Tumor-derived exosome miR-558 promotes angiogenesis in tongue squamous cell carcinoma by targeting HPSE

Tumor-derived exosome miR-558 promotes angiogenesis in tongue squamous cell carcinoma by targeting HPSE | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Short Report Tumor-derived exosome miR-558 promotes angiogenesis in tongue squamous cell carcinoma by targeting HPSE Bixiao Ding, Qingwen Chen, Xiaoguang Li, Zhen Wu, Yuancheng Ding, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3458216/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Purpose This study aimed to investigate the role of miR-558 in tumor angiogenesis by targeting heparinase (HPSE) in tongue squamous cell carcinoma (TSCC)-derived exosomes. Materials and Methods In the present study, the role of exosome miR-558 in angiogenesis in vitro and in vivo was investigated by cell proliferation, migration, tube formation, subcutaneous tumor formation in mice, and in vivo Matrigel plug assay. The target genes of miR-558 were detected by means of dual luciferase assay. Results It was found that TSCC cells secrete miR-558 into the extracellular environment, with exosome as the carrier. Human umbilical vein endothelial cells (HUVEC) ingested exosomes, which not only increased the expression level of miR-558, but also enhanced their proliferation, migration, and tube formation functions. In vivo Matrigel plug assay demonstrated that TSCC cell-derived exosome miR-558 promoted neovascularization in vivo. Compared with negative control cells, TSCC cells overexpressing miR-558 formed subcutaneous tumors in nude mice, with larger volume, heavier mass, and more vascularization. Dual luciferase assay confirmed that HPSE was the direct target gene regulated by miR-558. HPSE promoted the proliferation, migration, and tube formation of HUVECs, and the knockout of HPSE could down-regulate the pro-angiogenic effect of miR-558. Conclusion In summary, miR-558 in TSCC exosomes promotes the proliferation, migration and tube formation of HUVECs by targeting HPSE, and enhance tumor angiogenesis. Tongue squamous cell carcinoma Exosomes miR-558 HPSE Angiogenesis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Research Highlights miR-558 can be secreted by TSCC cells and delivered to HUVECs. miR-558 enhanced angiogenesis in TSCC tumors. miR-558 directly regulates the expression of HPSE in HUVECS. 1. Introduction Head and neck squamous cell carcinoma (HNSCC) is a common malignant tumor. According to A global cancer report in 2018, there were 890,000 new cases of HNSCC and 450,000 deaths annually [ 1 ] . TSCC is a relatively common HNSCC, accounting for about 41% of oral and maxillofacial malignant tumors [ 2 ] . Prone to early metastasis, the local and regional recurrence rates of TSCC are 23.9% and 20.4%, respectively, and its 5-year survival rate is 50.6% [ 3 ] . In recent years, some progress has been made in the surgery, radiotherapy, chemotherapy and immunotherapy of TSCC [ 4 – 6 ] . However, the overall survival rate of patients with locally advanced tumors and cervical lymph node metastasis remains low [ 7 ] . For patients who receive radical surgery, major surgical trauma can cause damage to swallowing, speech and even breathing functions, leading to a severe decline in quality of life. We detected and identified circRNA in fresh TSCC tissue samples through high-throughput sequencing in the early stage. Subsequent experiments confirmed that hsa_circ_0000231 was highly expressed in TSCC tumor tissue, and promoted the proliferation, migration, and invasion of TSCC cells through the Wnt/β-catenin signaling pathway [ 8 ] . Through online prediction websites Target-Scan and miRanda, we discovered that miR-558 might be one of the candidate miRNAs adsorbed by hsa_circ_0000231 [ 9 ] , and the downstream molecule miR-558 was related to the occurrence and development of tumor. Through a differential analysis of miRNA chips of TSCC tissues in the NCBI database (GSE28100), we found that miR-558 was highly expressed in human TSCC tissues. Through literature search, it was found that in bladder cancer, miR-558 can improve the proliferation, migration,and invasion abilities of tumor cells, and the supernatant of tumor cell lines can promote angiogenesis, but in-depth mechanism research is lacking [ 10 ] . Not only tumor angiogenesis provides the nutrients needed for its growth, but also pathological angiogenesis is probably an important factor that promotes distant tumor metastasis [ 11 ] . It has been reported that miR-558 promotes angiogenesis by regulating HPSE protein [ 12 ] . Accordingly, we carried out research on whether miR-558 from TSCC-derived exosomes promoted tumor angiogenesis in tumor microenvironment, and whether it was related to HPSE. 2. Materials and methods 2.1 Statement All methods were carried out in accordance with relevant guidelines and regulations, and all experimental protocols were approved by Nantong University. All relevant data are within the manuscript and its Additional files. 2.2 The isolation, characterization and quantification of exosome TSCC cell lines Tca-8113 and SCC-25 were cultured in serum-free medium for 48h, and the supernatant was collected and centrifuged at a low speed with differential gradients, that is, 300 rpm for 10 min, 2000 rpm for half an hour, and 10,000 rpm for 1 hour. The precipitate was discarded, the cell supernatant after differential centrifugation was placed in a ultracentrifuge, and ultracentrifuged at a speed of 54,000 rpm for 2 hours. All centrifugation procedures were carried out at 4°C, and the precipitate was resuspended in PBS solution. After the exosome suspension was diluted with PBS, the particle size of exosome was analyzed by a ZetaView particle size analyzer. The exosomes were lysed with RIPA and exosome protein was extracted, the concentration of exosome protein was measured with a BCA kit, and the surface proteins CD9 and CD63 of exosome were identified by means of western blot. The exosome suspension was placed under a transmission electron microscope (HT7800, JPN) to observe its microscopic morphology. 2.3 Cell culture The HUVEC was donated by the Clinical Experimental Center of the Affiliated Hospital of Nantong University and cultured in RPMI-1640 medium containing 15% fetal bovine serum (FBS). Human oral keratinocytes (HOK) and TSCC cell lines (CAL27, Tca-8113 and SCC-25) were donated by the Department of Head and Neck Surgery, the Ninth People’s Hospital of Shanghai Jiaotong University. SCC-25 cells were cultured in DMEM/F12 medium containing 10% fetal bovine serum (FBS). CAL27 and HOK cells were cultured in DMEM high glucose medium containing 10% FBS. Tca-8113 cells were cultured in 1640 medium containing 10% FBS. 2.4 Extracellular vesicle track experiment The PKH26 working solution was configured lucifugally, and the exsomes of Tca-8113 and SCC-25 were incubated with PKH26 working solution in darkness for 20 min. The exosomes stained by fluorescence were incubated using HUVEC lucifugally for 24 h. HUVEC cells were fixed with 4% paraformaldehyde for 20 min, and then stained with DAPI for 10 min. Images were obtained under an inverted microscope (OLYMPUS, JPN), and the images were synthesized by ImageJ software. 2.5 RNA oligonucleotides, plasmids and virus The following materials were synthesized by Genechem (Shanghai, China): lentiviral vectors expressing miR-141 (OE-miR-141) and their control (OE-NC), anti-miR-558 and its control (anti-NC). According to the instructions, the cells were planted in 6-well plates and transfected. After 24h, the virus-containing supernatant was discarded, antibiotic-free medium was added, and further incubated in a 5% CO 2 incubator at 37℃. After three passages, the medium containing puromycin was added to select stable strains. The plasmids of the overexpressing HPSE and control group were synthesized by Genechem Shanghai, China). HUVEC was planted in a 6-well plate, 2ml opti-MEM low-serum medium was added, 4µg plasmid was dissolved in 250µl opti-MEM low-serum medium, 10µl Lipofectamine 2000 was added to opti-MEM low-serum medium, plasmid mixed with Lipofectamine 2000 was added to HUEVC medium, and the cell supernatant was discarded after 6h, and further experiments were carried out after culturing for 24 h. Small interfering RNAs (siRNAs) of HPSE together with their negative control were synthesized with Ribobio (Guangzhou, China): siRNA-1,5’-CTAACAGTTTCCTTAAGAA-3’;siRNA-2,5’-GAAGGAAGCTTCGAGTATA-3’;siRNA-3,5’-CCATAAACCTCCATAATGT-3’. 100pmol siRNA was dissolved in 250µl opti-MEM low-serum medium, 5µl Lipofectamine 2000 was added to opti-MEM low-serum medium, and the plasmid was mixed with Lipofectamine 2000 and then added to HUEVC medium. After 6h, the supernatant was abandoned and cultured for 24 h. 2.6 RNA isolation, PCR The cells and exosomes were lysed with TRIzol reagent, and cel-miR-39-3p was added to the exosome mixture before chloroform was added. Total RNA was extracted and dissolved in DEPC water. The concentration of RNA was determined through a Nanodropone ultraviolet spectrophotometer. RT-PCR assay was done with a ThermoFisher K1622 reverse transcription kit to reverse transcribed RNA into cDNA for further study. cel-miR-39-3p was used as an exogenous reference for cellular exosomes and RNU6 was used as an endogenous reference for cell samples. qRT-PCR was done with a Ribobio primer kit. The primers of miRNAs or mRNAs used in this study were as follows: miR-558 was synthesized by Ribobio(Guangzhou,China);miR-39-3p(Forward:5’-TCACCGGGTGTAAATCAGCTTG-3’);RNU6(Forward:5’-CTCGCTTCGGCAGCACA-3’);GAPDH(Forward:5’-GTCTCCTCTGACTTCAACAGCG-3’;Reverse:5’-ACCACCCTGTTGCTGTAGCCAA-3’);HPSE(Forward:5’-CCATAAACCTCCATAATGTCACC-3’,Reverse:5’-CACCATCTTTAGAGTTAGACCATTG-3’). Set up the real-time fluorescent quantitative PCR instrument according to the instructions of the primer kit,and record the experimental results. 2.7 Western blot Cells and exosomes were lysed with RIPA, the total protein was extracted, and the protein concentration was determined by a BCA kit. The protein was dissolved in a 5× protein loading buffer at a ratio of 1:4. 