Abstract Background The role of exosomal circular RNAs (circRNAs) in Hepatocellular carcinoma (HCC) cells with high metastatic potential has been little studied. Methods Exosomal circRNA from cells with non-metastatic (HepG2), low metastatic (97L), and high metastatic (LM3) potential were sequencing. Metastatic-related circRNAs in serum from HCC patients were measured and their association with clinical prognosis was evaluated. Furthermore, candidate functional circRNAs in LM3-derived exosomes was assessed. Findings LM3 exosomes enhanced the cell migration and invasion potential of HepG2 and 97 L cells. CircPTGR1, a circRNA with three isoforms, was specifically expressed in exosomes from 97 L and LM3 cells, upregulated in serum exosomes from HCC patients and was associated with the clinical stage and prognosis. Knockdown of circPTGR1 expression suppressed the migration and invasion of HepG2 and 97L cells induced by co-culturing with LM3 exosomes. Bioinformatics, co-expression analysis, and a luciferase assay indicated that circPTGR1 competed with MET to target miR449a. Interpretation Higher metastatic HCC cells can confer this potential on those with lower or no metastatic potential via exosomes with circPTGR1, resulting in increased migratory and invasive abilities in those cells. Fund National Natural Science Foundation of China (No. 81470870, 81670601, 81570593), Guangdong Natural Science Foundation (No. 2015A030312013, 2015A030313038), Sci-tech Research Development Program of Guangdong Province (2014B020228003), Sci-tech Research Development Program of Guangzhou City (No. 201508020262, 201400000001-3, 201604020001, 201607010024), Innovative Funds for Small and Medium-Sized Enterprises of Guangdong Province (2016A010119103), Pearl River S&T Nova Program of Guangzhou (201710010178), and National 13th Five-Year Science and Technology Plan Major Projects of China (No. 2017ZX10203205-006-001). Keywords: circPTGR1, Hepatocellular carcinoma, Metastasis, miR449a __________________________________________________________________ Research in context. Evidence before this study Exosomes originating from various types of hepatocellular carcinoma (HCC) cells have been characterized, and their effects on cell growth, metastasis, and drug resistance have been determined. Circular RNA (circRNA) is a class of RNA derived from precursor mRNA, which has been reported to play roles in tumors, including HCC. However, exosomal circRNA and its biological function in HCC cells, especially those with high metastatic potential, have been little studied. Added value of this study This study has provided information about exosomal circRNA in HCC cells with different metastatic potentials. In particular, we identified three isoforms of circPTGR1 that are preferentially located in exosomes derived from LM3, an HCC cell line with high metastatic potential. These were found to be associated with the clinical stage and prognosis of HCC patients, indicating their prognostic value in the clinical setting. In addition, knockdown of LM3 exosomal circPTGR1 significantly reduced tumor progression in non- and low-metastatic cell lines both in vivo and in vitro, suggesting the involvement of the miR449a/MET pathway in this effect. Implications of all the available evidence HCC cells with higher metastatic potential may communicate with less metastatic and non-metastatic cells via exosomal cargo such as circPTGR1, thereby affecting cell fate. Exosomes from highly metastatic cells with a high abundance of circPTGR1 may influence cells with lower malignancy by downregulating miR449a-MET interactions in the recipient cells, leading to the disruption of tumor microenvironment homeostasis and the promotion of HCC progression. Because circPTGR1 is highly abundant and is aberrantly expressed in malignant cells and in cells from patients with metastases, it could function as a prognostic biomarker and therapeutic target for HCC. Alt-text: Unlabelled Box 1. Introduction Hepatocellular carcinoma (HCC) is the fifth most commonly diagnosed cancer in China and the third leading cause of cancer-related death worldwide [[43]1,[44]2], responsible for >600,000 deaths annually [[45]3]. Its high incidence (16 cases per 100,000 inhabitants) and poor prognosis have led to it being an increasing financial burden [[46]4]. There is therefore an urgent need for a better understanding of the pathology of HCC and for candidate biomarkers to allow its early detection and prognosis and for developing therapeutic strategies. Exosomes are small extracellular vesicles (30–150 nm in diameter) with an intact lipid bilayer; they are endocytic in origin are able to encapsulate cargo such as lipids, RNA, DNA, and proteins from the parent cell. There is evidence that exosomes exist in almost all mammalian cells, including tumor cells [[47]5]. RNA and proteomics analyses have clarified the role played by tumor-derived exosomes in tumor development and progression [[48]6]. Kogure et al., identified exosomal microRNAs (miRNAs) in HCC that were differentially expressed between a donor H3B cell and the recipient HepG2 cell and found that HCC cell-derived exosomes downregulated hepatocarcinogenesis-related transforming growth factor β activated kinase-1 (TAK1), as well as its associated signaling pathway, resulting in the promotion of HepG2 cell growth [[49]7]. A study that characterized the RNA and proteome contents of exosomes revealed that exosomes derived from metastatic HCC cell lines could enhance hepatocyte motility [[50]8]. Circular RNAs (circRNAs) are a class of endogenous noncoding RNAs with cell type-specific expression, which function as miRNA sponges to regulate gene expression. It is thought they are generated by the backsplicing of exons and/or introns during the process of precursor mRNA splicing. However, unlike the linear transcripts from which they are generated, circRNAs lack the 5′ cap and 3′ polyadenylated tail, making them more resistant than the linear mRNAs to RNAase [[51]9]. A recent study suggested that circRNAs are abundant and stable in exosomes derived from liver cancer cells [[52]10], and it has been suggested that their expression is modulated by endonucleic activity and exosomes [[53]11]. Together, these findings suggest a possible therapeutic role of exosomal circRNAs in controlling cancer progression, with a potential application as promising biomarkers in the diagnosis of HCC. However, most previous studies of HCC exosomes have focused on miRNAs and proteins, and research into the functional roles of exosomal circRNAs derived from HCC cells is limited. The aim of this study was to investigate the RNA profile of HCC-derived exosomes and the potential role of an HCC exosomal circRNA in tumor cell migration and invasion, and to clarify the underlying mechanisms. 2. Materials and methods 2.1. Cell lines and culture The human hepatocellular carcinoma cell lines HepG2, L-O2, SMCC-7721, HEP3B and HUH7 were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). MHCC97-L (97 L), MHCC 97H (97H) and HCC-LM3 (LM3) cells were kindly provided by Prof. Zhaoyou Tang (Liver Cancer Institute, Zhongshan Hospital, Fudan University, Shanghai, China). The cells were grown in high-glucose Dulbecco's Modified Eagle Medium (DMEM; Invitrogen, CA, USA) supplemented with 10% fetal bovine serum (FBS; Invitrogen) and 1% penicillin/streptomycin. All the cultures were incubated at 37 °C with 5% CO[2]. To construct both the circPTGR1 and the corresponding parent linear PTGR1 stable knockdown cell lines, short hairpin RNAs (shRNAs) containing the si-circPTGR1 (GAAGAAAGCGTCTCCTGAT), si-PTGR1(GGACCCTGAAGAAGCACTT) sequence were cloned into lentiviral vectors (Forevergen, Guangzhou, China). A vector containing the si-NC (CTTTCTCCGAACGTG TCAC) sequence was used as a negative control. All constructed lentivirus vectors were transfected with the packaging plasmids pGag/Pol, pRev, and pVSV-G into 293 T cells with Lipofectamine 2000. Viruses in cell supernatant were collected at 48 h and 72 h post transfection and transduced to LM3 cells, in order to generate the shcircPTGR1, shPTGR1, and control NC cell lines. 