Hepatocellular carcinoma-derived exosomal miRNA-21 contributes to tumor progression by converting hepatocyte stellate cells to cancer-associated fibroblasts

Zhou et al. Journal of Experimental & Clinical Cancer Research https://doi.org/10.1186/s13046-018-0965-2 (2018) 37:324 RESEARCH Open Access Hepatocellular carcinoma-derived exosomal miRNA-21 contributes to tumor progression by converting hepatocyte stellate cells to cancer-associated fibroblasts Yuan Zhou1†, Haozhen Ren1†, Bo Dai1†, Jun Li1, Longcheng Shang1, Jianfei Huang2* and Xiaolei Shi1* Abstract Background: Hepatocellular carcinoma (HCC) remains a global challenge due to its high morbidity and mortality rates as well as poor response to treatment. The communication between tumor-derived elements and stroma plays a critical role in facilitating cancer progression of HCC. Exosomes are small extracellular vesicles (EVs) that are released from the cells upon fusion of multivesicular bodies with the plasma membrane. There is emerging evidence indicating that exosomes play a central role in cell-to-cell communication. Much attention has been paid to exosomes since they are found to transport bioactive proteins, messenger RNA (mRNAs) and microRNA (miRNAs) that can be transferred in active form to adjacent cells or to distant organs. However, the mechanisms underlying such cancer progression remain largely unexplored. Methods: Exosomes were isolated by differential ultracentrifugation from conditioned medium of HCC cells and identified by electron microscopy and Western blotting analysis. Hepatic stellate cells (HSCs) were treated with different concentrations of exosomes, and the activation of HSCs was analyzed by Western blotting analysis, wound healing, migration assay, Edu assay, CCK-8 assay and flow cytometry. Moreover, the different miRNA levels of exosomes were tested by real-time quantitative PCR (RT-PCR). The angiogenic ability of activated HSCs was analyzed by qRT-PCR, CCK-8 assay and tube formation assay. In addition, the abnormal lipid metabolism of activated HSCs was analyzed by Western blotting analysis and Oil Red staining. Finally, the relationship between serum exosomal miRNA-21 and prognosis of HCC patients was evaluated. Results: We showed that HCC cells exhibited a great capacity to convert normal HSCs to cancer-associated fibroblasts (CAFs). Moreover, our data revealed that HCC cells secreted exosomal miRNA-21 that directly targeted PTEN, leading to activation of PDK1/AKT signaling in HSCs. Activated CAFs further promoted cancer progression by secreting angiogenic cytokines, including VEGF, MMP2, MMP9, bFGF and TGF-β. Clinical data indicated that high level of serum exosomal miRNA-21 was correlated with greater activation of CAFs and higher vessel density in HCC patients. Conclusions: Intercellular crosstalk between tumor cells and HSCs was mediated by tumor-derived exosomes that controlled progression of HCC. Our findings provided potential targets for prevention and treatment of live cancer. Keywords: Hepatocellular carcinoma, Hepatic stellate cells, Cancer associated fibroblasts, Exosome, miRNA-21, PTEN, AKT, Angiogenesis * Correspondence: ; † Yuan Zhou, Haozhen Ren and Bo Dai contributed equally to this work. 2 Department of Clinical Biobank, Nantong University Affiliated Hospital, 20, Xisi Road, Nantong 226001, Jiangsu Province, China 1 Department of Hepatobiliary Surgery, Affiliated Drum Tower Hospital of Nanjing University Medical School, 321, Zhongshan Road, Nanjing 210008, Jiangsu Province, China © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Zhou et al. Journal of Experimental & Clinical Cancer Research (2018) 37:324 Introduction Hepatocellular carcinoma (HCC) is responsible for the third most common cancer-related death worldwide [1]. Importantly, the vast majority of HCC is caused by liver fibrosis or cirrhosis [2]. Liver fibrosis occurs when inactive liver fibroblasts or hepatic stellate cells (HSCs) become activated after liver injury and turn into collagen-producing cells [3]. In HCC, cancer-associated fibroblasts (CAFs) are loosely defined as HSCs found within the tumor mass [4]. These CAFs have been implicated as etiologic players both in the cancer genesis and homeostasis. These data suggest that liver fibrosis, or activated HSCs, plays a crucial role in the development of HCC, which may be similar to the role of CAFs in desmoplastic tumors. How fibroblasts are activated in HCC remains controversial. Recent studies have revealed multiple potential origins, including activation of stellate cells or portal fibroblasts or transdifferentiation of hepatocytes through epithelial-mesenchymal transition (EMT) [5]. Stemmed from different origins, CAFs are highly heterogeneous, and they can be identified by different specific markers [6]. Among them, α-smooth muscle actin (α-SMA) is the most commonly used marker for CAFs [7]. Moreover, CAFs are believed to regulate the inflammatory microenvironment by expressing pro-inflammatory genes, such as IL-1β, IL-6, IL-8, TGF-β and CXCL12 as well as collagen [8, 9]. The crosstalk between tumor cells and CAFs has been extensively studied [10–12]. However, the mechanisms underlying the activation of HSCs by tumor cells remain largely unexplored in liver cancer. An important type of cell-cell communication occurs through exosomes. These small, nanometer-sized (50– 100 nM) vesicles of endocytic origin are released into the extracellular milieu by cells under physiological and pathological conditions, including antigen presentation and infectious agent transmission. Tumor exosomes are important mediators of the cross-talk between tumor cells and their microenvironment by sharing genetic information or functional proteins to modulate cellular behavior [13, 14]. Tumor cell-derived exosomes are involved in the regulation of EMT, tumor angiogenesis, tumor metastasis and radioresistance [15, 16]. It has been revealed that microRNA (miRNA) dysregulation greatly contributes to the activation of HSCs [17]. Interestingly, there is a significant overlap in the list of up-regulated or down-regulated miRNAs between HCC tumor tissues and normal tissues [18]. It has been demonstrated that secreted miRNAs can function in a paracrine manner in the surrounding microenvironment, promoting tumor development [19]. Meanwhile, studies have indicated that exosomes contain a high level of miRNAs, and exosomal miRNAs have been shown to contribute to immunomodulation, chemoresistance and Page 2 of 18 metastasis in multiple types of tumor [20, 21]. However, it remains (...truncated)