10% SDS-PAGE was used to separate proteins by electrophoresis at a constant voltage of 100V, and proteins were printed onto PVDF membrane at 4℃ with a constant current of 300mA. Primary antibodies, including anti-matrix metalloproteinase 9 (MMP9), anti- heparanase (HPSE; 1:1000; Proteintech), anti- glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:1000; Sangon Biotech) and anti-vascular endothelial growth factor A (VEGF-A; 1:1000; ABclonal) antibodies, Primary antibody was used to incubate overnight and the membrane was washed with TBST solution for 3 times, 10 min each time. After the HRP-conjugated secondary antibody (1:1000; Sangon Biotech) was incubated for 1 h, the membrane containing separated proteins was washed with TBST solution for 3 times, 5 min each time. With GAPDH as the internal reference protein, the image of protein band was captured by Gel Imager (Gel Doc XR, USA). 2.8 Cell proliferation assay The cells in logarithmic growth phase were digested and the centrifuged cells were re-suspended in medium. 2500 cells were added to each well of a 96-well plate, 10µl of Cell Counting Kit 8 (CCK8, Genomeditech) was added to each experimental well for three days in a row, and the OD value of the well was measured by a multifunctional microplate reader (Thermofisher, USA) after incubating in the dark for 3 hours. 2.9 Cell migration assay The cells in the logarithmic growth phase were digested, the HUVEC content was diluted to 1. 5×10 5 /ml, and the tumor cell content was diluted to 5×10 5 /ml. The 24-well plate was placed in a small chamber, 200 µl cell suspension was added to the upper chamber, and 600 µl medium containing 10% FBS was added to the lower chamber to ensure chemotaxis of the solution in the lower chamber. The chambers in the HUVEC group were recovered after 24 hours, while the chambers in the tumor cell group were recovered after 36 hours. Cells were fixed with paraformaldehyde for 20 min and stained with crystal violet (Solarbio, Beijing, China) for 10 min. The cells in the upper chamber were wiped off with a cotton swab, and the small chamber was washed with PBS solution and let dry. Pictures were taken under a microscope (OLYMPUS, JPN). 2.10 Tube formation assay 50µl of Matrigel (model: BD356234) was spread on the bottom of the precooled 96-well plate at 4℃ using a precooled pipette tip and placed at 37℃ for 1h. HUVEC cells were suspended in serum-free medium after the Matrigel was solidified. 1.5×104 HUVEC cells were added to each well, and placed in a cell incubator for 6 h, observed and photographed under an inverted microscope. 2.11 Luciferase reporter assay Dual luciferase reporter plasmids and miR-558 mimics were designed by Limibio. With Lipofectamine 2000 transfection reagent, HUVECs were co-transfected with the reporter plasmid of the luciferase vector containing the promoter sequence of HPSE and miR-558 mimics or NC. After 48 hours of incubation, cell lysates were collected and added to a 96-well plate, and the wavelength of luciferase was detected by a microplate reader (Thermofisher, USA). 2.12 In vivo Matrigel plug assay 500 µl of low growth factor Matrigel (BD: 356231) was mixed with exosomes from Tca-8113 and SCC-25 cells and injected into the right shoulder of c57BL/6 mice. 10 days later, the Matrigel plug was removed and stored in 4% paraformaldehyde. With the help of paraffin section and immunohistochemical staining techniques, we can stain CD31 molecule in the Matrigel plug and evaluate the internal vascular density. 2.13 Animal model TSCC tumor cells in the logarithmic growth phase were digested into single-cell suspension and injected into the right armpit of five-week-old nude mice, with 5 mice in each group (n = 5). After 14 days, fresh subcutaneous tumors were taken out and the weight of tumors was accurately measured with an electronic scale. The longest diameter (a) and the shortest diameter (b) of tumors were accurately measured with a vernier caliper. The approximate volume of tumors was estimated through the formula V = a×b 2 /2. The removed tumor tissues were sectioned and used for immunohistochemical staining experiment. 2.14 Statistical analysis Statistical analysis was performed using GraphPad Prism statistical software. The comparison between two groups was done with an unpaired Student’s t-test. The comparison between three or more groups was done with a one-way ANOVA test. Statistical results with * P < 0.05, ** P < 0.01, *** P < 0.001, or **** P < 0.0001, were considered to be statistically significant. 1. TSCC cell-derived exosomes promoted HUVEC proliferation Related studies have shown that overexpression of miR-558 cancer cell supernatant can promote HUVEC tube formation. In order to explore the expression level of miR-558 in TSCC cell lines, we selected HOK, Tca-8113, CAL27, SCC-25 cell lines, and qRT-PCR experiments indicated that the expressions of miR-558 in Tca-8113 and SCC-25 cells in wild-type TSCC cell lines were relatively low (Fig. 1A). Ultracentrifugation method was employed to obtain the supernatant precipitate of wild-type TSCC cells, and the nanoparticle tracking (NTA) technology was adopted to prove that the particle size range of the precipitate obtained was between 30 and 150 nm (Fig. 1B). The total protein was extracted by Western blot experiment. It was found that the expression levels of CD9 and CD63 proteins in the total protein were much higher than the total protein of cancer cells, and the expressions of protein were consistent with the characteristics of exosomes (Fig. 1C). The extracted exosomes showed typical cup-packed and spherical morphology under a transmission electron microscope (Fig. 1D). In an effort to study the biological function of TSCC exosomes on HUVEC, we co-incubated the exosomes of wild-type SCC-25 and Tca-8113 cell lines with HUVEC in a 96-well plate. CCK8 experiment showed that 10µg/ml and 100 µg/ml exosomes played a better role in promoting HUVEC proliferation at 48 hours and 72 hours than those of the control group mixed with PBS. Comparing the effects of exocrine at different concentrations on HUVEC proliferation, exosomes with a final concentration of 100 µg/ml had the best proliferation effect at 48 h and 72 h. However, the proliferation effect of HUVEC was not further enhanced by co-incubating with 500µg/ml exosome suspension (Fig. 1E). The above experiments proved that exosomes from Tca-8113 and SCC-25 cell lines can promote the proliferation of HUVECs. Under the conditions of this experiment, TSCC cell-derived exosomes did not show a concentration-dependent effect on the proliferation of HUVECs. We took 100µg/ml as the optimal concentration under the experimental conditions. 2. TSCC exosomal miR-558 promoted the proliferation, migration and tube formation of HUVEC in vitro. To determine the function of exosome miR-558 in TSCC angiogenesis, we first built Tca-8113 and SCC-25 cell lines that stably overexpressed miR-558, to obtain exosomes with high expression of miR-558. Then the total RNA of cancer cells and exsomes with stable overexpression of miR-558 and its negative control group were extracted, and the expression of miR-558 was determined by qRT-PCR. The results showed that overexpression of miR-558 lentivirus increased the expression of miR-558 in cells and exsomes. (Fig. 2A). After incubating HUVECs with TSCC cell supernatants, the HUVECs incubated with the supernatants of Tca-8113 and SCC-25 cell lines that overexpressed miR-558 had stronger proliferation ability than the control group, the number of migrating cells increased, and the tubes generated were markedly increased (Supplementary Figs. 1A ~ D). To further investigate whether the above results were associated with exosomes, we used exosomes from Tca-8113 and SCC-25 cell lines that overexpressed miR-558 to incubate with HUVECs, and the HUVECs after incubation showed stronger proliferation ability than the negative control group. The number of migrated cells was larger than that of the negative control group, and the number of tubes was greater (Figs. 2B ~ D). Anti-miR-558 lentivirus and its NC virus were transfected into HUVEC, and the treated cells were incubated with the exosomes of cancer cells overexpressing miR-558 for 24 h. The experimental results indicated that pre-transfection of anti-miR-558 could antagonize the promoting effect of exosomes overexpressing miR-558 on HUVEC proliferation, migration and tube formation (Figs. 2B ~ D). In order to further determine the direct effect of miR-558 on HUVEC, we directly transfected HUVEC with lentivirus overexpressing miR-558 and repeated the above experiment. The transfection of lentivirus raised the expression level of miR-558 in HUVEC (Fig. 2A), and the upregulation of miR-558 improved HUVEC proliferation, migration, and tube formation (Supplementary Figs. 2B ~ D). 3. miR-558 was delivered to HUVEC from TSCC cell lines by exosomes We speculated that TSCC-derived exosomal miR-558 was delivered into HUVECs to initiate TSCC-associated angiogenesis. Thus, after we incubated the exosomes of TSCC cells in the overexpression miR-558 group and its negative control group with HUVECs, qRT-PCR showed that the expression of miR-558 in HUVEC cells in the experimental group was heightened (Fig. 3A ~ B). The extracellular vesicle track experiment captured the fluorescent images of the uptake of PKH26-stained exosomes by HUVECs. The images of three different colors were fused with imageJ software to show that the red fluorescence was distributed within the cell outline and had a clear boundary with blue nucleus (Fig. 3C). The above experiments demonstrated that TSCC cell exosomes were successfully taken up by HUVECs. 4. TSCC cell line-derived exosomal miR-558 induced neovascularization in vivo We further explored the function of exosomal miR-558 in neovascularization in vivo. Exosomes were isolated from TSCC cell supernatants, then mixed with Matrigel and injected into the right shoulder of mice. After 10 days, the Matrigel plug was recovered, and the results indicated that the Matrigel mixed with overexpressed miR-558 had more neovascularization than the control group (Figs. 4A ~ B). Immunohistochemical staining results indicated that Matrigel plugs mixed with exosomes overexpressing miR-558 had more CD31 staining areas than those in the control group, suggesting that Matrigel plugs mixed with exosomes overexpressing miR-558 had higher vascular density (Figs. 4C ~ D). The above in vivo experiments demonstrated that TSCC-derived exosomal miR-558 had a better pro-angiogenic effect. 