2.2. Patients and specimens A total of 82 HCC patients and 47 healthy people at the Third Affiliated Hospital of Sun Yat-sen University (Guangzhou, China), were enrolled in this study between November 2015 and April 2017. All the patients had been diagnosed with primary HCC, and none had received any preoperative treatment. The patients underwent surgical resection, and serum samples were collected on the day of surgery. Their clinicopathological characteristics are presented in Supplementary Table 1. The protocol for collecting clinical samples was approved by the Ethics Committee of the Third Affiliated Hospital of Sun Yat-sen University (Guangzhou, China), and the patients provided informed consent before samples were collected. 2.3. Isolation and identification of cellular exosomes Cells cultured on 15 cm plates with DMEM containing 10% FBS were replenished with serum-free medium when they reached 80% confluence and were maintained in culture in a 37 °C incubator with 5% CO[2] until the cell medium was collected for exosome isolation after 48 h. The cell medium was centrifuged at 600 ×g for 5 min, followed by at 12,000 ×g for 25 min to remove any cell debris and possible apoptotic bodies. The supernatants were then incubated overnight with ExoQuick-TC exosome precipitation solution (System Biosciences, CA, USA) at 4 °C and were then centrifuged at 1500 ×g for 30 min to harvest the exosome pellet. The exosomes were resuspended in 100 μl 1× phosphate-buffered saline (PBS) and verified with electron microscopy JEM-1400 (JEOL, Tokyo, Japan). A NanoSight LM10 instrument (Nanosight, Malvern, UK) was used to analyze the size and number of exosomes, following the manufacturers' instructions. 2.4. Blood preparation and exosome isolation After centrifugation of the whole blood at 1600 ×g for 10 min at 4 °C, the aspirated serum was stored at −80 °C until use. Exosomes were isolated from the serum sample using ExoQuick Exosome Precipitation Solution (System Biosciences). Briefly, the serum sample was centrifuged at 3000 ×g for 15 min to remove cell debris. Next, 63 μl of ExoQuick Exosome Precipitation Solution was added to 250 μl of the serum sample and mixed well. Then, 125 μL ExoQuick Exosome Precipitation Solution was added to 500 μL serum and mixed well. After incubating at 4 °C for 30 min, the mixture was centrifuged at 1500 ×g for 30 min. The supernatant was then removed, and the tubes were centrifuged for another 5 min. Finally, the exosomes were resuspended with PBS. 2.5. Exosome fluorescence assay An exosome fluorescence assay was used to validate the internalization of labeled LM3-Exosome (0 ng, 10 ng, and 25 ng) by HepG2 cells. Firstly, we resuspended LM3-Exosome in 500 μl PBS in a 1.5 ml Eppendorf tube, and added 50 μl of 10× labeling dye Exo-Green to the LM3-Exosome preparation. The mixture was then incubated at 37 °C for 10 min without shaking. 100ul ExoQuick-TC was added to the solution and incubated at 4 °C for 30 min. The Eppendorf tube was spun at 14,000 rpm for 3 min and the supernatant was carefully aspirated from the corner of the tube and the LM3-Exosome was resuspended in 500 μl PBS for downstream applications. Cells were seeded at a density of 5 × 10 [[54]3] cells/well in 24-well plates and co-cultured with different concentrations of labeled LM3-Exosome for 2–24 h. Finally, the cells were observed under a fluorescence microscope (Excitation: 494 nm; Emission: 521 nm (green), Filter setting: Typical GFP filter set). 2.6. Cell proliferation, migration, invasion assays, and flow cytometry Cells were incubated with exosomes (10 μg/ml) or PBS for the indicated time periods. For the valuation of cell proliferation, the [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophen yl)-2H-tetrazolium] MTS assays were conducted according to a previous report [[55]12], but briefly, the cells (2 × 10^4 per well) were seeded into 96-well plates and cultured for 3 days. MTS reagents were added to assess cell proliferation at days 1, 2, and 3. For the cell invasion and migration analysis, 1 × 10^6 cells were seeded in the upper chambers that were pre-coated with or without Matrigel (Matrigel BD biosciences, NY, USA). Cells in the upper chambers were maintained in serum-free DMEM, whereas those in the lower chambers were maintained in DMEM with 10% FBS. At 48 h, all cells that had transferred to the lower chambers were stained with 0.5% crystal