The Function And Machanism Of LincRNA-UFC1 In Hepatocellular Carcinoma Progression

Background and objectivePrimary liver cancer (PLC) is one of the most common malignant tumors, and among PLC, more than 90% is hepatocellular carcinoma (HCC). Of all the malignant tumors, the clinical morbidity of HCC ranks second in male patients and sixth in female patients. Each year 782,500 patients are diagnosed with HCC, with a mortality rate of 95%. Half of the world’s share of HCC occurs in China, thus making HCC a severe health problem of the country.Rapid proliferation is a hallmark of HCC, which leads to poor prognosis in many patients, as evidenced by a 5-year survival rate. In order to improve patient prognosis it is imperative to elucidate the mechanism of HCC’s rapid proliferation.Long non-coding RNA (lncRNA) is a subgroup of non-coding RNAs greater than 200 nucleotide that is ubiquitously expressed in mammalian cells and function to modulate gene expression at epigenetic, transcriptional and post-transcriptional levels. Based on their structural or functional characteristics, lncRNAs can be classified further into several subgroups, including sense lncRNA, antisense lncRNA, intronic lncRNA, bidirectional lncRNA, and long intergenic noncoding RNAs (lincRNAs). In the whole genome transcript products, the percentage of lncRNA is much more than mRNA. Studying the role of lncRNA in the context of disease processes, will help us to better understand the pathology and develop better therapeutic strategies.In the current work, we used lncRNA microarray to explore the differentially expressed genes between HCC and the adjacent tissues and then confirmed this by qRT-PCR. Then we focused on lincRNA-UFCl, we used in situ hybridization, functional experiments, RIP and RNA pull-down to test the functions of lincRNA-UFC1 in HCC, illustrating the mechanism underlying HCC initiation and progression, finally laying the theoretical basis for HCC new prognostic molecular markers.Methods1. The identification of different expression lncRNAs by lncRNA microarray.We performed lncRNA microarray to identify the lncRNA and mRNA expression profiles in 7 pairs of HCC tissues and matched non-tumor tissues, and we used independent sample t-test to analyze the differences between the HCC tissues and matched non-tumor tissues. We randomly picked several genes from the microarray data and confirmed by qRT-PCR in 10 pairs of tumor tissues and matched non-tumor tissues. Then we expanded the sample size to 49 pairs.2. The expression of lincRNA-UFC1 and the biological significance in HCC.We tested lincRNA-UFC1 expression level in 131 paraffin embedded HCC samples and 72 non-tumor tissues by ISH and scored the staining images, and used chi-square to analyze the lincRNA-UFC1 expression differences between HCC samples and 72 non-tumor tissues. Then spearman rank correlation method was used to analyze the relationship between the lincRNA-UFC1 expression and clinical pathological parameters, and the effects of variables on survival were determined by univariate and multivariate Cox proportional hazards modeling. Finally, we demonstrated that lincRNA-UFC1 can be an independent risk factor for the prognosis of HCC.3. The biological effect of lincRNA-UFC1 in HCC.We used real time-PCR to detect lincRNA-UFC1 expression level in HCC cell lines. We used shRNA to knockdown lincRNA-UFC1 expression in the cell lines that lincRNA-UFC1 high-expression and used plasmid to overexpress lincRNA-UFC1 expression in cell lines that lincRNA-UFC1 low expression. We used MTT, clonogenic etc. in vitro assays and subcutaneous xenograft tumor etc. In vivo assays to detect lincRNA-UFC1 effect on HCC cells proliferation. We used flow cytometry, western blot, immunochemistry method and tunel to make clear whether lincRNA-UFC1 can regulate HCC cell cycle and apoptosis.4. The upstream of lincRNA-UFC1.We used bioinformatics analysis to indentify the miRNAs that can directly bind with lincRNA-UFC1 and dual luciferase gene reporter assay to confirm this. Then the level of lincRNA-UFC1 and this miRNA was