5. miR-558 promoted tumor growth and increased the vascular density of tumor in nude mice To further investigate whether miR-558 can promote tumorigenesis in vivo by inducing neovascularization, we injected Tca-8113 and SCC-25 cells subcutaneously into the right axilla of nude mice, and found that TSCC cells overexpressing miR-558 had larger tumor volume and heavier tumor mass that those in the negative control group (Fig. 5A ~ F). We assessed the number of microvessels in tumor nodules by CD31 staining, and the results showed that the expression of CD31 molecules was upregulated in tumor sections overexpressing miR-558, suggesting a high vascular density in neoplastic tumors (Fig. G ~ J). These results showed that miR-558 can promote the growth of TSCC tumors in vivo by inducing tumor angiogenesis. 6. miR-558 promoted angiogenesis by targeting HPSE In order to further investigate the molecular mechanism how exosomal miR-558 promoted angiogenesis, we designed a dual-luciferase experiment with reference to information on the sequence of the 5’ UTR binding site between miR-558 and HPSE reported in the study of gastric cancer. The results proved that miR-558 can bind to the promoter sequence of the 5’UTR of HPSE. Overexpression of miR-558 in HUVECs increased the promoter luciferase activity of HPSE, which was counteracted by mutation in the binding site sequence (Figs. 6A ~ B). The expression level of HPSE mRNA in HUVEC was verified by qRT-PCR. Compared with the control group, the expression level of HPSE mRNA in HUVEC overexpressing miR-558 increased (Fig. 6C). The expressions of miR-558 downstream protein HPSE and HPSE downstream protein MMP9 and VEGF-A were detected by western blot assay. The results showed that compared with the negative control group, the expression of HPSE in HUVEC that overexpressed miR-558 increased, while the expression of VEGF-An and MMP9 regulated by HPSE was significantly up-regulated (Fig. 6D). The above experiments suggested that the expression of HPSE was regulated by miR-558, but whether it promoted angiogenesis via HPSE remained unclear. We synthesized the overexpressed HPSE and negative control plasmids. In the experimental group, the HPSE expression of HUVEC significantly increased (Supplementary Fig. 3A), and the overexpression of HPSE can facilitate the proliferation, migration and tube formation of HUVEC (Supplementary Fig. 3B ~ D). We used three siRNAs to knock out the HPSE gene of HUVEC, and qRT-PCR was used to verify the knockout effect. The results showed that the knockout effect of siRNA-HPSE1 was the best (Fig. 6E). Therefore, we selected it for further experiment. We used siRNA-HPSE1 to transfect HUVEC that overexpressed miR-558. The results of CCK8 experiment indicated that HPSE knockout significantly down-regulated the effect of miR-558 on the promotion of HUVEC proliferation at 72 h (Fig. 6F). The results of Transwell experiment showed that the knockout of HPSE can reverse the promoting effect of miR-558 on HUVEC migration (Fig. 6G). The results of tube formation experiment also showed that the knockout of HPSE could down-regulate the promoting function of HUVEC tube formation by overexpressing miR-558 (Fig. 6H). The above results indicated that miR-558 promoted the proliferation, migration and angiogenesis of HUVECs by regulating the expression of HPSE. 3. Discussion Malignant tumor cells have strong proliferation ability. During the rapid proliferation, the tumor tissues are in a relatively hypoxic state, which will stimulate tumor cells to secrete pro-angiogenic factors and induce neovascularization in tumor tissues [ 13 ] . Neovascularization not only brings oxygen and nutrients into tumor cells, but also raises the pressure of tumor interstitial tissues and facilitates the drainage of interstitial materials into lymph nodes because of its high permeability. This mechanism is often one of the factors that promote tumor metastasis and regional recurrence [ 14 ] . It has been reported that the expression of angiogenic factors in patients with HNSCC is higher than that in healthy people, which may result in a poor prognosis due to the enhanced tumor resistance to traditional cytotoxic drugs [ 15 ] . At present, a variety of targeted drugs have been developed for tumor angiogenesis. Bevacizumab was the first antiangiogenic drug approved by FDA in the United States. By targeting the inhibition of VEGF-A down-regulation of angiogenesis, bevacizumab can effectively treat metastatic rectal cancer, advanced non-small cell lung cancer, ovarian cancer and other malignant tumors, but the effect is not ideal in HNSCC [ 16 ] . Although antiangiogenic targeted drugs, such as sorafenib and sumatinib developed based on tyrosine kinase receptors, showed relatively lower toxicity in HNSCC clinical trials, they did not achieve a marked anticancer effect [ 17 ] . Therefore, it is very important to carry out in-depth research on HNSCC and find a more reliable and safe therapeutic target. In previous studies, our team found that hsa_circ_0000231 was highly expressed in TSCC tumor tissues and promoted the proliferation, migration and invasion of TSCC cells. At the same time, it was predicted that miR-558 may be one of the candidate molecules regulated by its targeting. Through literature search, miR-558 can promote the occurrence of lung cancer and paclitaxel resistance [ 18 ] , and up-regulate the transcription of HPSE gene in retinoblastoma [ 19 ] . YaweiLi reported that the supernatant of bladder cancer cells overexpressing miR-558 had a promoting effect on HUVEC angiogenesis [ 10 ] , but no research on its relationship with HNSCC has been found. Due to extremely high stability of exosomal miRNA, it can not only be employed as an effective biomarker by expressing the out-of-control miRNA in cancer cells, but also act as a medium to deliver miRNA to target cells and regulate the corresponding functions, when it is secreted by cancer cells into the internal environment [ 20 – 23 ] . Therefore, we carried out a study on miR-558 of TSCC-derived exosomes. In this study, we found that the expression level of miR-588 increased in exosomes of TSCC cells that overexpressed miR-588. Extracellular vesicles track experiments showed that the exosomes of miR-558 were delivered from TSCC cells to HUVEC as an uptake carrier, and the expression level of miR-558 in HUVEC increased. Compared with the control group, the supernatant and exosome of TSCC cells that overexpressed miR-588 showed stronger promotion of HUVEC proliferation, migration, and tube formation, and HUVEC pretreated with anti-miR-558 could down-regulate the above results. These results demonstrated the ability of miR-558 to promote angiogenesis in vitro. To further explore the role of TSCC exosome miR-558 in vivo, we performed in vivo Matrigel plug assay and subcutaneous tumor formation experiment on c57BL/6 mice and nude mice, respectively. The results of in vivo Matrigel plug assay showed that Matrigel mixed with exosomes overexpressing miR-558 had a higher density of angiogenesis in mice, and the results of subcutaneous tumor formation experiment indicated that the TSCC cell line overexpressing miR-558 had larger tumor size, heavier weight, and higher density of neovasculum in nude mice. Based on the prediction that miR-558 probably targeted the regulation of HPSE, we carried out research on HPSE. HPSE is a protein endonuclease secreted by tumor cells, which has the activity of degrading and remodeling extracellular matrix (ECM). Its expression is increased in a variety of malignant tumors. It can promote angiogenesis, degrade the vascular basement membrane and extracellular matrix, and is an important factor for promoting invasion and metastasis of malignant tumors [ 12 ] . HPSE can activate ERK pathway and enhance the expression of matrix metallopeptidase 9 (MMP-9) [ 24 – 26 ] . MMP-9 can raise the concentration of VEGF in tissues and facilitate the binding of VEGF and VEGFR2 [ 27 , 28 ] . In addition, HPSE has been shown to significantly increase the expression of VEGF-An in many cell lines [ 29 ] . VEGF-A in tumor cells can not only promote tumor angiogenesis, but also enhance vascular permeability. This is probably one of the initiating factors of tumor distant metastasis [ 30 , 31 ] . In this study, we proved that HPSE was a direct target of miR-558 through dual luciferase experiment. The western blot assay showed that miR-558 promoted the expression of HPSE, MMP-9 and VEGF-An in HUVEC. HPSE overexpressing HUVEC could enhance the proliferation, migration and tube formation of HUVEC, which proved that HPSE could promote angiogenesis in vitro, and the knockout of HPSE could down-regulate the above results. Therefore, we believe that the targeted regulation of HPSE expression by miR-558 is one of the main drivers of angiogenesis. In conclusion, our study is the first to demonstrate that miR-558 in TSCC-derived exosomes can promote HUVEC proliferation, migration and tube formation by targeting HPSE, and lead to the formation of tumor neovasculum. Declarations Funding This work was supported by the the Postgraduate Research & Practice Innovation of Jiangsu Province#1(No.SJCX21_1474); Jiangsu Natural Science Foundation#2(BK20211107); Nantong 226 Talent Project#3(2020-9); Nantong Science and Technology Project#4(JCZ2022069); Nantong Health Commission Science and Technology Project#5(MS2022047,MS12021097); Special Project of Clinical Medicine of Nantong University#6(2022JQ003); Changshu Science and Technology Project#7(csws202001/cswsq201901). Conflict of interest: The authors declare no potential conflicts of interest. Author Contributions Statement Bixiao Ding participated in the experimental operation and image collection, Qingwen Chen participated in the experimental operation and data analysis, Xiaoguang Li participated in the animal experiment operation, Yuancheng Ding and Qiong Wu participated in the data analysis, Hao Wu participated in the drafting and review of the paper, and Liang Han participated in the critical revision of the