-5p was identified using a luciferase assay. Cell cycle distribution was also assessed using flow cytometry. The measurement of p21 protein expression, Hoechst 33342 staining and Annexin V/propidium iodide staining was used to evaluate apoptosis. The expression of miR-188-5p significantly increased in SW480/OXA compared with wild-type SW480 cells. The luciferase assay demonstrated that miR-188-5p inhibited Ras GTPase-activating protein 1 (RASA1; also known as p120/RasGAP) luciferase activity by binding to the 3'-untranslated region of RASA1 mRNA, suggesting that miR-188-5p could target RASA1. In addition, miR-188-5p downregulation or RASA1 overexpression promoted the chemosensitivity of SW480/OXA, as evidenced by increased apoptosis and G₁/S cell cycle arrest. Moreover, RASA1 silencing abrogated the increase in cell apoptosis induced by the miR-188-5p inhibitor. The findings of the present study suggested that miR-188-5p could enhance colon cancer cell chemosensitivity by promoting the expression of RASA1.

SUBMITTER: Zhu X

PROVIDER: S-EPMC8100964 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:PurposePediatric acute promyelocytic leukemia (APL) accounts for 10% of pediatric acute myelogenous leukemia (AML) case and is accompanied by a tendency to hemorrhage. miR-188-5p plays an important role in adult AML. Therefore, the purpose of this study was to explore the effects of miR-188-5p on cell proliferation and apoptosis and tumor growth, and its mechanism in pediatric APL patients.Materials and methodsSurvival-associated miRNAs or mRNAs from TCGA database associated with AML were identified via using the "survival R" package in R language. CCK8, clone formation, flow cytometry, RT-PCR, immunohistochemistry and Western blot assays were used to detect the viability, proliferation, apoptosis, cell cycle, and related gene expression in APL cell lines. The prognostic value of miR-188-5p was evaluated using a ROC curve. The tumorigenic ability of APL cell lines was determined using a nude mouse transplantation tumor experiment. Tumor cell apoptosis was determined by TUNEL assay in vivo. The target genes of miR-188-5p were predicted using the miRDB, miRTarBase, and TargetScan databases. A PPI network was constructed using STRING database and the hub gene was identified using the MCODE plug-in of the Cytoscape software. The DAVID database was used to perform GO and KEGG pathway enrichment analyses. A luciferase reporter assay was used to demonstrate the binding of miR-188-5p to CD2AP.ResultsmiR-188-5p overexpression or CD2 associated protein (CD2AP) inhibition was significantly associated with poor survival in pediatric APL patients. Upregulation of miR-188-5p was identified in the blood of pediatric APL patients and cell lines. Increased expression of miR-188-5p also promoted the viability, proliferation, and cell cycle progression, and reduced the apoptosis of APL cells. Additionally, upregulation of miR-188-5p regulated the expressions of cyclinD1, p53, Bax, Bcl-2 and cleaved caspase-3. The area under the ROC curve (AUC) of miR-188-5p was 0.661. miR-188-5p overexpression increased the tumorigenic ability of APL and Ki67 expression, and reduced cell apoptosis in vivo. CD2AP was identified as the only overlapping gene from the list of miR-188-5p target genes and survival-related mRNAs of the TCGA database. It was mainly enriched in the "biological process (BP)" and "cellular component (CC)" terms, and was downregulated in the blood of pediatric APL patients and cell lines. The luciferase reporter, RT-PCR, and Western blot assays demonstrated that the binding of miR-188-5p to CD2AP. CD2AP inhibition promoted the proliferation and inhibited the apoptosis of APL cells. Rescue experiments showed that inhibition of miR-188-5p inhibited cell proliferation, activated the PI3K/AKT/mTOR signaling pathway, induced G0/G1 phase arrest, regulated gene expression, and promoted cell apoptosis, which were reversed by CD2AP inhibition.ConclusionmiR-188-5p, an oncogene, promoted tumor growth and progression of pediatric APL in vitro and in vivo via targeting CD2AP and activating the PI3K/AKT/mTOR signaling pathway.

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MiR-188-5p promotes oxaliplatin resistance by targeting RASA1 in colon cancer cells.

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