tested by qRT-PCR in HCC tissues to analyze the correlation relation between them. We used actinomysin D treatment followed by over-expression or silence miRNA and then test lincRNA-UFC1 level.5. The downstream of lincRNA-UFC1.We used RNA pull-down and RIP assay to indentify the protein that can directly bind with lincRNA-UFC1. Then we observed whether the biological function can be altered after this protein was over-expressed or silenced to make sure lincRNA-UFC1 can function through this protein.Results1. IncRNA and mRNA was disregulated in HCC.To identify transcripts that potentially drive liver tumorigenesis, IncRNA and mRNA expression profiles were determined by microarray analysis. Hierarchical clustering showed systematic variations in transcript expression levels between paired tumor and non-tumor tissues from 7 HCC patients. (1493 lncRNAs was up-regulated and 820 IncRNAs was down regulated in HCC compared with non-tumor tissues, 1384 mRNAs was up-regulated and 1350 mRNAs was down regulated in HCC compared with non-tumor tissues). The microarray data have been deposited in NCBI Gene Expression Omnibus and are accessible through GEO Series Accession Number GSE58043 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE58043). To validate our microarray analysis findings, differentially expressed transcripts (10 lncRNAs and 8 mRNAs) were analyzed by qRT-PCR in 10 pairs of randomly selected, paired tumor and non-tumor liver tissues from 10 HCC patients. qRT-PCR analysis confirmed the microarray findings, revealing that a set of lncRNAs and mRNAs that are dysregulated in HCC tissues.2. lincRNA-UFC1 expression is correlated with HCC progression.We focused mainly on up-regulated lncRNAs because this set of lncRNAs can be more readily utilized than down-regulated lncRNAs as early diagnosis markers or therapeutic targets. lincRNA-UFCl was one of the highly up-regulated lncRNAs. To further validate the increase in lincRNA-UFC1, its expression was further analyzed by qRT-PCR in an additional 49 pairs of HCC and non-tumor tissues. We found that the lincRNA-UFC1 level was significantly higher in HCC tissues than adjacent non-tumor liver tissues (.P=0.006). In situ hybridization (ISH) studies, which involved 131 paraffin-embedded HCC specimens, also confirmed the over-expression of lincRNA-UFC1 in HCC (P<0.001). Collectively, these findings strongly suggested that lincRNA-UFC1 is up-regulated in HCC.To determine whether lincRNA-UFC1 expression levels are related to the HCC progression, we analyzed the association between lincRNA-UFC1 and clinicopathological status in HCC patients. Statistical analysis represented a strong correlation between lincRNA-UFC1 expression and portal vein tumor thrombus (PVTT; P=0.021), tumor size (P=0.013) and Barcelona Clinic Liver Cancer (BCLC) stage (P=0.015). These associations were confirmed by analysis of samples. Furthermore, Kaplan-Meier and log-rank test analyses suggested a positive correlation between the tumoral lincRNA-UFC1 expression and a significantly reduced overall survival (OS) and disease-free survival (DFS) rates (P<0.001 for both OS and DFS). Notably, lincRNA-UFC1 expression significantly correlated with OS in patient subgroups with early BCLC stage (n=69; P=0.002) or no PVTT (n=98; P=0.001), but not in subgroups with advanced BCLC stage (n=62;P=0.072) or PVTT (n=33; P=0.594). In addition, multivariate analysis showed that lincRNA-UFC1 expression (95% CI:1.924-5.75; P<0.001), BCLC stage (95% CI:1.017-2.579; P=0.043), tumor relapse (95% CI:1.07-2.579; P=0.024), alpha fetoprotein (AFP; 95% CI:1.033-2.896; P=0.037) and tumor size (95% CI:1.018-2.723; P=0.037) were independent prognostic factors for OS in HCC patients.3. lincRNA-UFC1 promotes HCC cell proliferation, inhibits cell apoptosis and induces cell cycle progression.To investigate the biological significance of lincRNA-UFC 1 expression in the development and progression of HCC, we next performed gain-and loss-of-function studies in HCC cells. Rapid amplification of cDNA ends (5’-and 3’-RACE) analysis was performed to determine the