content and the provision of research funds. All authors agree to take responsibility for all aspects of their work. References Xu Y, Hong M, Kong D, Deng J, Zhong Z, Liang J (2022) Ferroptosis-associated DNA methylation signature predicts overall survival in patients with head and neck squamous cell carcinoma. BMC Genomics 23(1):63 Patel RS, Clark JR, Dirven R, Wyten R, Gao K, O'Brien CJ Prognostic factors in the surgical treatment of patients with oral carcinoma. ANZ J Surg 2009 Jan-Feb ;79(1–2):19–22 Kokemueller H, Rana M, Rublack J, Eckardt A, Tavassol F, Schumann P, Lindhorst D, Ruecker M, Gellrich NC (2011) The Hannover experience: surgical treatment of tongue cancer–a clinical retrospective evaluation over a 30 years period. Head Neck Oncol 3:27 Colella G, Rauso R, De Cicco D, Boschetti CE, Iorio B, Spuntarelli C, Franco R, Tartaro G (2021) Clinical management of squamous cell carcinoma of the tongue: patients not eligible for free flaps, a systematic review of the literature. Expert Rev Anticancer Ther 21(1):9–22 Onidani K, Miura N, Sugiura Y, Abe Y, Watabe Y, Kakuya T, Mori T, Yoshimoto S, Adachi J, Kiyoi T, Kabe Y, Suematsu M, Tomonaga T, Shibahara T, Honda K (2021) Possible Therapeutic Strategy Involving the Purine Synthesis Pathway Regulated by ITK in Tongue Squamous Cell Carcinoma. Cancers (Basel) 13(13):3333 Xi L, Yang Y, Xu Y, Zhang F, Li J, Liu X, Zhang Z, Du Q (2022) The enhanced genomic 6 mA metabolism contributes to the proliferation and migration of TSCC cells. Int J Oral Sci 14(1):11 Chin D, Boyle GM, Porceddu S, Theile DR, Parsons PG, Coman WB (2006) Head and neck cancer: past, present and future. Expert Rev Anticancer Ther 6(7):1111–1118 Chen QW, Wang DQ, Ding BX, Tang MM, Li XG, Zhou JY, Xu K, Fang ZR, Han L, Wu H (2022) [hsa_circ_0000231 affects the progression of tongue squamous cell carcinoma by activating Wnt/β-catenin signaling pathway]. Zhonghua Er Bi Yan Hou Tou Jing Wai Ke Za Zhi 57(10):1230–1239 Chinese Qiu X, Ke X, Ma H, Han L, Chen Q, Zhang S, Da P, Wu H (2019) Profiling and bioinformatics analyses reveal differential expression of circular RNA in tongue cancer revealed by high-throughput sequencing. J Cell Biochem 120(3):4102–4112 Li Y, Zheng F, Xiao X, Xie F, Tao D, Huang C, Liu D, Wang M, Wang L, Zeng F, Jiang G (2017) CircHIPK3 sponges miR-558 to suppress heparanase expression in bladder cancer cells. EMBO Rep 18(9):1646–1659 Lugano R, Ramachandran M, Dimberg A (2020) Tumor angiogenesis: causes, consequences, challenges and opportunities. Cell Mol Life Sci 77(9):1745–1770 Vreys V, David G Mammalian heparanase: what is the message? J Cell Mol Med 2007 May-Jun ;11(3):427–452 Harris B, Saleem S, Cook N, Searle E (2022) Targeting hypoxia in solid and haematological malignancies. J Exp Clin Cancer Res 41(1):318 Leong SP, Naxerova K, Keller L, Pantel K, Witte M (2022) Molecular mechanisms of cancer metastasis via the lymphatic versus the blood vessels. Clin Exp Metastasis 39(1):159–179 Mineta H, Miura K, Ogino T, Takebayashi S, Misawa K, Ueda Y, Suzuki I, Dictor M, Borg A, Wennerberg J (2000) Prognostic value of vascular endothelial growth factor (VEGF) in head and neck squamous cell carcinomas. Br J Cancer 83(6):775–781 Argiris A, Li S, Savvides P, Ohr JP, Gilbert J, Levine MA, Chakravarti A, Haigentz M Jr, Saba NF, Ikpeazu CV, Schneider CJ, Pinto HA, Forastiere AA, Burtness B (2019) Phase III Randomized Trial of Chemotherapy With or Without Bevacizumab in Patients With Recurrent or Metastatic Head and Neck Cancer. J Clin Oncol 37(34):3266–3274 Hyytiäinen A, Wahbi W, Väyrynen O, Saarilahti K, Karihtala P, Salo T, Al-Samadi A (2021) Angiogenesis Inhibitors for Head and Neck Squamous Cell Carcinoma Treatment: Is There Still Hope? Front Oncol 11:683570 Li X, Feng Y, Yang B, Xiao T, Ren H, Yu X, Li L, Li M, Zhang W (2021) A novel circular RNA, hsa_circ_0030998 suppresses lung cancer tumorigenesis and Taxol resistance by sponging miR-558. Mol Oncol 15(8):2235–2248 Qu H, Zheng L, Pu J, Mei H, Xiang X, Zhao X, Li D, Li S, Mao L, Huang K, Tong Q (2015) miRNA-558 promotes tumorigenesis and aggressiveness of neuroblastoma cells through activating the transcription of heparanase. Hum Mol Genet 24(9):2539–2551 Boyiadzis M, Whiteside TL (2018) Exosomes in acute myeloid leukemia inhibit hematopoiesis. Curr Opin Hematol 25(4):279–284 Colombo M, Giannandrea D, Lesma E, Basile A, Chiaramonte R (2019) Extracellular Vesicles Enhance Multiple Myeloma Metastatic Dissemination. Int J Mol Sci 20(13):3236 Rak J (2013) Extracellular vesicles - biomarkers and effectors of the cellular interactome in cancer. Front Pharmacol 4:21 Zhou W, Fong MY, Min Y, Somlo G, Liu L, Palomares MR, Yu Y, Chow A, O'Connor ST, Chin AR, Yen Y, Wang Y, Marcusson EG, Chu P, Wu J, Wu X, Li AX, Li Z, Gao H, Ren X, Boldin MP, Lin PC, Wang SE (2014) Cancer-secreted miR-105 destroys vascular endothelial barriers to promote metastasis. Cancer Cell 25(4):501–515 Jiang G, Zheng L, Pu J, Mei H, Zhao J, Huang K, Zeng F, Tong Q (2012) Small RNAs targeting transcription start site induce heparanase silencing through interference with transcription initiation in human cancer cells. PLoS ONE 7(2):e31379 Sowmya SV, Rao RS, Prasad K (2020) Prediction of metastasis in oral squamous cell carcinoma through phenotypic evaluation and gene expression of E-cadherin, β-catenin, matrix metalloproteinase-2, and matrix metalloproteinase-9 biomarkers with clinical correlation. J Carcinog 19:8 Yang B, Dong K, Guo P, Guo P, Jie G, Zhang G, Li T (2020) Identification of Key Biomarkers and Potential Molecular Mechanisms in Oral Squamous Cell Carcinoma by Bioinformatics Analysis. J Comput Biol 27(1):40–54 Bergers G, Brekken R, McMahon G, Vu TH, Itoh T, Tamaki K, Tanzawa K, Thorpe P, Itohara S, Werb Z, Hanahan D (2000) Matrix metalloproteinase-9 triggers the angiogenic switch during carcinogenesis. Nat Cell Biol 2(10):737–744 Rodriguez-Manzaneque JC, Lane TF, Ortega MA, Hynes RO, Lawler J, Iruela-Arispe ML (2001) Thrombospondin-1 suppresses spontaneous tumor growth and inhibits activation of matrix metalloproteinase-9 and mobilization of vascular endothelial growth factor. Proc Natl Acad Sci U S A 98(22):12485–12490 Cohen-Kaplan V, Naroditsky I, Zetser A, Ilan N, Vlodavsky I, Doweck I (2008) Heparanase induces VEGF C and facilitates tumor lymphangiogenesis. Int J Cancer 123(11):2566–2573 Harney AS, Arwert EN, Entenberg D, Wang Y, Guo P, Qian BZ, Oktay MH, Pollard JW, Jones JG, Condeelis JS (2015) Real-Time Imaging Reveals Local, Transient Vascular Permeability, and Tumor Cell Intravasation Stimulated by TIE2hi Macrophage-Derived VEGFA. Cancer Discov 5(9):932–943 Stoletov K, Montel V, Lester RD, Gonias SL, Klemke R (2007) High-resolution imaging of the dynamic tumor cell vascular interface in transparent zebrafish. Proc Natl Acad Sci U S A 104(44):17406–17411 Additional Declarations No competing interests reported. Supplementary Files SupplementaryFigure1.png SupplementaryFigure2.png SupplementaryFigure3.png Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3458216","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":240966499,"identity":"4234baa0-60ee-4d77-9f38-e85d749140a9","order_by":0,"name":"Bixiao Ding","email":"","orcid":"","institution":"Affiliated Hospital of Nantong University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Bixiao","middleName":"","lastName":"Ding","suffix":""},{"id":240966500,"identity":"19a12495-4f27-4f0b-ace9-91d8a5fdfb5a","order_by":1,"name":"Qingwen Chen","email":"","orcid":"","institution":"Affiliated Tumor Hospital of 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secreted by TSCC cells and delivered to HUVECs.\u003c/li\u003e\n \u003cli\u003emiR-558 enhanced angiogenesis in TSCC tumors.\u003c/li\u003e\n \u003cli\u003emiR-558 directly regulates the expression of HPSE in HUVECS.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"1. Introduction","content":"\u003cp\u003eHead and neck squamous cell carcinoma (HNSCC) is a common malignant tumor. According to A global cancer report in 2018, there were 890,000 new cases of HNSCC and 450,000 deaths annually \u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. TSCC is a relatively common HNSCC, accounting for about 41% of oral and maxillofacial malignant tumors \u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. Prone to early metastasis, the local and regional recurrence rates of TSCC are 23.9% and 20.4%, respectively, and its 5-year survival rate is 50.6%\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. In recent years, some progress has been made in the surgery, radiotherapy, chemotherapy and immunotherapy of TSCC\u003csup\u003e[\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. However, the overall survival rate of patients with locally advanced tumors and cervical lymph node metastasis remains low\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. For patients who receive radical surgery, major surgical trauma can cause damage to swallowing, speech and even breathing functions, leading to a severe decline in quality of life.\u003c/p\u003e \u003cp\u003eWe detected and identified circRNA in fresh TSCC tissue samples through high-throughput sequencing in the early stage. Subsequent experiments confirmed that hsa_circ_0000231 was highly expressed in TSCC tumor tissue, and promoted the proliferation, migration, and invasion of TSCC cells through the Wnt/β-catenin signaling pathway\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. Through online prediction websites Target-Scan and miRanda, we discovered that miR-558 might be one of the candidate miRNAs adsorbed by hsa_circ_0000231\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e, and the downstream molecule miR-558 was related to the occurrence and development of tumor. Through a differential analysis of miRNA chips of TSCC tissues in the NCBI database (GSE28100), we found that miR-558 was highly expressed in human TSCC tissues. Through literature search, it was found that in bladder cancer, miR-558 can improve the proliferation, migration,and invasion abilities of tumor cells, and the supernatant of tumor cell lines can promote angiogenesis, but in-depth mechanism research is lacking \u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. Not only tumor angiogenesis provides the nutrients needed for its growth, but also pathological angiogenesis is probably an important factor that promotes distant tumor metastasis\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. It has been reported that miR-558 promotes angiogenesis by regulating HPSE protein\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. Accordingly, we carried out research on whether miR-558 from TSCC-derived exosomes promoted tumor angiogenesis in tumor microenvironment, and whether it was related to HPSE.