Background: Angiogenesis is considered as an important process in the development of malignancies and is associated with cancer progression and metastasis. Hepatocellular carcinoma (HCC) is the most common primary tumor of the liver and is recognized as a typical angiogenic tumor. Thus, it is of great importance to study the underlying mechanism of angiogenesis in HCC. The long non-coding RNA (lncRNA) ubiquitin conjugating enzyme E2C pseudogene 3 (UBE2CP3) has been reported as an oncogene that promotes tumor metastasis in HCC. However, the role and underlying mechanisms of UBE2CP3 in HCC angiogenesis are still unclear. Methods: We measured the expression levels of UBE2CP3 by in situ hybridization (ISH) and quantitative real-time polymerase chain reaction (qRT-PCR) in HCC patient samples. We also concomitantly used CD31/PAS double-staining to measure endothelial vessel (EV) density and used qRT-PCR to measure the CD31 mRNA level. HepG2 and SMMC- 7721 cells were transfected with Lv-UBE2CP3 or Sh-UBE2CP3 virus to obtain stably over-expressing or knocking-down UBE2CP3 cell lines. The indirect effects of UBE2CP3 on ECs were studied by establishing a co-culture system using Transwell chambers with a 0.4-μm pore size. HCC cells and ECs in the co-culture system were separated, but the cytokines and growth factors were able to communicate with each other. Following exposed to HCC cells, ECs were collected for functional studies. Finally, we studied the function of UBE2CP3 in vivo by chick embryo chorioallantoic membrane (CAM) angiogenesis assays and nude mouse tumorigenicity assays. Results: In this study, we found that UBE2CP3 expression was higher in HCC tissues than in para-tumor tissues and was up-regulated in tissues with high EV density. Functionally, we found that in the co-culture systems, HCC cells overexpressing UBE2CP3 promoted HUVEC proliferation, migration and tube formation via the activation of ERK/HIF-1α/p70S6K/VEGFA signalling, increasing the level of VEGFA in HCC cell supernatant. In addition, the opposite results appeared when the expression of UBE2CP3 in HCC cells was knocked down. Consistent with these results, CAM angiogenesis assays and nude mouse tumorigenicity assays showed that UBE2CP3 expression up-regulated EV density in vivo. (Continued on next page) * Correspondence: email@example.com; firstname.lastname@example.org Department of Laboratory Medicine Center, Nanfang Hospital, Southern Medical University/The First School of Clinical Medicine, Southern Medical University, Guangzhou, Guangdong 510515, China Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Lin et al. Journal of Experimental & Clinical Cancer Research (2018) 37:113 Page 2 of 13 (Continued from previous page) Conclusion: Our study suggests that UBE2CP3 can enhance the interaction between HCC tumor cells and HUVECs and promote HCC tumorigenicity by facilitating angiogenesis. Keywords: Long non-coding RNA, UBE2CP3, HCC, Angiogenesis, ERK, VEGFA, Co-culture Background [13, 14]. The lncRNA ubiquitin conjugating enzyme E2C Hepatocellular carcinoma (HCC) is one of the most pseudogene 3 (UBE2CP3) is recognized as an oncogene and common malignancies and is known for its poor prog- has been reported to promote tumor metastasis by inducing nosis ; worldwide, it is the second and sixth leading epithelial–mesenchymal transition (EMT) in HCC . It cause of cancer-related death in men and women, re- was recently reported that EMT can promote tumorigenicity spectively . Therefore, it is important to explore the in breast cancer cells by increasing tumor angiogenesis . underlying mechanisms involved in the pathogenesis of Therefore, we hypothesized that UBE2CP3 may play a major HCC. It is well-established that HCC, especially moder- role in angiogenesis. ately to poorly differentiated HCC, is a typical angiogenic In this study, we measured endothelial vessel (EV) density tumor . An increasing number of studies have verified by anti-CD31 immunohistochemistry (IHC) and quantita- that the dysregulation of angiogenesis is associated with tive real-time polymerase chain reaction (qRT-PCR). We cancer progression and metastasis . As such, studies on analysed the expression of UBE2CP3 by qRT-PCR and in the molecular mechanism of angiogenesis in HCC are situ hybridization (ISH). Our results showed that UBE2CP3 greatly needed. was frequently highly expressed in HCC tissues, especially Angiogenesis is the formation of new vessels from pre- in high EV density HCC tissues. To study the indirect ef- existing vasculature . More and more studies suggest fects of UBE2CP3 in ECs, we constructed a co-culture sys- that angiogenesis plays a key role in cancer development. tem to simulate the internal interaction by using Transwell To date, anti-angiogenic therapy has become to be one chambers with a 0.4-μm pore size. In the co-culture system, of the anti-cancer strategies. A considerable number of we co-cultured ECs with either UBE2CP3 overexpressing anti-angiogenic drugs have been approved by the FDA or UBE2CP3 knockdown HCC cells. The results showed and have been used in cancer treatment. Angiogenesis that in the co-culture system, overexpressing UBE2CP3 in has been confirmed as a complex process that involves HCC cells