full-length transcript of lincRNA-UFC1. We also confirmed that lincRNA-UFC1 has no coding capability. After detecting lincRNA-UFC1 expression in 6 HCC cell lines, we developed SK-Hep-1 and BEL-7402 cell lines with stably over-expressed lincRNA-UFC1, and Huh7 and MHCC-97H cell lines with stably silenced lincRNA-UFC1 expression. By performing MTT and colony formation assays in vitro, we found that lincRNA-UFC1 over-expression increased the proliferative capacity of SK-Hep-1 and BEL-7402 cells, compared to that of parallel stable cell lines containing the empty vector. In contrast, knockdown endogenous lincRNA-UFC1 expression dramatically reduced the proliferative capacity of HCC cells. The growth enhancing effect of lincRNA-UFC1 expression was confirmed by performing in vivo tumor growth assays. Xenograft tumors grown from cells over-expressing lincRNA-UFC1 had larger mean volumes and weights, and formed more rapidly, than tumors grown from control cells. Tumors grown from lincRNA-UFC1 knockdown were smaller than controls. These results demonstrated that lincRNA-UFC1 expression can promote HCC cell proliferation both in vitro and in vivo.To gain insights into the mechanism by which lincRNA-UFC1 enhances HCC cell proliferation, we analyzed differences in cell cycle distributions following lincRNA-UFC1 over-expression or silencing by fluorescence-activated cell sorting (FACS). We found that lincRNA-UFC1 over-expression drove progression beyond the G1/S transition in SK-Hep-1 and BEL-7402 cells, whereas significant G1/S arrest was observed in lincRNA-UFC1-knockdown Huh7 and MHCC-97H cells. Consistent with the FACS data, the expression of G1/S phase checkpoint proteins such as CDK4, CDK.6, c-myc, and cyclin D1 was markedly up-regulated in cells with lincRNA-UFC1 over-expression and decreased when lincRNA-UFC1 was silenced (Figure 3C). Moreover, xenografts in nude mice with cells over-expressing lincRNA-UFC1 showed strong staining for CDK4, CDK6, and cyclin D1 relative to vector-transduced xenografts. Conversely, lincRNA-UFC1 knockdown xenografts was linked to a weak staining for G1/S checkpoint markers, indicating that lincRNA-UFC1 is closely associated with the cell cycle distribution in vivo. Collectively, these data strongly demonstrated that lincRNA-UFC1 promotes cell proliferation by facilitating cell cycle progression.Because lincRNA-UFC1 exerts an oncogenic effect in HCC cells, we speculated that lincRNA-UFC1 may be critical for cell survival and apoptosis. To test this hypothesis, we detected apoptosis by FACS analysis in HCC cells stained for Annexin V and 7-AAD. The results showed that lincRNA-UFC 1-knockdown Huh7 and MHCC-97H cells had a significantly higher percentage of Annexin V-positive cells than did cells expressing a scrambled shRNA.lincRNA-UFC1 over-expression did not affect cellular apoptosis; however, protective effects were observed in lincRNA-UFC1-overexpressing SK-Hep-1 and BEL-7402 cells treated with 5-fluorouracil. Consistent with the FACS data, the expression levels of well-defined apoptosis protein markers, including Bax, cleaved PARP, and cleaved caspase 3, markedly increased in Huh7 and MHCC-97H cells with silenced lincRNA-UFC1 expression. Furthermore, lincRNA-UFC1 knockdown induced xenograft tumor apoptosis in vivo, as determined by performing TUNEL assays. Taken together, these data indicate that lincRNA-UFC1 affects HCC cell proliferation and HCC patient survival.4. lincRNA-UFC1 interacts with HuR and enhances β-catenin expression.To investigate the molecular mechanism by which lincRNA-UFC1 is associated with HCC progression, we studied correlations between lincRNA-UFC1 and differentially expressed mRNAs by qRT-PCR. We found that P-catenin was significantly induced in HCC compared to corresponding tumor adjacent non-tumor tissues. In addition, we observed a positive relationship between linRNA-UFC1 and β-catenin mRNA by qRT-PCR in 23 HCC specimens (r=0.495,P=0.016). However, lincRNA-UFC1 expression was not correlated with the mutation status of the β-catenin gene. The expression of β-catenin significantly