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. 2018 Jun 4;37(1):113.
doi: 10.1186/s13046-018-0727-1.

Long non-coding RNA UBE2CP3 enhances HCC cell secretion of VEGFA and promotes angiogenesis by activating ERK1/2/HIF-1α/VEGFA signalling in hepatocellular carcinoma

Jinduan Lin  1   2 , Shunwang Cao  1 , Yu Wang  3 , Yanwei Hu  1 , Hongwei Liu  2 , Jiehua Li  2 , Jing Chen  1 , Pan Li  1 , Jumei Liu  1 , Qian Wang  4 , Lei Zheng  5
Affiliations

Long non-coding RNA UBE2CP3 enhances HCC cell secretion of VEGFA and promotes angiogenesis by activating ERK1/2/HIF-1α/VEGFA signalling in hepatocellular carcinoma

V体育平台登录 - Jinduan Lin et al. J Exp Clin Cancer Res. .

Abstract

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 VSports手机版. 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 V体育安卓版. 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 V体育ios版. Consistent with these results, CAM angiogenesis assays and nude mouse tumorigenicity assays showed that UBE2CP3 expression up-regulated EV density in vivo. .

Conclusion: Our study suggests that UBE2CP3 can enhance the interaction between HCC tumor cells and HUVECs and promote HCC tumorigenicity by facilitating angiogenesis VSports最新版本. .

Keywords: Long non-coding RNA, UBE2CP3, HCC, Angiogenesis, ERK, VEGFA, Co-culture V体育平台登录. .

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Conflict of interest statement

Ethics approval and consent to participate

The research protocol was approved by the Ethics Committee Nanfang Hospital. All the patientsprovied written informed consent VSports注册入口.

Consent for publication

All authors have seen the manuscript and approved to submit to your journal.

Competing interests

The authors declare that they have no competing interests.

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Figures

Fig. 1
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)
Fig. 2
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 Transwell 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
Fig. 3
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
Fig. 4
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
Fig. 5
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
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