enhances EC proliferation, migration and tube multiple steps such as the degradation of the basement formation abilities, and knocking down UBE2CP3 induced membrane near the original vessels and endothelial cell the opposite results. In addition, we investigated the bio- (EC) proliferation, migration, aggregation and new tube logical function of UBE2CP3 in angiogenesis by chick em- formation, which eventually forms a new blood vessel sys- bryo chorioallantoic membrane (CAM) angiogenesis and tem. Tumor angiogenesis involves interactions among nude mouse tumorigenicity assay, which showed that tumor cells, ECs and mesenchymal cells through growth UBE2CP3 up-regulates EV density in vivo. Our study sug- factors or cytokines and their corresponding receptors. gests that UBE2CP3 may play an important role in the Among these growth factors, vascular endothelial growth angiogenesis of HCC. factor A (VEGFA) plays a key role in the pathophysio- logical process of angiogenesis . Normally, the secretion Methods levels of VEGFA are strictly controlled; when the levels of Antibodies, inhibitor and neutralizing antibody VEGFA are dysregulated, abnormal vessel formation oc- Antibodies against ERK (Proteintech, USA), p-ERK (Cell curs. Recently, studies have shown that many cancer cells Signalling Technology, Beverly, MA, USA), P70S6K (Cell can upregulate the expression of VEGFA to promote Signalling Technology), p-P70S6K (Cell Signalling Tech- tumor angiogenesis . However, the upstream mecha- nology), HIF-1α (Abcam, Cambridge, UK), VEGFA (ABclo- nisms in VEGFA expression regulations are still incom- nal, Wuhan, China) were used in Western blotting (WB). pletely understood. CD31 (Abcam) antibody was used for IHC. PD98059(Sel- Long non-coding RNAs (lncRNAs) are defined as a leck Chemicals, Houston, USA) were used to inhibit p- kind of RNA that is more than 200 nucleotides long and ERK. Neutralizing antibody to VEGFA165 was used to that lacks an open reading frame (ORF) with little or no block the effects of VEGFA in cell supernatant (R&D sys- protein-coding capacity. Multiple studies have shown tems, Minneapolis, USA). that lncRNA participates in essential physiological and pathological processes including tumorigenesis and Patient samples and inclusion criteria tumor progression [8–12]. Furthermore, lncRNAs have Two independent cohorts comprising a total of 94 HCC been reported to play a regulatory role in tumor angiogenesis patients were enrolled in this study. In cohort 1, formalin- Lin et al. Journal of Experimental & Clinical Cancer Research (2018) 37:113 Page 3 of 13 fixed, paraffin-embedded tissues from 48 HCC patients Table 2 Correlation among UBE2CP3, CD31 mRNA and clinicopathological parameters of HCC patients in cohort 2 were used for IHC and ISH. All patients in cohort 1 were followed up for 5 years after surgery; the detailed clinical UBE2CP3 CD31 mRNA information for these patients is shown in Table 1.In Low(n) High(n) P Low(n) High(n) P cohort 2, fresh HCC samples and corresponding para- All cases 22 24 29 17 tumor tissues were obtained from 46 HCC patients Age(year) who were undergoing routine surgery at Nangfang Hos- >=55 13 14 0.958 17 10 0.989 pital, Southern Medical University (Guangzhou, China); the <55 9 10 12 7 clinical information for these patients is shown in Table Sex 2. All patients provided written informed consent, and the research protocol was approved by the Nang- Male 16 20 0.384 21 15 0.376 fang Hospital (Guangzhou, China) Ethics Committee. Female 6 4 8 2 Edmondson grade IHC and ISH I~II 14 12 0.351 15 11 0.391 IHC assays were performed with anti-VEGFA antibody III~IV 8 12 14 6 and CD31/periodic acid-Schiff (PAS) double-staining. Tumor invasion The ISH probe used for detecting UBE2CP3-labelled T1~T2 14 7 0.019* 16 5 0.090 T3~T4 8 17 13 12 Table 1 Correlation among UBE2CP3, EV density and clinicopathological parameters of HCC patients in cohort1 Cirrhosis UBE2CP3 EV Without 13 12 0.536 18 7 0.170 Low(n) High(n) P Low(n) High(n) P With 9 12 11 10 All cases 26 22 31 17 Tumor number Age(year) Single 21 16 0.014* 27 10 0.015* >=55 16 13 0.863 17 12 0.286 Multiple 1 8 2 7 <55 10 9 14 5 Tumor size,cm Sex <=10 15 11 0.127 20 6 0.026* Male 21 21 0.274 28 14 0.732 >10 7 13 9 11 Female 5 1 3 3 The median expression level was used as the cut off. Low expression of th UBE2CP3 in 22 patients was classified as values below the 50 percentile. Edmondson grade High UBE2CP3 expression in 24 patients was classified as values at or above th I~II 20 13 0.184 21 12 0.839 the 50 percentile. Abbreviations: EV endothelial vessel, UBE2CP3 ubiquitin conjugating enzyme III~IV 6 9 10 5 E2 C pseudogene 3. *P < 0.05, ** P < 0.01 Tumor invasion T1~T2 15 7 0.037* 16 6 0.193 digoxin was designed and synthesized by Exiqon T3~T4 9 15 13 11 (Shanghai, Chia). The probe sequence is listed in Cirrhosis Additional file 1: Table S1. ISH was performed using an Without 17 11 0.281 18 10 0.959 ISH Kit (Boster Bio-Engineering Company, Wuhan, With 9 11 13 7 China) in accordance with the manufacturer’s instructions. The scoring for staining intensity was as follows: 0 (nega- Tumor number tive staining), 1 (weak), 2 (medium), 3 (strong) (Fig. 1c). Single 26 14 0.003** 29 11 0.031* The score of staining extent