violet. Positive staining cells from six to eight fields of chambers in each group were counted under a microscope (Olympus, Tokyo, Japan). Cells were seeded into 6-well plates at a density of 1 × 10^6 per well and cultured for 12 h prior to exosome incubation. Cells were incubated with exosomes (10 μg/ml) for 48 h, collected by trypsin treatment, and resuspended in cold PBS. For measurement of the cell cycle, cells were fixed overnight in cold 70% ethanol and stained with propidium iodide (PI) in staining buffer (50 μg/ml PtdIns (Sigma, CA, USA) and 20 μg/ml RNase in PBS for 2 h at 4 °C. In order to detect apoptosis, cells were stained with Annexin V-APC and 7-AAD (BioLegend, CA, USA) according to the manufacturer's instructions. The nuclear DNA content at each phase of the cell cycle and the apoptotic cell proportion were assessed with flow cytometry (Becton-Dickinson, CA, USA). 2.7. Western blot analysis Exosome pellets were lysed in lysis buffer (8 M Urea/2.5% SDS containing 5 μg.ml^−1 leupeptin, 1 μg.ml^−1 pepstatin, and 1 mM phenylmethylsulphonyl fluoride) as previously described [[56]13]. The Bradford method was used for protein quantification and Western blot was conducted as previously reported [[57]12]. The following primary antibodies were used: Alix, HSPA8, MET, and GAPDH (Cell Signaling Technology, Inc., MA, USA), Tsg101, CD63 (Abcam, CA, UK), and PTGR1 (Sigma-Aldrich, St. Louis). Blots were visualized with enhanced chemiluminescent agents (Forevergen biosciences, GZ, China). 2.8. RNA isolation and qRT-PCR detection Trizol reagent (Life Technologies, CA, USA) was used to isolate the total RNA from cells, following the standard protocol. Exosomal RNA was extracted using a SeraMir™ Exosome RNA Amplification Kit (System Biosciences). RNA quantification was performed with Qubit 3.0 (Thermo Fisher, MA, USA). To compare expression levels between groups, quantitative real-time PCR (qRT-PCR) was performed using an RNA-direct SYBR Realtime PCR Master Mix (TOYOBO, Osaka, Japan) and an ABI 7500 real-time PCR system (Applied Biosystems, CA, USA). The qRT-PCR results were analyzed with the 2-ΔΔCt method. To validate the backspliced circRNAs predicted by RNA-seq, RT-PCR was carried out using circRNA-specific divergent primers. The primers used are listed in Supplementary Table 2. 2.9. RNA library preparation and sequencing RNA integrity and size distributions were assessed using Agilent 2100 Bioanalyzer pico-RNA chips (Agilent, CA, USA). RNA libraries were prepared according to the instructions for the VAHTS Total RNA-seq (H/M/R) Library Prep Kit from Illumina® (VAZYME, Nanjing, China). RNA-seq was performed as previously described [[58]12], and the libraries were created and sequenced using the Illumina HiSeq 2500 platform. The RNA-seq data were uploaded to NCBI database ([59]https://trace.ncbi.nlm.nih.gov/Traces/sra/?study=SRP165940). The accession number was NCBI: SRP165940. 2.10. Bioinformatics analysis The reads from the Illumina sequencer were subjected to mRNA, lncRNA, and circRNA analyses and the data were aligned with the human reference genome GRCh37/hg19 using TOPHAT v2.1.0. Counts of mapped reads for each gene were normalized by the number of reads per kilobase per million mapped reads, which allowed the comparison of expression levels between genes. A differential expression analysis of two samples was performed using a previous statistical model [[60]14]. The P-values were adjusted using Benjamini and Hochberg's approach for controlling the false discovery rate and an adjusted P-value of <0.05 was considered to indicate differential expression. To construct the hierarchical clustering analysis, the heatmap package in R version 1.0.8 ([61]https://cran.r-project.org/web/packages/pheatmap/) was used to cluster the differentially expressed circRNAs that overlapped between the 97 L and LM3 cells. The biological processes involving these genes were obtained from the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways database ([62]http://www.genome.jp/kegg/) (adjusted P-value <.05; gene count ≥2). The miRanda ([63]http://www.microrna.org/) and TargetScan ([64]http://www.targetscan.org/) software packages were used to predict the circPRTG1 miRNA targets and the potential mRNA targets of the miRNAs. The circRNAs–miRNAs–mRNA interaction network was constructed by merging the common targets of the circRNAs and mRNAs, as previously described [[65]15]. Finally, the network obtained was visualized with Cytoscape software (V 2.8.3, [66]http://www.cytoscape.org/). 