The Role And Mechanism Of Long Non-coding RNA UBE2CP3 On Angiogenesis In Hepatocellular Carcinoma

ObjectiveHepatocellular carcinoma(HCC)is one of the most common malignancies and is known for its poor prognosis worldwide,it is the second and sixth leading cause of cancer-related death in men and women,respectively.Therefore,it is important to explore the underlying mechanisms involved in the pathogenesis of HCC.It is well-established that HCC,especially moderately to poorly differentiated HCC,is a typical angiogenic tumor.An increasing number of studies have verified that the dysregulation of angiogenesis is associated with cancer progression and metastasis.As such,studies on the molecular mechanism of angiogenesis in HCC are greatly needed.More and more studies suggest that angiogenesis plays a key role in cancer development.Tumor angiogenesis involves interactions among tumor cells,endothelial cell and mesenchymal cells through growth factors or cytokines and their corresponding receptors.However,the upstream mechanisms in the growth factors or cytokines expression regulations are still incompletely understood.Long non-coding RNAs(IncRNAs)are defined as a kind of RNA that is more than 200 nucleotides long and that lacks an open reading frame(ORF)with little or no protein-coding capacity.Multiple studies have shown that IncRNA participates in essential physiological and pathological processes including tumorigenesis and tumor progression.Furthermore,lncRNAs have been reported to play a regulatory role in tumor angiogenesis.The IncRNA ubiquitin conjugating enzyme E2C pseudogene 3(UBE2CP3)is recognized as an oncogene and has been reported to promote tumor metastasis by inducing epithelial-mesenchymal transition(EMT)in HCC.It was recently reported that EMT can promote tumorigenicity in breast cancer cells by increasing tumor angiogenesis.Therefore,we hypothesized that UBE2CP3 may play a major role in angiogenesis.Further study on the role of UBE2CP3 in HCC angiogenesis will help us to gain a sight into the mechanism in HCC development.MethodsIn this study,we measured intraturclormicrovessel density(MVD)by anti-CD31 immunohistochemistry(IHC)and quantitative real-time polymerase chain reaction(qRT-PCR).We analysed the expression of UBE2CP3 by qRT-PCR and in situ hybridization(ISH).Our results showed that UBE2CP3 was frequently highly expressed in HCC tissues,especially in high MVD HCC tissues.To study the indirect effects of UBE2CP3 in endothelial cells(EC s),after constructed the stable over-expressing or knocking down UBE2CP3 HCC cell lines by Lentivirus infection,we constructed a co-culture system to simulate the internal interaction by using Transwell chambers with a 0.4-?m pore size.In the co-culture system,we co-cultured ECs with either UBE2CP3-overexpressing or UBE2CP3 knocking down HCC cells.After co-culture with HCC cells,ECs were clollected from the co-culture system for futher study.To gain a sight into indirect effects of UBE2CP3 on ECs,we study the functional alteration in ECs by cell cycle analysis,EDU assy,Transwell assy,wounding healing assay and tube formation assay to determine the ability of cell proliferation,migration and tube formation.Finally,we study the function of UBE2CP3 in vivo by CAM angiogenesis assays and nude mouse tumorigenicity assays.ResultsTo identify the relationship between UBE2CP3 expression and HCC angiogenesis,we examined UBE2CP3 expression levels by ISH and qRT-PCR.We also concomitantly used CD31/PAS double-staining to measure MVD and used qRT-PCR to measure the CD31 mRNA level.UBE2CP3 expression was higher in HCC tissues than in para-tumor tissues and was up-regulated in high MVD tissues compared with its level in low MVD tissues.Moreover,the levels of UBE2CP3 were positively correlated with CD31 mRNA levels(R2=0.3063,p<0.001).Kaplan-Meier and log-rank test analyses revealed that the high level of UBE2CP3 and high MVD were associated with reduced overall survival time(OS).When grouped by both UBE2CP3 expression and MVD,HCC patients in the high category had poor OS.Chi-square test revealed that both the levels of UBE2CP3 and MVD were significantly correlated with tumor numbers.Furthermore,the levels of UBE2CP3 were positively correlated with mortality and tumor invasion;and MVD was significantly correlated with tumor size.To further study on the indirect effects of UBE2CP3 on ECs,we construct a HCC cells and ECs co-culture system.The results showed that in the co-culture system,overexpressing UBE2CP3 in HCC cells enhances EC proliferation,migration and tube formation abilities,and knocking down UBE2CP3 induced the opposite results.In addition,we investigated the biological function of UBE2CP3 in angiogenesis by CAM angiogenesis and nude mouse tumorigenicity assay,which showed that UBE2CP3 up-regulates MVD in vivo.VEGFA has been proved to play an important role in angiogenesis.To investigate the molecular mechanisms of IncRNA UBE2CP3 in angiogenesis,the levels of VEGFA in HCC cell and in HCC cell supernatant were measured by western blot,we found that the levels of VEGFA in HCC cell and in HCC cell supernatant were up-regulated in the UBE2CP3 over-expressing cell lines,and the oppsite results were appeared when UBE2CP3 was knocking down.To further investigate the molecular mechanisms,several VEGFA-related signaling pathway key protien were detected by western blot analysis.Our results showed that lncRNA UBE2CP3 could up-regulate the expression of phosphor-ERK(p-ERK),HIF-1?,and VEGFA in the UBE2CP3-overexpressing cells than in the control cells.In contrast,knocking down the expression of UBE2CP3 reduced the levels of p-ERK,HIF-1?,and VEGFA compared with their expression in the control cells.ConclusionOur results indicate that IncRNA UBE2CP3 is frequently up-regulated in high MVD HCC tissue.LncRNA UBE2CP3 can promote ECs proloferarion,migration and tuble formation by activating ERKHIF-1?/VEGFA signaling and up-regulating the VEGFA level in HCC cell supernatant in vitro.In vivo,IncRNA UBE2CP3 can enhance MVD desity.Our findings suggest that IncRNA UBE2CP3 promotes angiogenesis in an direct way.Specifically,UBE2CP3 promotes HCC cell secretion of VEGFA into the tumor microenvironment by activating the ERK/HIF-1? pathway;this VEGFA alters EC proliferation,migration and tube formation capacities.Modulating tumor angiogenesis by inhibiting UBE2CP3 expression may be a potential strategy for HCC prevention and treatment.Our results suggest that in HCC,UBE2CP3 indirectly enhances tumor cell-induced angiogenesis.UBE2CP3 is a potential oncogene that participates in HCC tumorigenicity by facilitating angiogenesis.