was as follows: 0 (<10%), 1 Multiple 0 8 2 6 (11%-25%), 2 (26%-50%), 3 (51%-75%), and 4 (76%-100%). Tumor size,cm The final UBE2CP3 expression score was calculated as the <=10 20 12 0.101 24 8 0.033* intensity score × the extent score, and it ranged from 0 to >10 6 10 7 9 12. Sections with a total score of 6 or higher were consid- ered as the high expression group, and those with a score Mortality less than 6 were categorized as the low expression group. Survive 15 5 0.014* 16 4 0.059 The IHC and ISH scores were evaluated by two patholo- Die 11 17 15 13 gists in a blinded manner. When their opinions were in- Abbreviations: EV endothelial vessel, UBE2CP3 ubiquitin conjugating enzyme consistence, a third pathologist who was also blinded to E2 C pseudogene 3. *P < 0.05, ** P < 0.01 Remarks: 2 records of tumor invasion were missing the patient information was asked to give the final score. Lin et al. Journal of Experimental & Clinical Cancer Research (2018) 37:113 Page 4 of 13 Fig. 1 UBE2CP3 is frequently up-regulated in HCC tissues and in tissues with high EV density and is associated with HCC patient prognosis. a Representative images of different intensities of UBE2CP3 ISH staining and of CD31/PAS double-staining for EV (CD31+). b, c, d Serial sections were stained with haematoxylin and eosin for H&E. ISH was used to examine UBE2CP3 expression and orientation. CD31/PAS double-staining was used to determine the expression of EV density. The results showed that UBE2CP3 was upregulated. e, f qRT-PCR analysis showed that UBE2CP3 expression was higher in HCC tissues than in para-tumor tissues (e) and was upregulated in HCC tissues with high CD31 mRNA expression (f). g The correlation between UBE2CP3 expression level and CD31 mRNA level in 46 HCC tissues. h, i Patients with high UBE2CP3 expression (h) and EV density (i)had a shorter overall survival time (OS) (P=0.0040, and P=0.0069, respectively). j Log-rank (Mantel-Cox) tests showed that when grouped by both UBE2CP3 and EV expression, HCC patients with high UBE2CP3 expression and EV density had a worse OS (P=0.0003) Cell lines and cultures vitro, HepG2 and SMMC7721 cells were transfected Human HCC cell lines (HepG2 and SMMC-7721) and with shRNA-UBE2CP3 (Sh-UBE2CP3) or control (Sh- HUVECs were purchased from the Cell Bank of Type control) viruses (Obio Technology, Shanghai, China). Culture Collection (CBTCC, Chinese Academy of The infection efficiency was confirmed by qRT-PCR. Sciences, Shanghai, China). Cells were grown in Dulbec- co’s modified Eagle’s medium (DMEM, Gibco, Gaithers- burg, MD, USA) containing 10% foetal bovine serum Cell co-culture (FBS) in a humidified incubator at 37 °C with 5% CO . Co-culture inserts (0.4-μm pores; Corning, USA) were placed into 6-well culture plates. HUVECs were added  Construction of stable cell lines to the lower culture wells (2 × 10 cells per well), and  To obtain cell lines stably overexpressing UBE2CP3, HepG2 and SMMC7721 cells (2 × 10 cells per well) HepG2 and SMMC7721 cells were infected with Lv- were placed in the inserts and cultured in DMEM sup- UBE2CP3 and Lv-control viruses (Land, Guangzhou, plemented with 5% FBS for 48 h; then, the HUVECs China). To study the knockdown effects of UBE2CP3 in were harvested for biological function studies. Lin et al. Journal of Experimental & Clinical Cancer Research (2018) 37:113 Page 5 of 13 Cell culture supernatant concentration determination 5-Ethynyl-2′-deoxyuridine (EdU) incorporation assay The supernatants from either UBE2CP3 overexpressing Following the manufacturer’s instructions, we used the or UBE2CP3 knockdown HepG2 and SMMC-7721 cells Cell-Light EdU imaging detection kit (RiboBio, Guangzhou, were collected. Cell culture supernatants were centri- China) for EdU incorporation assays. HUVECs were har- fuged at 1,000 g for 10 min to remove the cells and cell vested from the co-culture system, and after being washed debris; then, the supernatants were added to an Amicon twice with PBS, the cells were seeded into 96-well plates at Ultra filter device (Millipore, Billerica, MA, USA) and a density of 5000 cells/well. Six h later, we added EdU label- were centrifuged at 4,000 g for 30 min to obtain the ling medium and incubated the cells for approximately 60 concentrated, protein-containing liquid. min after they adhered to the plate surface; then, the cells were fixed with 4% formaldehyde for 15 min, treated with 0.5% Triton X-100 for 20 min and exposed to Apollo Enzyme-linked immunoassay (ELISA) reaction cocktail for 30 min. Next, in each well, the VEGFAin the medium was measuredbyusing theQuanti- cellular DNA was stained with Hoechst 33342 (5 up/mL) kine human VEGF ELISA kit (R&D Systems, Minneapolis, for 30 min. Finally, we visualized the results under a fluor- USA) according to manufacturer’sinstruction. escence microscope. Cell cycle analysis RNA isolation and RT-PCR Cells were harvested from the co-culture system; after Total RNA from cultured cells or human tissue was ex- being washed twice with PBS, the cells were fixed in 1 tracted using TRlzol reagent (Takara, Dalian, China) ac- ml of 70% ice-cold ethanol and were stored overnight at cording to the manufacturer’s instructions. The mRNA 4°C. Subsequently, the cells were stained with