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MicroRNAs carried by exosomes promote epithelial-mesenchymal transition and metastasis of liver cancer cells.

ABSTRACT: In this study, transforming growth factor-?1 treatment effectively induced epithelial-mesenchymal transition (EMT) of SMMC-7721 cells, and the expression and function of microRNAs (miRNAs) were determined to understand the processes involved in liver cancer metastasis. Nanoparticle tracking analysis and western blotting were performed to identify exosomes. Transwell and MTS assays were used to assess cell migration and proliferation, respectively. Immunofluorescence microscopy was used to identify the metastasis of exosomes in cells. High-throughput sequencing was used to identify mRNAs and miRNAs in cells and exosomes, respectively. The identified differentially expressed miRNAs (DEmis) were further confirmed using quantitative real-time polymerase chain reaction. An miRNA-target mRNA interaction network was constructed using Cytoscape_V2_8_3. SPSS version 16.0 software with one-way analysis of variance was used for statistical analysis. P < 0.05 was considered statistically significant. The overall size of exosomes in EMT SMMC-7721 cells was smaller than that in normal SMMC-7721 cells. Exosomes of EMT SMMC-7721 cells could promote cell migration and invasion in several cell lines. We identified differentially expressed mRNAs (DEms) and DEmis. Among them, a total of 60 and 78 DEms were upregulated and downregulated, respectively, in EMT SMMC-7721 cells compared with those in SMMC-7721 cells. A total of 709 and 123 DEmis were upregulated and downregulated, respectively, in exosomes in EMT SMMC-7721 cells compared with those in SMMC-7721 cells. hsa-miR-24-3p and hsa-miR-21-5p were further selected for knockdown experiments. Exosomes in cells with hsa-miR-24-3p knockdown could effectively inhibit EMT. hsa-miR-24-3p may be one of the most important molecular markers for EMT in liver cancer, which provides novel clues for the mechanisms involved in liver cancer metastasis.

SUBMITTER: Chen QL

PROVIDER: S-EPMC7653584 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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microRNAs carried by exosomes promote epithelial-mesenchymal transition and metastasis of liver cancer cells.

Chen Qin-Lian QL Xie Chun-Feng CF Feng Kun-Liang KL Cui Dong-Ying DY Sun Shui-Lian SL Zhang Jun-Chang JC Xiong Cheng-Ming CM Huang Jun-Hai JH Chong Zhong Z

American journal of translational research 20201015 10

In this study, transforming growth factor-β1 treatment effectively induced epithelial-mesenchymal transition (EMT) of SMMC-7721 cells, and the expression and function of microRNAs (miRNAs) were determined to understand the processes involved in liver cancer metastasis. Nanoparticle tracking analysis and western blotting were performed to identify exosomes. Transwell and MTS assays were used to assess cell migration and proliferation, respectively. Immunofluorescence microscopy was used to identi ...[more]

PMID: 33194074

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Project description:BackgroundCancer associated fibroblasts (CAFs) are key stroma cells that play dominant roles in tumor progression. However, the CAFs-derived molecular determinants that regulate colorectal cancer (CRC) metastasis and chemoresistance have not been fully characterized.MethodsCAFs and NFs were obtained from fresh CRC and adjacent normal tissues. Exosomes were isolated from conditioned medium and serum of CRC patients using ultracentrifugation method and ExoQuick Exosome Precipitation Solution kit, and characterized by transmission electronic microscopy, nanosight and western blot. MicroRNA microarray was employed to identify differentially expressed miRNAs in exosomes secreted by CAFs or NFs. The internalization of exosomes, transfer of miR-92a-3p was observed by immunofluorescence. Boyden chamber migration and invasion, cell counting kit-8, flow cytometry, plate colony formation, sphere formation assays, tail vein injection and primary colon cancer liver metastasis assays were employed to explore the effect of NFs, CAFs and exosomes secreted by them on epithelial-mesenchymal transition, stemness, metastasis and chemotherapy resistance of CRC. Luciferase report assay, real-time qPCR, western blot, immunofluorescence, and immunohistochemistry staining were employed to explore the regulation of CRC metastasis and chemotherapy resistance by miR-92a-3p, FBXW7 and MOAP1.ResultsCAFs promote the stemness, epithelial-mesenchymal transition (EMT), metastasis and chemotherapy resistance of CRC cells. Importantly, CAFs exert their roles by directly transferring exosomes to CRC cells, leading to a significant increase of miR-92a-3p level in CRC cells. Mechanically, increased expression of miR-92a-3p activates Wnt/β-catenin pathway and inhibits mitochondrial apoptosis by directly inhibiting FBXW7 and MOAP1, contributing to cell stemness, EMT, metastasis and 5-FU/L-OHP resistance in CRC. Clinically, miR-92a-3p expression is significantly increased in CRC tissues and negatively correlated with the levels of FBXW7 and MOAP1 in CRC specimens, and high expression of exosomal miR-92a-3p in serum was highly linked with metastasis and chemotherapy resistance in CRC patients.ConclusionsCAFs secreted exosomes promote metastasis and chemotherapy resistance of CRC. Inhibiting exosomal miR-92a-3p provides an alternative modality for the prediction and treatment of metastasis and chemotherapy resistance in CRC.

Dataset Information

MicroRNAs carried by exosomes promote epithelial-mesenchymal transition and metastasis of liver cancer cells.

Publications

microRNAs carried by exosomes promote epithelial-mesenchymal transition and metastasis of liver cancer cells.

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