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Statement\u003c/h2\u003e \u003cp\u003eAll methods were carried out in accordance with relevant guidelines and regulations, and all experimental protocols were approved by Nantong University.\u003c/p\u003e \u003cp\u003eAll relevant data are within the manuscript and its Additional files.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 The isolation, characterization and quantification of exosome\u003c/h2\u003e \u003cp\u003eTSCC cell lines Tca-8113 and SCC-25 were cultured in serum-free medium for 48h, and the supernatant was collected and centrifuged at a low speed with differential gradients, that is, 300 rpm for 10 min, 2000 rpm for half an hour, and 10,000 rpm for 1 hour. The precipitate was discarded, the cell supernatant after differential centrifugation was placed in a ultracentrifuge, and ultracentrifuged at a speed of 54,000 rpm for 2 hours. All centrifugation procedures were carried out at 4°C, and the precipitate was resuspended in PBS solution. After the exosome suspension was diluted with PBS, the particle size of exosome was analyzed by a ZetaView particle size analyzer. The exosomes were lysed with RIPA and exosome protein was extracted, the concentration of exosome protein was measured with a BCA kit, and the surface proteins CD9 and CD63 of exosome were identified by means of western blot. The exosome suspension was placed under a transmission electron microscope (HT7800, JPN) to observe its microscopic morphology.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Cell culture\u003c/h2\u003e \u003cp\u003eThe HUVEC was donated by the Clinical Experimental Center of the Affiliated Hospital of Nantong University and cultured in RPMI-1640 medium containing 15% fetal bovine serum (FBS). Human oral keratinocytes (HOK) and TSCC cell lines (CAL27, Tca-8113 and SCC-25) were donated by the Department of Head and Neck Surgery, the Ninth People’s Hospital of Shanghai Jiaotong University. SCC-25 cells were cultured in DMEM/F12 medium containing 10% fetal bovine serum (FBS). CAL27 and HOK cells were cultured in DMEM high glucose medium containing 10% FBS. Tca-8113 cells were cultured in 1640 medium containing 10% FBS.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Extracellular vesicle track experiment\u003c/h2\u003e \u003cp\u003eThe PKH26 working solution was configured lucifugally, and the exsomes of Tca-8113 and SCC-25 were incubated with PKH26 working solution in darkness for 20 min. The exosomes stained by fluorescence were incubated using HUVEC lucifugally for 24 h. HUVEC cells were fixed with 4% paraformaldehyde for 20 min, and then stained with DAPI for 10 min. Images were obtained under an inverted microscope (OLYMPUS, JPN), and the images were synthesized by ImageJ software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 RNA oligonucleotides, plasmids and virus\u003c/h2\u003e \u003cp\u003eThe following materials were synthesized by Genechem (Shanghai, China): lentiviral vectors expressing miR-141 (OE-miR-141) and their control (OE-NC), anti-miR-558 and its control (anti-NC). According to the instructions, the cells were planted in 6-well plates and transfected. After 24h, the virus-containing supernatant was discarded, antibiotic-free medium was added, and further incubated in a 5% CO\u003csub\u003e2\u003c/sub\u003e incubator at 37℃. After three passages, the medium containing puromycin was added to select stable strains. The plasmids of the overexpressing HPSE and control group were synthesized by Genechem Shanghai, China). HUVEC was planted in a 6-well plate, 2ml opti-MEM low-serum medium was added, 4µg plasmid was dissolved in 250µl opti-MEM low-serum medium, 10µl Lipofectamine 2000 was added to opti-MEM low-serum medium, plasmid mixed with Lipofectamine 2000 was added to HUEVC medium, and the cell supernatant was discarded after 6h, and further experiments were carried out after culturing for 24 h.\u003c/p\u003e \u003cp\u003eSmall interfering RNAs (siRNAs) of HPSE together with their negative control were synthesized with Ribobio (Guangzhou, China): siRNA-1,5’-CTAACAGTTTCCTTAAGAA-3’;siRNA-2,5’-GAAGGAAGCTTCGAGTATA-3’;siRNA-3,5’-CCATAAACCTCCATAATGT-3’. 100pmol siRNA was dissolved in 250µl opti-MEM low-serum medium, 5µl Lipofectamine 2000 was added to opti-MEM low-serum medium, and the plasmid was mixed with Lipofectamine 2000 and then added to HUEVC medium. After 6h, the supernatant was abandoned and cultured for 24 h.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 RNA isolation, PCR\u003c/h2\u003e \u003cp\u003eThe cells and exosomes were lysed with TRIzol reagent, and cel-miR-39-3p was added to the exosome mixture before chloroform was added. Total RNA was extracted and dissolved in DEPC water. The concentration of RNA was determined through a Nanodropone ultraviolet spectrophotometer. RT-PCR assay was done with a ThermoFisher K1622 reverse transcription kit to reverse transcribed RNA into cDNA for further study. cel-miR-39-3p was used as an exogenous reference for cellular exosomes and RNU6 was used as an endogenous reference for cell samples. qRT-PCR was done with a Ribobio primer kit. The primers of miRNAs or mRNAs used in this study were as follows: miR-558 was synthesized by Ribobio(Guangzhou,China);miR-39-3p(Forward:5’-TCACCGGGTGTAAATCAGCTTG-3’);RNU6(Forward:5’-CTCGCTTCGGCAGCACA-3’);GAPDH(Forward:5’-GTCTCCTCTGACTTCAACAGCG-3’;Reverse:5’-ACCACCCTGTTGCTGTAGCCAA-3’);HPSE(Forward:5’-CCATAAACCTCCATAATGTCACC-3’,Reverse:5’-CACCATCTTTAGAGTTAGACCATTG-3’). Set up the real-time fluorescent quantitative PCR instrument according to the instructions of the primer kit,and record the experimental results.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Western blot\u003c/h2\u003e \u003cp\u003eCells and exosomes were lysed with RIPA, the total protein was extracted, and the protein concentration was determined by a BCA kit. The protein was dissolved in a 5× protein loading buffer at a ratio of 1:4. 10% SDS-PAGE was used to separate proteins by electrophoresis at a constant voltage of 100V, and proteins were printed onto PVDF membrane at 4℃ with a constant current of 300mA. Primary antibodies, including anti-matrix metalloproteinase 9 (MMP9), anti- heparanase (HPSE; 1:1000; Proteintech), anti- glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:1000; Sangon Biotech) and anti-vascular endothelial growth factor A (VEGF-A; 1:1000; ABclonal) antibodies, Primary antibody was used to incubate overnight and the membrane was washed with TBST solution for 3 times, 10 min each time. After the HRP-conjugated secondary antibody (1:1000; Sangon Biotech) was incubated for 1 h, the membrane containing separated proteins was washed with TBST solution for 3 times, 5 min each time. With GAPDH as the internal reference protein, the image of protein band was captured by Gel Imager (Gel Doc XR, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Cell proliferation assay\u003c/h2\u003e \u003cp\u003eThe cells in logarithmic growth phase were digested and the centrifuged cells were re-suspended in medium. 2500 cells were added to each well of a 96-well plate, 10µl of Cell Counting Kit 8 (CCK8, Genomeditech) was added to each experimental well for three days in a row, and the OD value of the well was measured by a multifunctional microplate reader (Thermofisher, USA) after incubating in the dark for 3 hours.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Cell migration assay\u003c/h2\u003e \u003cp\u003eThe cells in the logarithmic growth phase were digested, the HUVEC content was diluted to 1. 5×10\u003csup\u003e5\u003c/sup\u003e/ml, and the tumor cell content was diluted to 5×10\u003csup\u003e5\u003c/sup\u003e/ml. The 24-well plate was placed in a small chamber, 200 µl cell suspension was added to the upper chamber, and 600 µl medium containing 10% FBS was added to the lower chamber to ensure chemotaxis of the solution in the lower chamber. The chambers in the HUVEC group were recovered after 24 hours, while the chambers in the tumor cell group were recovered after 36 hours. Cells were fixed with paraformaldehyde for 20 min and stained with crystal violet (Solarbio, Beijing, China) for 10 min. The cells in the upper chamber were wiped off with a cotton swab, and the small chamber was washed with PBS solution and let dry. Pictures were taken under a microscope (OLYMPUS, JPN).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Tube formation assay\u003c/h2\u003e \u003cp\u003e50µl of Matrigel (model: BD356234) was spread on the bottom of the precooled 96-well plate at 4℃ using a precooled pipette tip and placed at 37℃ for 1h. HUVEC cells were suspended in serum-free medium after the Matrigel was solidified. 1.5×104 HUVEC cells were added to each well, and placed in a cell incubator for 6 h, observed and photographed under an inverted microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Luciferase reporter assay\u003c/h2\u003e \u003cp\u003eDual luciferase reporter plasmids and miR-558 mimics were designed by Limibio. With Lipofectamine 2000 transfection reagent, HUVECs were co-transfected with the reporter plasmid of the luciferase vector containing the promoter sequence of HPSE and miR-558 mimics or NC. After 48 hours of incubation, cell lysates were collected and added to a 96-well plate, and the wavelength of luciferase was detected by a microplate reader (Thermofisher, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.12 In vivo Matrigel plug assay\u003c/h2\u003e \u003cp\u003e500 µl of low growth factor Matrigel (BD: 356231) was mixed with exosomes from Tca-8113 and SCC-25 cells and injected into the right shoulder of c57BL/6 mice. 10 days later, the Matrigel plug was removed and stored in 4% paraformaldehyde. With the help of paraffin section and immunohistochemical staining techniques, we can stain CD31 molecule in the Matrigel plug and evaluate the internal vascular density.