propidium levels were measured by qRT-PCR using SYBR Green iodide supplemented with RNaseA (Keygen Biotech) for PCR Master Mix (Takara) by with an ABI 7500 Fast 30 min at 37°C. The DNA content of the labelled cells Real-Time PCR system. The expression of β-actin was was analysed using FACS flow cytometry (BD Biosci- used as the internal control for analysing the mRNA ences Inc., Franklin Lakes, NJ, USA). Each experiment level of VEGFA. The expression of U6 was used as the was performed three times. internal control for analysing lncRNA UBE2CP3 expres- sion in tissues and cells. Comparative quantification was -ΔΔ Tube formation assay determined using the 2 CT method. All samples were For tube formation assay, 50 μl of Matrigel (BD Biosci- measured with at least three independent experiments, ences, San Jose, CA, USA) was added to each well of a and the results are expressed as the means ± SD for 96-well plate. After polymerizing at 37°C for 30 min, comparative analysis. The primers used are listed in HUVECs were harvested from the co-culture system Additional file 1: Table S1. and were seeded onto the Matrigel at the density of 30,000 cells per well. After incubation for 6 h in a hu- Western blot analysis midified incubator at 37°C with 5% CO , tubules were Proteins from HepG2 and SMMC7721 cells and from visualized under an IX71 inverted microscope. the concentrated cell culture supernatant were extracted with protein extraction kits (Keygen Biotech, Jiangsu, Cell migration assay China) containing protease and phosphatase inhibitors. All cell migration assays in vitro were performed using The protein concentration was quantified using a Transwell chambers (8-μm pore size; Corning) in 24- bicinchoninic acid (BCA) protein assay kit (Keygen Bio- well plates. After being suspended in DMEM with 10% tech). Then, proteins were separated on a 10% sodium FBS, HCC cells with UBE2CP3 overexpressed or dodecyl sulfate-polyacrylamide gel and transferred onto knocked down along with the corresponding control polyvinylidene difluoride membranes. The membranes groups were seeded at 50,000 cells per well into the bot- were blocked with 5% BSA for 1 h at room temperature tom chamber. These HCC cells were cultured in the and incubated with primary antibodies overnight at 4°C. lower chamber for 24 h; then, in the upper chamber, Next, the membranes were washed with TBST three we added a total of 30,000 HUVECs that had been times and incubated with goat anti-rabbit secondary harvested from the co-culture system. After an18-h antibodies for 1 h at room temperature; the protein incubation, the cells in the chamber were fixed with bands were visualized using a chemiluminescence detec- methanol, followed by staining with 0.1% crystal vio- tion system (ECL Plus Western Blot Detection System; let. Cells on the upper side of the Transwell mem- Amersham Biosciences, Foster City, CA) according to brane were wiped off with a cotton swab, and cells the manufacturer's protocol. Quantification was com- on the underside were photographed under a micro- pleted by Image J software. scope and quantified. Lin et al. Journal of Experimental & Clinical Cancer Research (2018) 37:113 Page 6 of 13 Wound healing assay Results HUVECs were harvested from the co-culture system, The expression of UBE2CP3 was frequently up-regulated and each group was washed, suspended in DMEM, and in HCC tissues, especially in high EV density tissues, and seeded at a density of 500,000 cells per well in 6-well UBE2CP3 expression combined with EV density was culture plates. After culturing for 24 h and reaching associated with HCC patient prognosis 100% confluence, wounds were made with a 200-μl plastic To identify the relationship between UBE2CP3 expres- pipette tip. After being photographed at h 0, co-culture in- sion and HCC angiogenesis, we examined UBE2CP3 serts with HepG2 or SMMC-7721 cells (at a density of expression levels by ISH and qRT-PCR. We also con- 200,000 cells per insert) were placed into 6-well culture comitantly used CD31/PAS double-staining to measure plates. After incubating in a humidified incubator at 37°C EV density and used qRT-PCR to measure the CD31 with 5% CO for 18 h, the wound width was measured mRNA level. The representative images are shown in and recorded using an IX71 inverted microscope. Lastly, Fig. 1a. UBE2CP3 expression was higher in HCC tissues the results from three wound healing assays were calcu- than in para-tumor tissues (Fig. 1b, c and e) and was up- lated and analysed. regulated in high EV density tissues compared with its level in low EV density tissues (Fig. 1b, d and f). More- over, the levels of UBE2CP3 were positively correlated Xenograft tumors in vivo  with CD31 mRNA levels (R =0.3063, P<0.001 Fig. 1g). Our animal investigations were performed in accordance Kaplan-Meier and log–rank test analyses revealed that with the institutional guidelines and were approved by the the high level of UBE2CP3 (Fig. 1h) and high EV density Animal Experimental Committee of Nanfang Hospital. (Fig. 1i) were associated with reduced overall survival Four-week-old male BALB/c nude mice were purchased time (OS). When grouped by both UBE2CP3 expression from the Guangdong Experimental Animal Center of the and EV