increased in cells overexpressing lincRNA-UFC1. In contrast, β-catenin levels were down-regulated in cells with silenced lincRNA-UFC1 expression, indicating that β-catenin is a downstream effector of lincRNA-UFC1. In addition, nuclear accumulation of β-catenin was observed in cells with over-expressed lincRNA-UFC1. lincRNA-UFC1 expression also affected the expression levels of downstream effectors of P-catenin, such as c-myc and cyclin D1, which also varied with the expression of lincRNA-UFC 1. However, the other downstream molecules of β-catenin, such as VEGFA, MMP9, survivin, and c-Jun, were not affected by lincRNA-UFC1 expression.However, a direct binding between lincRNA-UFC1 and β-catenin was not found by RIP assays. To investigate how lincRNA-UFC 1 regulates P-catenin expression, we studied lincRNA-UFC1 localization firstly because the activities and functions of lncRNAs depend on their subcellular distribution. Using cytoplasmic and nuclear RNA fractions from HCC cells, we observed that lincRNA-UFC1 is expressed in relative abundance in the cytoplasm. This finding was consistent with our ISH data. Cytoplasmic lncRNAs are best known for modulating gene transcription through interaction with RNA-binding proteins (RBPs). Thus, we tested the hypothesis that functional interactions may occur between lincRNA-UFC1 and RBPs, such as HuR, hnRNPAB, CUGBP2, TTP, and TIA1, which may stabilize p-catenin mRNA to increase its expression. The results showed that HuR directly bound to both lincRNA-UFC1 and β-catenin mRNA in RIP assays. We further performed RNA pull-down assays to confirm the interaction between linRNA-UFC1 and HuR and found that lincRNA-UFC1 is associated with HuR. Notably, deletion analysis indicated that the 5’-end (1102-1613 nt) of lincRNA-UFC1 was essential for this association. Interestingly, it was noted that the HuR protein accumulated in the cytoplasm with over-expression of lincRNA-UFC1 by immunofluorescence staining and western blot, suggesting that lincRNA-UFC1 induces HuR translocation in complex with β-catenin mRNA. Moreover, HuR silencing abrogated the effect of lincRNA-UFC1 in inducing cell proliferation and β-catenin expression in SK-Hep-1 and BEL-7402 cells, indicating that the function of lincRNA-UFC1 depends on HuR. Collectively, these data indicated that lincRNA-UFC1 promotes tumorigenesis by activating β-catenin in a HuR-dependent manner.5. lincRNA-UFC1 is a direct target of miR-34a.Using microinspector software, we found a set of miRNAs that putatively bind to lincRNA-UFC1. Among these miRNA candidates, we found that miR-34a directly binds to lincRNA-UFC1. Dual-luciferase assays showed a significant decrease in luciferase activities following co-transfection of miR-34a and the wild type lincRNA-UFC1 expression vector, but not a mutant lincRNA-UFC1. We further clarified the regulatory relationship between lincRNA-UFC1 and miR-34a. Over-expression of miR-34a significantly inhibited lincRNA-UFC1 expression, whereas over-expression of lincRNA-UFC1 did not affect miR-34a expression, suggesting that lincRNA-UFC1 is targeted by miR-34a. Over-expression of miR-34a also decreased the lincRNA-UFC1 half-life. In addition, miR-34a over-expression significantly suppressed cellular proliferation, expression levels of cell cycle related proteins, and HuR expression in lincRNA-UFC1-overexpressing cells. Moreover, an inverse correlation was found between lincRNA-UFC1 and miR-34a expression levels in 24 pairs of HCC tissues by qRT-PCR (r=-0.541, P=0.006). Collectively, these data demonstrated that miR-34a directly binds to lincRNA-UFC1 and serves as a negative upstream regulator of lincRNA-UFC1-mediated proliferation and cell cycle progression.Conclusions1. lincRNA-UFC1 was up-regulated in HCC, and was correlated to the outcome of HCC, and it can be used as an independent prognostic risk factor of HCC.2. lincRNA-UFC1 promotes HCC proliferation through induction cell cycle progression and inhibition apoptosis.3. lincRNA-UFC1 interacts with HuR and enhances β-catenin expression.4. lincRNA-UFC1 is a direct target of miR-34a.