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.13 Animal model\u003c/h2\u003e \u003cp\u003eTSCC tumor cells in the logarithmic growth phase were digested into single-cell suspension and injected into the right armpit of five-week-old nude mice, with 5 mice in each group (n = 5). After 14 days, fresh subcutaneous tumors were taken out and the weight of tumors was accurately measured with an electronic scale. The longest diameter (a) and the shortest diameter (b) of tumors were accurately measured with a vernier caliper. The approximate volume of tumors was estimated through the formula V = a×b\u003csup\u003e2\u003c/sup\u003e/2. The removed tumor tissues were sectioned and used for immunohistochemical staining experiment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.14 Statistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was performed using GraphPad Prism statistical software. The comparison between two groups was done with an unpaired Student’s t-test. The comparison between three or more groups was done with a one-way ANOVA test. Statistical results with *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, or ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001, were considered to be statistically significant.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003e1. TSCC cell-derived exosomes promoted HUVEC proliferation\u003c/h3\u003e\n\u003cp\u003eRelated studies have shown that overexpression of miR-558 cancer cell supernatant can promote HUVEC tube formation.\u003c/p\u003e \u003cp\u003eIn order to explore the expression level of miR-558 in TSCC cell lines, we selected HOK, Tca-8113, CAL27, SCC-25 cell lines, and qRT-PCR experiments indicated that the expressions of miR-558 in Tca-8113 and SCC-25 cells in wild-type TSCC cell lines were relatively low (Fig.\u0026nbsp;1A). Ultracentrifugation method was employed to obtain the supernatant precipitate of wild-type TSCC cells, and the nanoparticle tracking (NTA) technology was adopted to prove that the particle size range of the precipitate obtained was between 30 and 150 nm (Fig.\u0026nbsp;1B). The total protein was extracted by Western blot experiment. It was found that the expression levels of CD9 and CD63 proteins in the total protein were much higher than the total protein of cancer cells, and the expressions of protein were consistent with the characteristics of exosomes (Fig.\u0026nbsp;1C). The extracted exosomes showed typical cup-packed and spherical morphology under a transmission electron microscope (Fig.\u0026nbsp;1D).\u003c/p\u003e \u003cp\u003eIn an effort to study the biological function of TSCC exosomes on HUVEC, we co-incubated the exosomes of wild-type SCC-25 and Tca-8113 cell lines with HUVEC in a 96-well plate. CCK8 experiment showed that 10µg/ml and 100 µg/ml exosomes played a better role in promoting HUVEC proliferation at 48 hours and 72 hours than those of the control group mixed with PBS.\u003c/p\u003e \u003cp\u003eComparing the effects of exocrine at different concentrations on HUVEC proliferation, exosomes with a final concentration of 100 µg/ml had the best proliferation effect at 48 h and 72 h. However, the proliferation effect of HUVEC was not further enhanced by co-incubating with 500µg/ml exosome suspension (Fig.\u0026nbsp;1E). The above experiments proved that exosomes from Tca-8113 and SCC-25 cell lines can promote the proliferation of HUVECs. Under the conditions of this experiment, TSCC cell-derived exosomes did not show a concentration-dependent effect on the proliferation of HUVECs. We took 100µg/ml as the optimal concentration under the experimental conditions.\u003c/p\u003e\n\u003ch3\u003e2. TSCC exosomal miR-558 promoted the proliferation, migration and tube formation of HUVEC in vitro.\u003c/h3\u003e\n\u003cp\u003eTo determine the function of exosome miR-558 in TSCC angiogenesis, we first built Tca-8113 and SCC-25 cell lines that stably overexpressed miR-558, to obtain exosomes with high expression of miR-558. Then the total RNA of cancer cells and exsomes with stable overexpression of miR-558 and its negative control group were extracted, and the expression of miR-558 was determined by qRT-PCR. The results showed that overexpression of miR-558 lentivirus increased the expression of miR-558 in cells and exsomes. (Fig.\u0026nbsp;2A).\u003c/p\u003e \u003cp\u003eAfter incubating HUVECs with TSCC cell supernatants, the HUVECs incubated with the supernatants of Tca-8113 and SCC-25 cell lines that overexpressed miR-558 had stronger proliferation ability than the control group, the number of migrating cells increased, and the tubes generated were markedly increased (Supplementary Figs.\u0026nbsp;1A ~ D). To further investigate whether the above results were associated with exosomes, we used exosomes from Tca-8113 and SCC-25 cell lines that overexpressed miR-558 to incubate with HUVECs, and the HUVECs after incubation showed stronger proliferation ability than the negative control group. The number of migrated cells was larger than that of the negative control group, and the number of tubes was greater (Figs.\u0026nbsp;2B ~ D).\u003c/p\u003e \u003cp\u003eAnti-miR-558 lentivirus and its NC virus were transfected into HUVEC, and the treated cells were incubated with the exosomes of cancer cells overexpressing miR-558 for 24 h. The experimental results indicated that pre-transfection of anti-miR-558 could antagonize the promoting effect of exosomes overexpressing miR-558 on HUVEC proliferation, migration and tube formation (Figs.\u0026nbsp;2B ~ D).\u003c/p\u003e \u003cp\u003eIn order to further determine the direct effect of miR-558 on HUVEC, we directly transfected HUVEC with lentivirus overexpressing miR-558 and repeated the above experiment. The transfection of lentivirus raised the expression level of miR-558 in HUVEC (Fig.\u0026nbsp;2A), and the upregulation of miR-558 improved HUVEC proliferation, migration, and tube formation (Supplementary Figs.\u0026nbsp;2B ~ D).\u003c/p\u003e\n\u003ch3\u003e3. miR-558 was delivered to HUVEC from TSCC cell lines by exosomes\u003c/h3\u003e\n\u003cp\u003eWe speculated that TSCC-derived exosomal miR-558 was delivered into HUVECs to initiate TSCC-associated angiogenesis. Thus, after we incubated the exosomes of TSCC cells in the overexpression miR-558 group and its negative control group with HUVECs, qRT-PCR showed that the expression of miR-558 in HUVEC cells in the experimental group was heightened (Fig.\u0026nbsp;3A ~ B). The extracellular vesicle track experiment captured the fluorescent images of the uptake of PKH26-stained exosomes by HUVECs. The images of three different colors were fused with imageJ software to show that the red fluorescence was distributed within the cell outline and had a clear boundary with blue nucleus (Fig.\u0026nbsp;3C). The above experiments demonstrated that TSCC cell exosomes were successfully taken up by HUVECs.\u003c/p\u003e\n\u003ch3\u003e4. TSCC cell line-derived exosomal miR-558 induced neovascularization in vivo\u003c/h3\u003e\n\u003cp\u003eWe further explored the function of exosomal miR-558 in neovascularization in vivo. Exosomes were isolated from TSCC cell supernatants, then mixed with Matrigel and injected into the right shoulder of mice. After 10 days, the Matrigel plug was recovered, and the results indicated that the Matrigel mixed with overexpressed miR-558 had more neovascularization than the control group (Figs.\u0026nbsp;4A ~ B). Immunohistochemical staining results indicated that Matrigel plugs mixed with exosomes overexpressing miR-558 had more CD31 staining areas than those in the control group, suggesting that Matrigel plugs mixed with exosomes overexpressing miR-558 had higher vascular density (Figs.\u0026nbsp;4C ~ D). The above in vivo experiments demonstrated that TSCC-derived exosomal miR-558 had a better pro-angiogenic effect.\u003c/p\u003e\n\u003ch3\u003e5. miR-558 promoted tumor growth and increased the vascular density of tumor in nude mice\u003c/h3\u003e\n\u003cp\u003eTo further investigate whether miR-558 can promote tumorigenesis in vivo by inducing neovascularization, we injected Tca-8113 and SCC-25 cells subcutaneously into the right axilla of nude mice, and found that TSCC cells overexpressing miR-558 had larger tumor volume and heavier tumor mass that those in the negative control group (Fig.\u0026nbsp;5A ~ F). We assessed the number of microvessels in tumor nodules by CD31 staining, and the results showed that the expression of CD31 molecules was upregulated in tumor sections overexpressing miR-558, suggesting a high vascular density in neoplastic tumors (Fig. G ~ J). These results showed that miR-558 can promote the growth of TSCC tumors in vivo by inducing tumor angiogenesis.\u003c/p\u003e\n\u003ch3\u003e6. miR-558 promoted angiogenesis by targeting HPSE\u003c/h3\u003e\n\u003cp\u003eIn order to further investigate the molecular mechanism how exosomal miR-558 promoted angiogenesis, we designed a dual-luciferase experiment with reference to information on the sequence of the 5’ UTR binding site between miR-558 and HPSE reported in the study of gastric cancer. The results proved that miR-558 can bind to the promoter sequence of the 5’UTR of HPSE. Overexpression of miR-558 in HUVECs increased the promoter luciferase activity of HPSE, which was counteracted by mutation in the binding site sequence (Figs.\u0026nbsp;6A ~ B). The expression level of HPSE mRNA in HUVEC was verified by qRT-PCR. Compared with the control group, the expression level of HPSE mRNA in HUVEC overexpressing miR-558 increased (Fig.\u0026nbsp;6C). The expressions of miR-558 downstream protein HPSE and HPSE downstream protein MMP9 and VEGF-A were detected by western blot assay. The results showed that compared with the negative control group, the expression of HPSE in HUVEC that overexpressed miR-558 increased, while the expression of VEGF-An and MMP9 regulated by HPSE was significantly up-regulated (Fig.\u0026nbsp;6D). The above experiments suggested that the expression of HPSE was regulated by miR-558, but whether it promoted angiogenesis via HPSE remained unclear.