density, HCC patients in the high category had Chinese Academy of Sciences and were bred and main- poor OS (Fig. 1j). The correlations among UBE2CP3 ex- tained in specific pathogen-free conditions. Cells from pression levels, EV density and the clinicopathological each group were resuspended in serum-free DMEM at a parameters of the HCC patients in cohort 1 are shown density of 50,000,000 cells per ml and then 0.1 ml of the in Table 1. The correlations among UBE2CP3 expression suspension was injected into the back of the nude mice. levels, CD31 mRNA level and the clinicopathological pa- Tumor tissues were obtained 5 weeks later for IHC. rameters of the HCC patients in cohort 2 are shown in Table 2. Chi-square test revealed that both the levels of UBE2CP3 (P=0.003,Table 1; P=0.014, Table 2) and EV Angiogenesis assay in chick chorioallantoic membranes density (P=0.031,Table 1; P=0.015, Table 2) were signifi- (CAM) cantly correlated with tumor numbers. Furthermore, the After being cultured at 37 °C for 7 days, a window was levels of UBE2CP3 were positively correlated with opened on the shell of fertilized eggs to expose the mortality (P=0.014,Table 1) and tumor invasion (P=0.037, CAM and then was covered with a filter paper disc (0.5 Table 1; P=0.019, Table 2); and EV density was significantly cm in diameter) containing HepG2 cells (18,000/disc) or correlated with tumor size (P=0.033, Table 1; P=0.026, PBS on the surface. Next, tape was used to cover the Table 2). window for further incubation. Two days later, the CAM was fixed in 3.7% formaldehyde, and we visualized the UBE2CP3 in HCC cells indirectly affected EC proliferation, results under a stereoscope. migration, and tube formation in the co-culture system Our ISH and IHC results indicated that UBE2CP3 may Statistical analysis participate in the HCC angiogenic process. The indirect Data were shown as the means±SD of at least three in- effects of UBE2CP3 on ECs were studied by establishing dependent experiments. Statistical analysis was per- a co-culture system using Transwell chambers with a formed using SPSS 13.0 software (SPSS Inc., Chicago, 0.4-μm pore size. HCC cells that had been transfected IL, USA) and GraphPad Prism (GraphPad Software, Inc., with Lv-UBE2CP3 or Sh-UBE2CP3 virus (the infection La Jolla, CA, USA). The chi-Square test was used to efficiencies are shown in Additional file 2: Figure S1A) examine the relationship between EV density, UBE2CP3 were seeded into the upper chamber, and ECs were expression and clinicopathological characteristics. Differ- seeded into the lower chamber. HCC cells and ECs from ences between experimental groups were assessed by the co-culture system were separated, but the cytokines Student’s t-test or one-way ANOVA. Kaplan-Meier and and growth factors were able to communicate with each log-rank tests were used to analyse survival time. Statistical other (Additional file 2: Figure S1B). In the co-culture significance was set at *P <0.05, **P < 0.01, ***P < 0.001. system, ECs were exposed to HCC cells for 48 h. Follow- P < 0.05 was considered statistically significant. ing this exposure, ECs were removed from the co-culture Lin et al. Journal of Experimental & Clinical Cancer Research (2018) 37:113 Page 7 of 13 system for further studies. Since angiogenesis involves EC culture system were significantly reduced. (Fig. 3c, d, e). proliferation, migration and tube formation, we analysed Meanwhile, the WB assays with HCC cell protein showed the cell cycle and performed EdU, Transwell, wound heal- higher levels of phosphor -ERK (p-ERK), phosphor-p70S6K ing and tube formation assays to gain insight into the role (p-p70S6K), HIF-1α, and VEGFA in the UBE2CP3 overex- of UBE2CP3 in cell proliferation, migration and tube for- pressing cellsthaninthe control cells. In contrast, knock- mation. We observed that compared with the control ing down the expression of UBE2CP3 reduced the levels of group, EC cells co-cultured with HCC cells stably overex- p-ERK, p-p70S6K, HIF-1α,and VEGFA(Fig. 3b, c)com- pressing UBE2CP3 had significantly enhanced prolifera- pared with their expression in the control cells (Fig. 3f). tion (Fig. 2a, b), migration (Fig. 2c, d) and tube formation After treated with 100μM PD98059 for 48h, the level potential (Fig. 2e). Conversely, ECs co-cultured with the of p-ERK was significantly reduced, and the levels of UBE2CP3-knockdown HCC cells had decreased prolifera- p-p70S6K, HIF-1α, and VEGFA can also be reduced tion (Fig. 2a, b), migration (Fig. 2c, d) and tube formation by the inhibitor (Fig. 3g). abilities (Fig. 2e). Our results suggested that the dysregula- tion of UBE2CP3 in HCC cells may play an indirect effect UBE2CP3 promoted angiogenesis in vivo on ECs proliferation, migration and tube formation in vitro. To determine the effects of UBE2CP3 on HCC angiogen- esis, we injected HepG2 cells that were stably transfected UBE2CP3 in HCC cells promoted EC proliferation, migration, with Lv-UBE2CP3 or Lv-control into chick chorioallantoic and tube formation by enhancing the secretion of VEGFA membranes (CAM). Chick embryos injected with cells into the supernatant via activation of the ERK/HIF-1α overexpressing UBE2CP3 had an increase in new vessel signalling pathway density (Fig. 