2.11. In vivo metastasis assay An experimental metastasis model was developed using athymic nude mice and the LM3 HCC cell line. The mice were anesthetized with pentobarbital and a small transverse incision was made below the sternum to expose the liver. After carefully exposing the liver, 2 × 10^6 viable cells were preincubated with PBS, shcircPTGR1, or negative control exosomes and were slowly injected into the upper left lobe of the liver with a 28-gage needle. Four weeks after the injection, the mice were sacrificed under anesthesia and the tumor metastasis was examined with a stereo microscope. 2.12. Statistical analysis The statistical analyses were performed with SPSS version 16.0 (SPSS, Inc.) and GraphPad Prism version 6.0 (GraphPad Software). Data from three or more independent experiments are presented as means ± standard error of the mean. Data that were not distributed normally were transformed into a normal distribution before analysis. The relationship between the circRNAs and the clinicopathological parameters of the HCC patients was analyzed using one-way ANOVA. Differences between two groups were evaluated using Student's t-test. P < .05 was considered to indicate a statistically significant difference. The expression in liver tissues of PTGR1 mRNA and the patient survival data were obtained from The Cancer Genome Atlas Liver Hepatocellular Carcinoma (TCGA LIHC) dataset ([67]https://tcga-data.nci.nih.gov/) and the OncoLnc Anaya dataset [[68]16]. Kaplan–Meier survival curves were created with GraphPad Prism and compared using the log-rank test. 3. Results 3.1. LM3-derived exosomes promoted migration and invasion in HepG2 and 97 L cells Exosomes isolated from cells with metastatic potential were verified and used to evaluate whether such cells could affect the biological functions of cells with little or no metastatic potential. The identity of the exosomes was confirmed by electron microscopy, which revealed that exosomes with cup-shape morphology were 50–100 nm in diameter ([69]Fig. 1a). Nanoparticle Tracking Analysis showed a size range of 50–250 nm. There were no obvious differences in the shape or size of exosomes secreted by the HepG2, 97 L, and LM3 cells. In addition, western blot analysis confirmed the presence of the exosome-associated markers Alix, Tsg101, and CD63 in isolated exosomes ([70]Fig. 1a). We labeled LM3-derived exosomes with ExoGlow-Protein and followed their uptake by HepG2 cells. The punctate fluorescence signal confirmed the internalization of the labeled LM3 exosomes by HepG2 cells ([71]Supplementary Fig. 1a). Fig. 1. [72]Fig. 1 [73]Open in a new tab LM3-derived exosomes promoted the cell migratory and invasive ability of HepG2 and 97 L cells. (a) Exosome morphology viewed by transmission electron microscopy. Scale bar, 100 nm. NanoSight tracking analysis was used to measure the diameters of exosomes isolated from LM3 cells. The exosomal markers Alix, Tsg101, CD63, and HSPA8 were detected by western blot. An MTS assay, cytometry, and transwell assays were used to analyze the effects of LM3-derived exosomes, compared with treatment with phosphate-buffered saline (PBS), on HepG2 and 97 L cells for the following characteristics: (b) cell proliferation; (c) apoptosis; (d) cycle distribution; (e) cell migration (HepG2, P < .0001; 97 L, P < .0001); and (f) invasion (HepG2, P = .0004; 97 L, P < .0001). Scale bars: 100 μm. Error bars indicate standard deviation. **P < .01 vs. PBS. Supplementary Fig. 1. [74]Supplementary Fig. 1 [75]Open in a new tab (a) ExoGlow-Protein provided a clear visualization of the internalization by HepG2 cells of labeled LM3 exosomes. Scale bars: 100 μm. (b) MTS assay (HepG2: BSA vs. bigger vesicle, P = .0074; BSA vs. HepG2 exosomes (HepG2 exo), P = .0034; 97 L: BSA vs. bigger VC, P = .0042; BSA vs. HepG2 exo, P = .0107). (c) Flow cytometry (P < .0001 vs. BSA). (d) Flow cytometry (P = .001 vs. BSA). (e) Migration assay (HepG2: BSA vs. HepG2 exo, P = .0037; BSA vs. LM3 exo, P < .0001; BSA vs. 97H exo, P < .0001; HepG2 exo vs. LM3 exo, P = .0002; HepG2 exo vs. 97H exo, P < .0001; 97 L: BSA vs. big vesicle, P = .0002; BSA vs. HepG2 exo, P < .0001; BSA vs. LM3 exo, P < .0001; BSA vs. 97H exo, P < .0001; HepG2 exo vs. LM3 exo, P = .0006; HepG2 exo vs. 97H exo, P = .0011). (f) Invasion assay (HepG2: BSA vs. LM3 exo, P = .0073; BSA vs. 97H exo, P = .0002; 97 L: BSA vs. HepG2 exo, P < .0001; BSA vs. LM3 exo, P < .0001; BSA vs. 97H exo, P < .0001; HepG2 exo vs. LM3 exo, P < .0001; HepG2 exo vs. 97H exo, P < .0001). Transwell assays were used to evaluate the effects of LM3-derived exosomes on cell proliferation, apoptosis, cycle distribution, cell migration, and invasion in HepG2 and 97 L cells treated with BSA, big vesicles, HepG2 exosomes, LM3 exosomes, and 97H exosomes. Error bars indicated standard deviations. * P < .05 vs. BSA; ** P < .01 vs. BSA. Next, HepG2 and 97 L cells were incubated with LM3 exosomes and subjected to MTS, flow cytometry, and transwell assays to determine cell proliferation, apoptosis, the cell cycle, migration, and invasion. Cells treated with phosphate-buffered saline (PBS) were used as a control. The MTS assay showed no significant differences between the LM3 exosomes and the PBS treatment in either the HepG2 or the 97 L cell lines ([76]Fig. 1b), and there were no statistically significant differences in the apoptosis levels or cycle distribution between the exosomes and PBS-treated HepG2 and 97 L cells ([77]Fig. 1c, d). However, there were significant differences in cell numbers based on the transwell migration and invasion assays. The number of migrated HepG2 cells was more than two-fold higher in the group incubated with LM3 exosomes compared to the PBS-treated control (P < .0001) ([78]Fig. 1e). A similar increase was also clearly detected in the 97 L cells incubated with LM3 exosomes (P < .0001). Similar results were obtained in the invasion assay (P < .0001), in which, compared with the controls, more than double the number of cells in both of the cell lines treated with LM3 exosomes migrated into the lower compartment (HepG2, P = .0004; 97 L, P < .0001; [79]Fig. 1f). In addition, bovine serum albumin, big vesicles, and exosomes derived from HepG2 were used as further controls to rule out contamination from the culture conditions or the exosome isolation process ([80]Supplementary Fig. 1b–f). Exosomes were also isolated from another highly metastatic cell line, 97H, with similar results; there were no significant differences in cell viability, apoptosis level, or cycle distribution between the LM3 or 97H exosomes incubated with the HepG2 or 97 L cell lines. However, there were significant differences in cell numbers based on the transwell migration and invasion assays ([81]Supplementary Fig. 1b–f). 3.2. RNA deep sequencing revealed different RNA profiles for exosomes from HepG2, 97 L, and LM3 cells An Agilent Bioanalyzer was used to analyze the exosomal total RNA profiles of HepG2, 97 L, and LM3 cells. This demonstrated that exosomes derived from LM3 and 97 L cells had relatively lower levels of 28S and 18S ribosomal RNA (rRNA) compared with those from HepG2 cells. Compared with HepG2 exosomal RNA, 97 L and LM3 exosomal RNA included a greater proportion of short-length RNAs ([82]Fig. 2a). Fig. 2. [83]Fig. 2 [84]Open in a new tab RNA profiles of HepG2, 97 L, and LM3-derived exosomes revealed by RNA sequencing. (a) Bioanalyzer analyses of RNA isolated from HepG2, 97 L, and LM3-derived exosomes. (b) Transcript length distribution of mRNAs, lncRNAs, and circRNAs in HepG2, 97 L, and LM3-derived exosomes. The dots indicate discrete values. (c) Abundance of mRNAs, lncRNAs, and circRNAs in HepG2, 97 L, and LM3-derived exosomes. (d) Circos plots representing the distribution of all the mRNAs (black), lncRNAs (red), and circRNAs (blue) from HepG2, 97 L, and LM3-derived exosomes on different chromosomes. The outermost track (yellow) shows the different chromosomes. (For interpretation of the references to colour in this