| AimsHepatocellular carcinoma(HCC)is the most common form of liver caner.It has attracted lots of attention for its high morbidity and high mortality because of the lack of early diagnosis and effective treatment.HCC is a result of multiple deregulations of gene networks and the molecular mechanism remains to be elucidated.Long non-coding RNAs(lncRNAs)are defined as transcribed RNA molecules greater than 200 nucleotides in length,which show no protein-coding capacity.LncRNAs once have been deemed to be simply transcriptional "noise" or cloning artifacts for a long time.However,recent mounting evidence has linked deregulations of lncRNAs to many types of diseases,including HCC.Thus,it is of great value to find HCC-related lncRNAs in order to further understand the pathogenic mechanisms of HCC and to identify novel early biomarkers and therapeutic targets for HCC patients.MethodsIn this study,differences in the expression of lncRNAs between 3 HCC and paired non-cancerous tissue were identified using microarrays.The expression level of a specific differentially expressed lncRNA UBE2CP3 was determined by quantitative real-time polymerase chain reaction(qRT-PCR)in 46 HCC and paired non-cancerous tissue.Additional,we determined the expression level of lncRNA UBE2CP3 in 85 HCC and paired non-cancerous tissue by in situ hybridization(ISH)experiments.The associations between lncRNA UBE2CP3 expression and clinicopathologic characteristics were analyzed.We constructed stably over-expressing and silenced lncRNA UBE2CP3 cell lines to explore the effect of lncRNA UBE2CP3 on tumor development in vitro and in vivo.Wound healing assay and trans-well migration assay were used to determine the ability of cell migration,and trans-well invasion assay was used to determine the ability of cell invasion.In order to determine the effect of lncRNA UBE2CP3 on metastasis in vivo,we constructed a metastasis model in vivo by injecting cells into the spleen of nude mice and intrahepatic metastasis was assessed.qRT-PCR and western blot were applied to detect the expression of epithelial-mesenchymal transition(EMT)-related genes in liver cancer cells and tissue.Serum concentration of lncRNA UBE2CP3 was confirmed by qRT-PCR in HCC patients.We also compared the concentration of lncRNA UBE3CP3 in HCC patients pre and post surgical resection.ResultsThe differentially expressed lncRNA profiles between 3 paired HCC tumor tissue and adjacent tissue were analyzed by microarrays.As compared with adjacent tissue,722 lncRNAs exhibited more than 1.5-fold change(P<0.05);513 were up-regulated,and 209 were down-regulated.Among all the differentially expressed lncRNAs,we noticed a novel lncRNA UBE2CP3 with 11.15 fold elevation in HCC tumor tissue.The microarray data have been deposited in NCBI Gene Expression Omnibus and are accessible through GEO Series Accession Number GSE89186(http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE89186).The levels of lncRNA UBE2CP3 were assessed in 46 HCC tumor tissue and adjacent tissue by qRT-PCR.The results showed that the levels of lncRNA UBE2CP3 were higher in HCC tumor tissue than adjacent tissue.The ISH studies,which involved 85 paraffin-embedded HCC specimens,also confirmed the expression of lncRNA UBE2CP3 in HCC and suggested that lncRNA UBE2CP3 was localized to the cytoplasm of HCC tissue.Statistical analysis revealed that high lncRNA UBE2CP3 expression levels were significantly associated with Edmondson grade,smoking and alcohol abuse.However,no significant correlation between lncRNA UBE2CP3 expression levels and other clinicopathological features,including gender,age,liver cirrhosis,tumor number and tumor size were observed.Moreover,Kaplan-Meier and log-rank test analysis suggested a positive correlation between the lncRNA UBE2CP3 expression and a significantly reduced overall survival(OS)in cohort 2.Additionally,univariate analysis of prognostic variables revealed that lncRNA UBE2CP3 expression(P=0.001),Edmondson grade(P=0.015),and tumor size(P=0.012)were significantly related to overall survival in patients with HCC.Multivariate analysis was performed using the Cox Proportional hazards model.The results showed that lncRNA UBE2CP3 expression(P=0.006)was an independent prognostic factor for HCC patients.These data strongly suggest that lncRNA UBE2CP3 may contribute to HCC malignant progression and may serve as a predictor for HCC prognosis.We constructed successfully stably over-expressing and silenced lncRNA UBE2CP3 cell lines to perform the effect of lncRNA UBE2CP3 on tumor development in vitro and in vivo.Wound healing assay and trans-well migration assay showed that lncRNA UBE2CP3 could enhance the ability of cell migration.Meanwhile,trans-well invasion assay showed that lncRNA UBE2CP3 could enhance the ability of cell invasion.Besides,the metastasis model results showed that lncRNA UBE2CP3 could promote tumor metastasis in vivo.EMT has been proved to play an important role in cell migration and invasion.To further investigate the molecular mechanisms of lncRNA UBE2CP3 in cell migration and invasion,the levels of several EMT-related regulators were detected by qRT-PCR and western blot analysis.Our results showed that lncRNA UBE2CP3 could up-regulate the expression of Snail1 and N-cadherin while decrease E-cadherin in HCC cells.Consistent with our findings at the protein level,N-cadherin and Snail1 were markedly elevated in HCC tissue while E-cadherin was down-regulated compared to the noncancerous liver tissue.The expression levels of E-cadherin were markedly decreased,while N-cadherin were highly detected by analysis of the liver tissue from the metastasis model in the lncRNA UBE2CP3 over-expressing group.Oppositely,E-cadherin was up-regulated while N-cadherin was reduced in the silenced lncRNA UBE2CP3 group compared to the sh-controls.In a pilot experiment,blood samples obtained from healthy volunteers(n=75),HCC patients(n=80)were tested by qRT-PCR for the presence of lncRNA UBE2CP3 in serum.The lncRNA UBE2CP3 concentration was markedly increased in HCC patients compared with healthy volunteers.The value of the area under the ROC curve(AUC)was 0,839.Moreover,the combination of lncRNA UBE2CP3 and AFP yielded an AUC of 0.933 which was improved as compared to lncRNA UBE2CP3 alone.Additionally,we compared the concentration of lncRNA UBE2CP3 in 40 paired HCC patients pre and post surgical resection and found that the concentration of lncRNA UBE2CP3 was significantly lower in postoperative patients.ConclusionOur results indicate that lncRNA UBE2CP3 is frequently up-regulated in HCC.LncRNA UBE2CP3 can promote cell migration and invasion in vitro and in vivo.Mechanistically,lncRNA UBE2CP3 was found to enhance the expression of Snail1,and thus repress the expression of E-cadherin and increase N-cadherin.It may promote tumor metastasis by inducing the process of EMT.Moreover,serum expression of lncRNA UBE2CP3 was detected highly expressed in HCC patients compared with the healthy controls;however,its levels decreased following surgery.Therefore,serum lncRNA UBE2CP3 may be a new biomarker for HCC,which may open new avenues for early monitoring of HCC. |
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