4a). In contrast, when chick embryos were ECs co-cultured with UBE2CP3-dysregulated HCC cells injected with cells with inhibited UBE2CP3 expression, a had alterations in cell proliferation, migration and tube decrease in new vessel density was observed (Fig. 4b). formation abilities. We hypothesized that cytokines and/ Consistent with these results, mice injected with cells or growth factors may participate in this process. As overexpressing UBE2CP3 had a higher EV density in the shown in Additional file 2:FigureS1C,12kinds of com- tumor tissue than the mice injected with Lv-control cells mon angiogenic factors were detected using qRT-PCR, the (Fig. 4c). Mice injected with cells knocking down mRNA levels of VEGFA (up-regulated fold=4.04, P=0.008), UBE2CP3 had a lower EV density in the tumor tissue than Ang2(up-regulated fold=1.82, P=0.031) were increased the mice injected with sh-control cells (Fig. 4d). in UBE2CP3 overexpressing HepG2 cells and the mRNA levels of VEGFA(down-regulated folds=0.31, Discussion P=0.008), Ang2(down-regulated folds=0.63, P=0.037), Angiogenesis is one hallmark of cancer , when grow- and PDGFC(down-regulated folds=0.49, P=0.038) were ing to a certain size, a tumor mass requires angiogenesis decreased in UBE2CP3 knocking down HepG2 cells. to maintain its nutritional supply for continued develop- Since VEGFA is one of the most important growth ment. In most instances, tumor cells secrete angiogenic factors in angiogenesis and is the most obvious chan- substances and initiate angiogenesis process . Tumor ging cytokine in UBE2CP3 overexpressing or knock- angiogenesis requires interactions among tumor cells, ing down HepG2 cells, we decided to investigate the ECs and mesenchymal cells through growth factors or role and the underlying mechanism of VEGFA in cytokines and their corresponding receptors. Recent UBE2CP3 inducing angiogenesis in HCC. The VEGFA studies have suggested that lncRNAs can modulate the levels in the Lv-control, Lv-UBE2CP3, sh-control, and process of angiogenesis by regulating the expression sh-UBE2CP3 cell supernatants from the co-culture level of angiogenic molecules and the functions in ECs system after concentrated by an Amicon Ultra filter [19, 20]. LncRNAs are a type of RNA that are longer device were determined by WB and ELISA assay. As than 200 nucleotides and do not have protein-coding expected, a higher level of VEGFA was observed in the capacity. An increased amount of evidence suggests that UBE2CP3 overexpressing HCC cell supernatant than in lncRNAs play an important role in regulating a wide var- the supernatant of the control cells. In contrast, in the co- iety of biological processes related to hepatocarcinogen- culture system, knocking down UBE2CP3 induced a esis, including cell survival, apoptosis, metastasis, and decrease in the supernatant VEGFA level (Fig. 3a, b). To angiogenesis [9, 21, 22]. Although several angiogenesis- investigate whether VEGFA take part in UBE2CP3 inducing associated lncRNAs have been identified, the function EC proliferation, migration and tube formation, VEGFA and clinical significance of most angiogenic lncRNAs neutralizing antibodies were used to block the VEGFA ef- in HCC angiogenesis remain largely unknown. In fects in the co-culture system. When adding VEGFA neu- addition, the effects of lncRNAs on the regulation of tralizing antibodies, the indirect effects of UBE2CP3 on EC the interactions between tumor cells and ECs have proliferation, migration, and tube formation in the co- not been identified. Lin et al. Journal of Experimental & Clinical Cancer Research (2018) 37:113 Page 8 of 13 de Fig. 2 (See legend on next page.) Lin et al. Journal of Experimental & Clinical Cancer Research (2018) 37:113 Page 9 of 13 (See figure on previous page.) Fig. 2 UBE2CP3 enhanced the crosstalk between HCC cells and HUVECs, and promoted cell cycle progression, migration and tube formation in HUVECs which co-cultured with HepG2/SMMC-7721 cells. a Analysis of cell cycle progression in HUVECs after co-culturing with UBE2CP3 overexpressing HepG2/SMMC-7721 cells or with UBE2CP3 knockdown HepG2/SMMC-7721 cells. b HepG2/SMMC-7721 cells with increased UBE2CP3 expression were seeded onto 24-well plates, HUVECs were seeded into the co-culture inserts, and cell proliferation was examined by EdU immunofluorescence staining. The effect of UBE2CP3 knockdown on HepG2/SMMC-7721 cell proliferation was also measured by EdU immunofluorescence staining. The graph on the right shows the percentage of EdU-positive nuclei. c, d UBE2CP3 promoted tumor-induced HUVEC migration according to wound healing (c)and Trans- well migration assays (d). e UBE2CP3 promoted tumor-induced HUVEC angiogenesis according to tube formation assays. The results show the means ± SD from at least three separate experiments. *P < 0.05, **P < 0.01, ***P < 0.001 In this study, we found that the expression of density. The clinical results showed that UBE2CP3 UBE2CP3 was up-regulated in HCC tissues compared to expression and EV density are both significantly corre- the levels in para-tumor tissues, and compared to its lated with metastasis (i.e., positively correlated with expression in tissues with low EV density, UBE2CP3 tumor number) and poor prognosis