\u003c/p\u003e \u003cp\u003eWe synthesized the overexpressed HPSE and negative control plasmids. In the experimental group, the HPSE expression of HUVEC significantly increased (Supplementary Fig.\u0026nbsp;3A), and the overexpression of HPSE can facilitate the proliferation, migration and tube formation of HUVEC (Supplementary Fig.\u0026nbsp;3B ~ D). We used three siRNAs to knock out the HPSE gene of HUVEC, and qRT-PCR was used to verify the knockout effect. The results showed that the knockout effect of siRNA-HPSE1 was the best (Fig.\u0026nbsp;6E). Therefore, we selected it for further experiment. We used siRNA-HPSE1 to transfect HUVEC that overexpressed miR-558. The results of CCK8 experiment indicated that HPSE knockout significantly down-regulated the effect of miR-558 on the promotion of HUVEC proliferation at 72 h (Fig.\u0026nbsp;6F). The results of Transwell experiment showed that the knockout of HPSE can reverse the promoting effect of miR-558 on HUVEC migration (Fig.\u0026nbsp;6G). The results of tube formation experiment also showed that the knockout of HPSE could down-regulate the promoting function of HUVEC tube formation by overexpressing miR-558 (Fig.\u0026nbsp;6H). The above results indicated that miR-558 promoted the proliferation, migration and angiogenesis of HUVECs by regulating the expression of HPSE.\u003c/p\u003e "},{"header":"3. Discussion","content":"\u003cp\u003eMalignant tumor cells have strong proliferation ability. During the rapid proliferation, the tumor tissues are in a relatively hypoxic state, which will stimulate tumor cells to secrete pro-angiogenic factors and induce neovascularization in tumor tissues\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. Neovascularization not only brings oxygen and nutrients into tumor cells, but also raises the pressure of tumor interstitial tissues and facilitates the drainage of interstitial materials into lymph nodes because of its high permeability. This mechanism is often one of the factors that promote tumor metastasis and regional recurrence\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. It has been reported that the expression of angiogenic factors in patients with HNSCC is higher than that in healthy people, which may result in a poor prognosis due to the enhanced tumor resistance to traditional cytotoxic drugs\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. At present, a variety of targeted drugs have been developed for tumor angiogenesis. Bevacizumab was the first antiangiogenic drug approved by FDA in the United States. By targeting the inhibition of VEGF-A down-regulation of angiogenesis, bevacizumab can effectively treat metastatic rectal cancer, advanced non-small cell lung cancer, ovarian cancer and other malignant tumors, but the effect is not ideal in HNSCC \u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. Although antiangiogenic targeted drugs, such as sorafenib and sumatinib developed based on tyrosine kinase receptors, showed relatively lower toxicity in HNSCC clinical trials, they did not achieve a marked anticancer effect \u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. Therefore, it is very important to carry out in-depth research on HNSCC and find a more reliable and safe therapeutic target.\u003c/p\u003e\u003cp\u003eIn previous studies, our team found that hsa_circ_0000231 was highly expressed in TSCC tumor tissues and promoted the proliferation, migration and invasion of TSCC cells. At the same time, it was predicted that miR-558 may be one of the candidate molecules regulated by its targeting. Through literature search, miR-558 can promote the occurrence of lung cancer and paclitaxel resistance \u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e, and up-regulate the transcription of HPSE gene in retinoblastoma \u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. YaweiLi reported that the supernatant of bladder cancer cells overexpressing miR-558 had a promoting effect on HUVEC angiogenesis \u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e, but no research on its relationship with HNSCC has been found. Due to extremely high stability of exosomal miRNA, it can not only be employed as an effective biomarker by expressing the out-of-control miRNA in cancer cells, but also act as a medium to deliver miRNA to target cells and regulate the corresponding functions, when it is secreted by cancer cells into the internal environment \u003csup\u003e[\u003cspan additionalcitationids=\"CR21 CR22\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e–\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. Therefore, we carried out a study on miR-558 of TSCC-derived exosomes.\u003c/p\u003e\u003cp\u003eIn this study, we found that the expression level of miR-588 increased in exosomes of TSCC cells that overexpressed miR-588. Extracellular vesicles track experiments showed that the exosomes of miR-558 were delivered from TSCC cells to HUVEC as an uptake carrier, and the expression level of miR-558 in HUVEC increased. Compared with the control group, the supernatant and exosome of TSCC cells that overexpressed miR-588 showed stronger promotion of HUVEC proliferation, migration, and tube formation, and HUVEC pretreated with anti-miR-558 could down-regulate the above results. These results demonstrated the ability of miR-558 to promote angiogenesis in vitro. To further explore the role of TSCC exosome miR-558 in vivo, we performed in vivo Matrigel plug assay and subcutaneous tumor formation experiment on c57BL/6 mice and nude mice, respectively. The results of in vivo Matrigel plug assay showed that Matrigel mixed with exosomes overexpressing miR-558 had a higher density of angiogenesis in mice, and the results of subcutaneous tumor formation experiment indicated that the TSCC cell line overexpressing miR-558 had larger tumor size, heavier weight, and higher density of neovasculum in nude mice.\u003c/p\u003e\u003cp\u003eBased on the prediction that miR-558 probably targeted the regulation of HPSE, we carried out research on HPSE. HPSE is a protein endonuclease secreted by tumor cells, which has the activity of degrading and remodeling extracellular matrix (ECM). Its expression is increased in a variety of malignant tumors. It can promote angiogenesis, degrade the vascular basement membrane and extracellular matrix, and is an important factor for promoting invasion and metastasis of malignant tumors\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. HPSE can activate ERK pathway and enhance the expression of matrix metallopeptidase 9 (MMP-9) \u003csup\u003e[\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e–\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. MMP-9 can raise the concentration of VEGF in tissues and facilitate the binding of VEGF and VEGFR2\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. In addition, HPSE has been shown to significantly increase the expression of VEGF-An in many cell lines\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. VEGF-A in tumor cells can not only promote tumor angiogenesis, but also enhance vascular permeability. This is probably one of the initiating factors of tumor distant metastasis\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. In this study, we proved that HPSE was a direct target of miR-558 through dual luciferase experiment. The western blot assay showed that miR-558 promoted the expression of HPSE, MMP-9 and VEGF-An in HUVEC. HPSE overexpressing HUVEC could enhance the proliferation, migration and tube formation of HUVEC, which proved that HPSE could promote angiogenesis in vitro, and the knockout of HPSE could down-regulate the above results. Therefore, we believe that the targeted regulation of HPSE expression by miR-558 is one of the main drivers of angiogenesis.\u003c/p\u003e\u003cp\u003eIn conclusion, our study is the first to demonstrate that miR-558 in TSCC-derived exosomes can promote HUVEC proliferation, migration and tube formation by targeting HPSE, and lead to the formation of tumor neovasculum.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the the\u0026nbsp;Postgraduate Research \u0026amp; Practice Innovation of Jiangsu Province#1(No.SJCX21_1474); Jiangsu Natural Science Foundation#2(BK20211107); Nantong 226 Talent Project#3(2020-9);\u0026nbsp;Nantong Science and Technology Project#4(JCZ2022069);\u0026nbsp;Nantong Health Commission Science and Technology Project#5(MS2022047,MS12021097);\u0026nbsp;Special Project of Clinical Medicine of Nantong University#6(2022JQ003);\u0026nbsp;Changshu Science and Technology Project#7(csws202001/cswsq201901).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest:\u0026nbsp;\u003c/strong\u003eThe authors declare no potential conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBixiao Ding participated in the experimental operation and image collection, Qingwen Chen participated in the experimental operation and data analysis, Xiaoguang Li participated in the animal experiment operation, Yuancheng Ding and Qiong Wu participated in the data analysis, Hao Wu participated in the drafting and review of the paper, and Liang Han participated in the critical revision of the content and the provision of research funds. All authors agree to take responsibility for all aspects of their work.