Exosomal miR-99a-5p is elevated in sera of ovarian cancer patients and promotes cancer cell invasion by increasing fibronectin and vitronectin expression in neighboring peritoneal mesothelial cells

Abstract

BackgroundmicroRNAs (miRNAs) stably exist in circulating blood and are encapsulated in extracellular vesicles such as exosomes. The aims of this study were to identify which exosomal miRNAs are highly produced from epithelial ovarian cancer (EOC) cells, to analyze whether serum miRNA can be used to discriminate patients with EOC from healthy volunteers, and to investigate the functional role of exosomal miRNAs in ovarian cancer progression.MethodsExosomes were collected from the culture media of serous ovarian cancer cell lines, namely TYK-nu and HeyA8 cells. An exosomal miRNA microarray revealed that several miRNAs including miR-99a-5p were specifically elevated in EOC-derived exosomes. Expression levels of serum miR-99a-5p in 62 patients with EOC, 26 patients with benign ovarian tumors, and 20 healthy volunteers were determined by miRNA quantitative reverse transcription-polymerase chain reaction. To investigate the role of exosomal miR-99a-5p in peritoneal dissemination, neighboring human peritoneal mesothelial cells (HPMCs) were treated with EOC-derived exosomes and then expression levels of miR-99a-5p were examined. Furthermore, mimics of miR-99a-5p were transfected into HPMCs and the effect of miR-99a-5p on cancer invasion was analyzed using a 3D culture model. Proteomic analysis with the tandem mass tag method was performed on HPMCs transfected with miR-99a-5p and then potential target genes of miR-99a-5p were examined.ResultsThe serum miR-99a-5p levels were significantly increased in patients with EOC, compared with those in benign tumor patients and healthy volunteers (1.7-fold and 2.8-fold, respectively). A receiver operating characteristic curve analysis showed with a cut-off of 1.41 showed sensitivity and specificity of 0.85 and 0.75, respectively, for detecting EOC (area under the curve = 0.88). Serum miR-99a-5p expression levels were significantly decreased after EOC surgeries (1.8 to 1.3, p = 0.002), indicating that miR-99a-5p reflects tumor burden. Treatment with EOC-derived exosomes significantly increased miR-99a-5p expression in HPMCs. HPMCs transfected with miR-99a-5p promoted ovarian cancer invasion and exhibited increased expression levels of fibronectin and vitronectin.ConclusionsSerum miR-99a-5p is significantly elevated in ovarian cancer patients. Exosomal miR-99a-5p from EOC cells promotes cell invasion by affecting HPMCs through fibronectin and vitronectin upregulation and may serve as a target for inhibiting ovarian cancer progression.Electronic supplementary materialThe online version of this article (10.1186/s12885-018-4974-5) contains supplementary material, which is available to authorized users.

Exosomes promote pre-metastatic niche formation in ovarian cancer

“…A growing body of evidence suggests exosomes derived from ovarian cancer cells can silence these immune cells in the tumor microenvironment and are critical in pre-metastatic niche formation [50][51][52]. In addition to reprogramming the immune cell gene profile, ovarian cancer cells release Fas ligand (FasL)-carrying exosomes which downregulate the expression of the TAMs M2 phenotype polarization, EOC proliferation and migration [39][40][41] miRNA 21 and 29a ES2 OC cellsLP9 mesothelial cells Mesothelial cell clearance [42] miR-99a-5p HPMC Cell invasion through fibronectin and vitronectin upregulation [43] MMP1 mRNAs MeT-5A and HPMC Destruction of peritoneal mesothelium barrier [44] let-7a-f and miR-200a-c N/A Correlates with ovarian cancer invasiveness [45] Abbreviations: TAMs Tumor-associated macrophages, HPMC Human peritoneal mesothelial cells, OC ovarian cancer, EOC Epithelial ovarian cancer, MMP Matrix metallopeptidase surface T-cell receptor/CD3-zeta (ζ) and promote T-cell apoptosis [53,54]. At the cellular level, lysophosphatidic acid elevates the expression of FasL on the surface of ovarian cancer cells, thus prompting the release of FasLcarrying exosomes [55].…”

“…2e). Serum miR-99a-5p is significantly elevated in ovarian cancer patients and promotes cell invasion by affecting human peritoneal mesothelial cells (HPMCs) via fibronectin and vitronectin upregulation [43]. CD44 is overexpressed in the peritoneal mesothelial cells of ovarian cancer patients with omental metastasis.…”

Pathophysiological Role and Potential Therapeutic Exploitation of Exosomes in Ovarian Cancer

“…The increased CD44 expression in PMCs promote cancer invasion by inducing PMCs to secrete matrix metalloproteinase (MMP) 9 and by destroying the mesothelial barrier for improved cancer cell invasion [47]. Exosomal miR-99a-5p upregulate fibronectin and vitronectin expression in PMCs and enhance cancer cell invasion [46]. Exosomal MMP1 mRNAs loaded in ovarian cancer-derived exosomes induce apoptotic changes in PMCs and disrupt the peritoneal mesothelial barrier, promoting the invasion of ovarian cancer cells [21].…”

Exosomes and cancer: From oncogenic roles to therapeutic applications

“…The study suggested the possible role of miR-99a-5p in cell invasion by affecting human peritoneal mesothelial cells (HPMCs) through upregulation of the fibronectin and vitronectin. 101 HPMCs produce many cytokines, and their roles in the fibrosis and progression have been reported in gastric and ovarian cancers. [102][103][104][105] The serum levels of seven miRNAs (let-7a, miR-1,229, miR-1,246, miR-150, miR-21, miR-223, and miR-23a) in exosomes were significantly elevated in patients with primary colon cancer only at the early stage, suggesting that the level of these miRNAs could be a potential biomarker for the early detection of colon cancer in patients.…”