in HCC patients. expression was up-regulated in tissues with high EV When categorized according to both UBE2CP3 Fig. 3 UBE2CP3 in HCC cells promoted EC proliferation, migration, and tube formation by enhancing the secretion of VEGFA into the supernatant via activation of the ERK/HIF-1α signalling pathway. a, b The levels of VEGFA in HepG2 concentrated supernatant were analysed by ELSIA (a) and western blot (b). For western blot, β-actin served as the internal control, β-Actin in the cell supernatant served as the quantity control. c, d, e Using VEGFA neutralizing antibody markedly reduce the effects of UBE2CP3 in HCC cells on EC proliferation (c), migration (d) and tube formation (e) in the co-culture system, IgG antibodies were used for negative control. f The levels of ERK1/2, p-ERK, p70S6K, p-p70S6K, HIF-1α, and VEGFA were examined by Western blot analysis in HepG2 cells overexpressing UBE2CP3 and in HepG2 cells with UBE2CP3 expression silenced. g Treatment with p-ERK inhibitor (PD98059) markedly reduce the levels of p-ERK, p-p70S6K, HIF-1α and VEGFA in UBE2CP3 overexpressing HepG2 cells. The data are expressed as the means ± SD. *P <0.05, **P < 0.01, ***P < 0.001 Lin et al. Journal of Experimental & Clinical Cancer Research (2018) 37:113 Page 10 of 13 ab Fig. 4 UBE2CP3 promoted angiogenesis in vivo. a CAM angiogenesis assays showed that when injected with HCC cells overexpressing UBE2CP3, the density of new chick embryo vessels increased. b Chick embryos injected with UBE2CP3 knockdown cells had decreased new vessel density. c CD31/PAS double-staining results showed that mice injected with cells overexpressing UBE2CP3 had higher EV density in the tumor tissue than those injected with Lv-control cells. d Mice injected with UBE2CP3 knockdown cells had lower EV density in the tumor tissue than those injected with the sh-control cells expression and EV density, HCC patients in the high angiogenesis and EMT; therefore, the role of UBE2CP3 category presented with poor OS. These results sug- in HCC angiogenesis is clearly indicated. gested that a high level of UBE2CP3 may be closely re- Since the EMT process has been suggested to be closely lated to HCC angiogenesis and that UBE2CP3 might related to the tumor extracellular microenvironment, we serve as a valuable therapeutic target in HCC. Consistent hypothesized that UBE2CP3 may play an important role with our results, Cao SW et al  found that UBE2CP3 in the tumor extracellular microenvironment. Thus, in plays an important role in promoting HCC metastasis by this study, we performed gain- and loss-of-function exper- inducing EMT. An increasing amount of evidence sug- iments in HCC cells. We first investigated the indirect role gests that EMT is closely related to angiogenesis [23– of UBE2CP3 on EC proliferation, migration, and tube for- 25]; angiogenesis is promoted by EMT-induced VEGFA mation by constructing a co-culture system with HCC in human breast tumors  and is enhanced by the cells and ECs by using a Transwell chamber with a 0.4-μm EMT-mediated increase in the secretion of factors pore size; in the co-culture system, HCC cells and ECs known to enhance angiogenesis (e.g., TGF-β, CSF-1, were separated, but cytokines and growth factors were still NGF, VGF, ADAM9 and ADAM17) into the extracellular able to communicate with each other. We demonstrated microenvironment . Considering the above findings, that HCC cells stably overexpressing UBE2CP3 in the co- there is a strong internal relationship between culture system promoted EC proliferation, migration and Lin et al. Journal of Experimental & Clinical Cancer Research (2018) 37:113 Page 11 of 13 Fig. 5 Diagrammatic sketches of UBE2CP3 promotion of tumor cell-induced angiogenesis. a UBE2CP3 promoted the secretion of VEGFA in HCC cells by activating ERK/HIF-1α signalling. b The HCC-induced process of forming new vessels from pre-existing ones tube formation by enhancing the secretion of VEGFA into important mediator of UBE2CP3-regulated angiogenesis. the supernatant. It has been well defined that VEGFA is However, which signalling pathways are activated in ECs one of the most important growth factors for angiogenesis by the ectopic expression of UBE2CP3 in HCC cells re- . VEGFA can activate relevant angiogenic signalling quires further investigation. pathways such as those of ERK, phosphoinositide 3 kinase An increasing amount of evidence demonstrates that (PI3K)/Akt , phospholipase Cγ (PLCγ)[29, 30], Src, hypoxia inducible factor-1α (HIF-1α) is the main up- focal adhesion kinase (FAK) , p38 mitogen-activated stream inducer of VEGFA, which plays a key role in tumor protein kinase (MAPK) , the Rho family , GTPases angiogenesis [36, 37]. Recent studies have characterized a  and endothelial nitric oxide (NO) . These signal- series of HIF-1α related pathways, such as the ERK , ling pathways eventually alter EC proliferation, migration, TNF-α , phosphatidylinositol-3-kinase (PI3K), mam- invasion and tube formation capacity and vascular perme- malian homologue target of rapamycin (mTOR), and ability. In summary, the secretion of VEGFA into the AKT [40, 41] pathways. Here, we found that UBE2CP3 HCC cell extracellular microenvironment might be an up-regulated the levels of