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eXu Y, Hong M, Kong D, Deng J, Zhong Z, Liang J (2022) Ferroptosis-associated DNA methylation signature predicts overall survival in patients with head and neck squamous cell carcinoma. BMC Genomics 23(1):63\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePatel RS, Clark JR, Dirven R, Wyten R, Gao K, O'Brien CJ Prognostic factors in the surgical treatment of patients with oral carcinoma. ANZ J Surg 2009 Jan-Feb ;79(1\u0026ndash;2):19\u0026ndash;22\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKokemueller H, Rana M, Rublack J, Eckardt A, Tavassol F, Schumann P, Lindhorst D, Ruecker M, Gellrich NC (2011) The Hannover experience: surgical treatment of tongue cancer\u0026ndash;a clinical retrospective evaluation over a 30 years period. Head Neck Oncol 3:27\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eColella G, Rauso R, De Cicco D, Boschetti CE, Iorio B, Spuntarelli C, Franco R, Tartaro G (2021) Clinical management of squamous cell carcinoma of the tongue: patients not eligible for free flaps, a systematic review of the literature. Expert Rev Anticancer Ther 21(1):9\u0026ndash;22\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOnidani K, Miura N, Sugiura Y, Abe Y, Watabe Y, Kakuya T, Mori T, Yoshimoto S, Adachi J, Kiyoi T, Kabe Y, Suematsu M, Tomonaga T, Shibahara T, Honda K (2021) Possible Therapeutic Strategy Involving the Purine Synthesis Pathway Regulated by ITK in Tongue Squamous Cell Carcinoma. Cancers (Basel) 13(13):3333\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXi L, Yang Y, Xu Y, Zhang F, Li J, Liu X, Zhang Z, Du Q (2022) The enhanced genomic 6 mA metabolism contributes to the proliferation and migration of TSCC cells. Int J Oral Sci 14(1):11\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChin D, Boyle GM, Porceddu S, Theile DR, Parsons PG, Coman WB (2006) Head and neck cancer: past, present and future. Expert Rev Anticancer Ther 6(7):1111\u0026ndash;1118\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen QW, Wang DQ, Ding BX, Tang MM, Li XG, Zhou JY, Xu K, Fang ZR, Han L, Wu H (2022) [hsa_circ_0000231 affects the progression of tongue squamous cell carcinoma by activating Wnt/β-catenin signaling pathway]. Zhonghua Er Bi Yan Hou Tou Jing Wai Ke Za Zhi 57(10):1230\u0026ndash;1239 Chinese\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQiu X, Ke X, Ma H, Han L, Chen Q, Zhang S, Da P, Wu H (2019) Profiling and bioinformatics analyses reveal differential expression of circular RNA in tongue cancer revealed by high-throughput sequencing. J Cell Biochem 120(3):4102\u0026ndash;4112\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi Y, Zheng F, Xiao X, Xie F, Tao D, Huang C, Liu D, Wang M, Wang L, Zeng F, Jiang G (2017) CircHIPK3 sponges miR-558 to suppress heparanase expression in bladder cancer cells. EMBO Rep 18(9):1646\u0026ndash;1659\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLugano R, Ramachandran M, Dimberg A (2020) Tumor angiogenesis: causes, consequences, challenges and opportunities. Cell Mol Life Sci 77(9):1745\u0026ndash;1770\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVreys V, David G Mammalian heparanase: what is the message? J Cell Mol Med 2007 May-Jun ;11(3):427\u0026ndash;452\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHarris B, Saleem S, Cook N, Searle E (2022) Targeting hypoxia in solid and haematological malignancies. J Exp Clin Cancer Res 41(1):318\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLeong SP, Naxerova K, Keller L, Pantel K, Witte M (2022) Molecular mechanisms of cancer metastasis via the lymphatic versus the blood vessels. Clin Exp Metastasis 39(1):159\u0026ndash;179\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMineta H, Miura K, Ogino T, Takebayashi S, Misawa K, Ueda Y, Suzuki I, Dictor M, Borg A, Wennerberg J (2000) Prognostic value of vascular endothelial growth factor (VEGF) in head and neck squamous cell carcinomas. Br J Cancer 83(6):775\u0026ndash;781\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArgiris A, Li S, Savvides P, Ohr JP, Gilbert J, Levine MA, Chakravarti A, Haigentz M Jr, Saba NF, Ikpeazu CV, Schneider CJ, Pinto HA, Forastiere AA, Burtness B (2019) Phase III Randomized Trial of Chemotherapy With or Without Bevacizumab in Patients With Recurrent or Metastatic Head and Neck Cancer. J Clin Oncol 37(34):3266\u0026ndash;3274\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHyyti\u0026auml;inen A, Wahbi W, V\u0026auml;yrynen O, Saarilahti K, Karihtala P, Salo T, Al-Samadi A (2021) Angiogenesis Inhibitors for Head and Neck Squamous Cell Carcinoma Treatment: Is There Still Hope? Front Oncol 11:683570\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi X, Feng Y, Yang B, Xiao T, Ren H, Yu X, Li L, Li M, Zhang W (2021) A novel circular RNA, hsa_circ_0030998 suppresses lung cancer tumorigenesis and Taxol resistance by sponging miR-558. Mol Oncol 15(8):2235\u0026ndash;2248\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQu H, Zheng L, Pu J, Mei H, Xiang X, Zhao X, Li D, Li S, Mao L, Huang K, Tong Q (2015) miRNA-558 promotes tumorigenesis and aggressiveness of neuroblastoma cells through activating the transcription of heparanase. Hum Mol Genet 24(9):2539\u0026ndash;2551\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBoyiadzis M, Whiteside TL (2018) Exosomes in acute myeloid leukemia inhibit hematopoiesis. Curr Opin Hematol 25(4):279\u0026ndash;284\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eColombo M, Giannandrea D, Lesma E, Basile A, Chiaramonte R (2019) Extracellular Vesicles Enhance Multiple Myeloma Metastatic Dissemination. Int J Mol Sci 20(13):3236\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRak J (2013) Extracellular vesicles - biomarkers and effectors of the cellular interactome in cancer. Front Pharmacol 4:21\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou W, Fong MY, Min Y, Somlo G, Liu L, Palomares MR, Yu Y, Chow A, O'Connor ST, Chin AR, Yen Y, Wang Y, Marcusson EG, Chu P, Wu J, Wu X, Li AX, Li Z, Gao H, Ren X, Boldin MP, Lin PC, Wang SE (2014) Cancer-secreted miR-105 destroys vascular endothelial barriers to promote metastasis. Cancer Cell 25(4):501\u0026ndash;515\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJiang G, Zheng L, Pu J, Mei H, Zhao J, Huang K, Zeng F, Tong Q (2012) Small RNAs targeting transcription start site induce heparanase silencing through interference with transcription initiation in human cancer cells. PLoS ONE 7(2):e31379\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSowmya SV, Rao RS, Prasad K (2020) Prediction of metastasis in oral squamous cell carcinoma through phenotypic evaluation and gene expression of E-cadherin, β-catenin, matrix metalloproteinase-2, and matrix metalloproteinase-9 biomarkers with clinical correlation. J Carcinog 19:8\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang B, Dong K, Guo P, Guo P, Jie G, Zhang G, Li T (2020) Identification of Key Biomarkers and Potential Molecular Mechanisms in Oral Squamous Cell Carcinoma by Bioinformatics Analysis. J Comput Biol 27(1):40\u0026ndash;54\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBergers G, Brekken R, McMahon G, Vu TH, Itoh T, Tamaki K, Tanzawa K, Thorpe P, Itohara S, Werb Z, Hanahan D (2000) Matrix metalloproteinase-9 triggers the angiogenic switch during carcinogenesis. Nat Cell Biol 2(10):737\u0026ndash;744\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRodriguez-Manzaneque JC, Lane TF, Ortega MA, Hynes RO, Lawler J, Iruela-Arispe ML (2001) Thrombospondin-1 suppresses spontaneous tumor growth and inhibits activation of matrix metalloproteinase-9 and mobilization of vascular endothelial growth factor. Proc Natl Acad Sci U S A 98(22):12485\u0026ndash;12490\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCohen-Kaplan V, Naroditsky I, Zetser A, Ilan N, Vlodavsky I, Doweck I (2008) Heparanase induces VEGF C and facilitates tumor lymphangiogenesis. Int J Cancer 123(11):2566\u0026ndash;2573\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHarney AS, Arwert EN, Entenberg D, Wang Y, Guo P, Qian BZ, Oktay MH, Pollard JW, Jones JG, Condeelis JS (2015) Real-Time Imaging Reveals Local, Transient Vascular Permeability, and Tumor Cell Intravasation Stimulated by TIE2hi Macrophage-Derived VEGFA. Cancer Discov 5(9):932\u0026ndash;943\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStoletov K, Montel V, Lester RD, Gonias SL, Klemke R (2007) High-resolution imaging of the dynamic tumor cell vascular interface in transparent zebrafish. Proc Natl Acad Sci U S A 104(44):17406\u0026ndash;17411\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Tongue squamous cell carcinoma, Exosomes, miR-558, HPSE, Angiogenesis","lastPublishedDoi":"10.21203/rs.3.rs-3458216/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3458216/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e \u003cp\u003eThis study aimed to investigate the role of miR-558 in tumor angiogenesis by targeting heparinase (HPSE) in tongue squamous cell carcinoma (TSCC)-derived exosomes.\u003c/p\u003e\u003ch2\u003eMaterials and Methods\u003c/h2\u003e \u003cp\u003eIn the present study, the role of exosome miR-558 in angiogenesis in vitro and in vivo was investigated by cell proliferation, migration, tube formation, subcutaneous tumor formation in mice, and in vivo Matrigel plug assay. The target genes of miR-558 were detected by means of dual luciferase assay.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eIt was found that TSCC cells secrete miR-558 into the extracellular environment, with exosome as the carrier. Human umbilical vein endothelial cells (HUVEC) ingested exosomes, which not only increased the expression level of miR-558, but also enhanced their proliferation, migration, and tube formation functions. In vivo Matrigel plug assay demonstrated that TSCC cell-derived exosome miR-558 promoted neovascularization in vivo. Compared with negative control cells, TSCC cells overexpressing miR-558 formed subcutaneous tumors in nude mice, with larger volume, heavier mass, and more vascularization. Dual luciferase assay confirmed that HPSE was the direct target gene regulated by miR-558. HPSE promoted the proliferation, migration, and tube formation of HUVECs, and the knockout of HPSE could down-regulate the pro-angiogenic effect of miR-558.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eIn summary, miR-558 in TSCC exosomes promotes the proliferation, migration and tube formation of HUVECs by targeting HPSE, and enhance tumor angiogenesis.\u003c/p\u003e","manuscriptTitle":"Tumor-derived exosome miR-558 promotes angiogenesis in tongue squamous cell carcinoma by targeting HPSE","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-10-19 21:39:24","doi":"10.21203/rs.3.rs-3458216/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"56175ebc-83c4-4bf6-88ff-b4e893bd0b5c","owner":[],"postedDate":"October 19th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-11-01T17:14:59+00:00","versionOfRecord":[],"versionCreatedAt":"2023-10-19 21:39:24","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3458216","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3458216","identity":"rs-3458216","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}