p-ERK, phosphor-p70S6K Lin et al. Journal of Experimental & Clinical Cancer Research (2018) 37:113 Page 12 of 13 (p-p70S6K), HIF-1α, and VEGFA in HCC cells. In con- Additional file 2: Figure S1. (A) The infection efficiencies of lncRNA trast, knocking down the expression of UBE2CP3 reduced UBE2CP3 in HepG2 and SMMC-7721 were screened by qRT-PCR. (B) The schematic diagram for the co-culture system. (C) 12 kinds of common the levels of p-ERK, p-p70S6K, HIF-1α and VEGFA. ERK angiogenic factors were detected by qRT-PCR.. (TIF 24 kb) signalling has been reported to not only participate in various tumor transitions for cell survival and invasion Abbreviations [42, 43] but also modulate the transcription factors for CAM: chick embryo chorioallantoic membrane; EMT: epithelial–mesenchymal tumor EMT and angiogenesis [44, 45]. P70S6K is one of transition; EV: endothelial vessel; HCC: hepatocellular carcinoma; HUVEC: human umbilical vein endothelial cell; IHC: immunohistochemistry; the ERK pathway effectors, and an increasing amount ISH: in situ hybridization; lncRNA: long non-coding RNA; ORF: open reading of evidence demonstrates that ERK/p70S6K signalling frame; OS: overall survival time; qRT-PCR: quantitative real-time polymerase participates in several pathological processes, such as chain reaction; UBE2CP3: ubiquitin conjugating enzyme E2C pseudogene 3 cell drug-resistance and angiogenesis [46, 47]. In addition, Acknowledgements p70S6K modulates the expression and activation of We are thankful for the department of Hepatobiliary Surgery, Nanfang HIF-1α [48, 49]. Our results demonstrated that the Hospital, Southern Medical University, Guangzhou, China for providing the HCC tissue samples and the clinical data. ERK/p70S6K/HIF-1α signalling pathway may be par- tially responsible for the UBE2CP3-induced accumula- Funding tion of VEGFA and leads to tumor cell-mediated This work was partly supported by a grant from the National Natural Science Foundation of China (No. 81672076) and Medical Scientific Research angiogenesis. Foundation of Guangdong Province, China (B2018133). Although we confirmed that UBE2CP3 may act as an oncogene to promote the secretion of VEGFA from Availability of data and materials All data generated or analysed during this study are included in this published HCC cells into the tumor microenvironment by activat- article [and its Additional files 1 and 2 ing the ERK/p70S6K/HIF-1α pathway and enhance tumor cell-induced angiogenesis, the underlying mech- Authors’ contributions JL performed the experimental work and drafted the manuscript. SC, YH, HL, anism of how ECs respond to UBE2CP3 dysregulation in JL, YW, JC, PL and JL participated in the experiments and performed the HCC cells is still unclear. Moreover, because our study statistical analysis. LZ and QW conceived of the study and participated in its was based on the co-culture system, adding ERK signal- design and coordination. All authors read and approved the final manuscript. ling inhibitors in the co-culture system will not only Ethics approval and consent to participate inhibited the ERK pathway in HCC cells but also reduce The research protocol was approved by the Ethics Committee Nanfang the activity of ERK in endothelial cells, a more appropri- Hospital. All the patientsprovied written informed consent. ate experimental design is needed to study the function Consent for publication of UBE2CP3 when ERK signalling is inhibited. Addition- All authors have seen the manuscript and approved to submit to your journal. ally, the molecules functioning directly downstream of Competing interests UBE2CP3 remain unknown; further research, such as The authors declare that they have no competing interests. pull-down assays, is needed to determine the direct tar- get molecules of UBE2CP3. Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Conclusions As summarized in Fig. 5, this study demonstrates that Author details Department of Laboratory Medicine Center, Nanfang Hospital, Southern lncRNA UBE2CP3 promotes angiogenesis indirectly. Medical University/The First School of Clinical Medicine, Southern Medical Specifically, UBE2CP3 promotes HCC cell secretion of 2 University, Guangzhou, Guangdong 510515, China. Department of Clinical VEGFA into the tumor microenvironment by activating Laboratory, The Sixth Affiliated Hospital of Guangzhou Medical University, Qingyuan People’s Hospital, Qingyuan, Guangdong, China. Department of the ERK/p70S6K/HIF-1α pathway; this VEGFA alters EC Hepatobiliary Surgery, Nanfang Hospital, Southern Medical University/The proliferation, migration and tube formation capacities. First School of Clinical Medicine, Southern Medical University, Guangzhou, Modulating tumor angiogenesis by inhibiting UBE2CP3 Guangdong, China. expression may be a potential strategy for HCC preven- Received: 16 November 2017 Accepted: 6 March 2018 tion and treatment. Our results suggest that in HCC, UBE2CP3 indirectly enhances tumor cell-induced angio- References genesis. UBE2CP3 is a potential oncogene that partici- 1. Greten TF, Wang XW, Korangy F. 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Journal of Experimental & Clinical Cancer Research
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Published: Jun 4, 2018