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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References
  8. Supporting Information

In recent years, long noncoding RNAs (lncRNAs) have been investigated as a new class of regulators of biological function. A recent study reported that lncRNAs control cell proliferation in hepatocellular carcinoma (HCC). However, the role of lncRNAs in liver regeneration and the overall mechanisms remain largely unknown. To address this issue, we carried out a genome-wide lncRNA microarray analysis during liver regeneration in mice after 2/3 partial hepatectomy (PH) at various timepoints. The results revealed differential expression of a subset of lncRNAs, notably a specific differentially expressed lncRNA associated with Wnt/β-catenin signaling during liver regeneration (an lncRNA associated with liver regeneration, termed lncRNA-LALR1). The functions of lncRNA-LALR1 were assessed by silencing and overexpressing this lncRNA in vitro and in vivo. We found that lncRNA-LALR1 enhanced hepatocyte proliferation by promoting progression of the cell cycle in vitro. Furthermore, we showed that lncRNA-LALR1 accelerated mouse hepatocyte proliferation and cell cycle progression during liver regeneration in vivo. Mechanistically, we discovered that lncRNA-LALR1 facilitated cyclin D1 expression through activation of Wnt/β-catenin signaling by way of suppression of Axin1. In addition, lncRNA-LALR1 inhibited the expression of Axin1 mainly by recruiting CTCF to the AXIN1 promoter region. We also identified a human ortholog RNA of lncRNA-LALR1 (lncRNA-hLALR1) and found that it was expressed in human liver tissues. Conclusion: lncRNA-LALR1 promotes cell cycle progression and accelerates hepatocyte proliferation during liver regeneration by activating Wnt/β-catenin signaling. Pharmacological intervention targeting lncRNA-LALR1 may be therapeutically beneficial in liver failure and liver transplantation by inducing liver regeneration. (Hepatology 2013;58:739–751)

Abbreviations
ChIP

chromatin immunoprecipitation

HCC

hepatocellular carcinoma

HGF

hepatocyte growth factor

lncRNAs

long noncoding RNAs

PCNA

proliferating cell nuclear antigen

PH

partial hepatectomy

qRT-PCR

quantitative real-time polymerase chain reaction

RIP

RNA immunoprecipitation

Liver regeneration is a series of physiopathological phenomena resulting in quantitative recovery from the loss of liver mass to compensate for decreased hepatic volume and impaired function. Clinically, liver regeneration has important implications because many therapeutic strategies for the surgical treatment of liver diseases, such as removal of liver tumors and liver transplantation, depend on the ability of the liver to regenerate physically and functionally. Insufficient liver regeneration may be potentially fatal for these patients.[1] Therefore, a better understanding of the mechanisms of liver regeneration could lead to clinical benefits.

A complex network of cytokine and growth factor signaling involving molecules such as interleukin-6 (IL-6)[2] and hepatocyte growth factor (HGF)[3] regulates the hepatocyte cell cycle to ensure that liver regeneration occurs quickly.[4] Recent studies have shown the critical role of microRNAs (miRNAs), such as miR-221[5] and miR-21,[6] in liver regeneration. Although various cytokines, growth factors, and miRNAs have been shown to regulate genes that orchestrate proliferation during liver regeneration, new molecular therapeutic targets for liver failure and liver transplantation are still urgently needed. It is important to understand the overall molecular changes that occur during liver regeneration to enhance the effectiveness of current regenerative technology.

The mammalian genome encodes thousands of noncoding transcripts that have structural, regulatory, or unknown functions.[7] Although studies of small noncoding RNAs have dominated the field of RNA biology in recent years,[8] long noncoding RNAs (lncRNAs)—defined as noncoding RNA molecules greater than 200 nucleotides in length—have been shown to play significant regulatory roles in X chromosomal inactivation,[9] chromatin remodeling,[10] and transcriptional repression.[11] LncRNAs also regulate multiple major biological processes, including development,[12] differentiation,[13] and carcinogenesis.[10] In our previous work, we showed that lncRNA-HEIH facilitates tumor cell growth through enhancer of zeste homolog 2.[14] A recent study has implicated lncRNAs involved in liver regeneration.[15] However, only preliminary studies have been conducted on the role of lncRNAs in liver regeneration, and the overall mechanisms remain largely unknown.

In this study we performed a comprehensive expression profiling analysis of lncRNAs in mouse livers at various timepoints after 2/3 partial hepatectomy (PH). The overall changes in lncRNA expression are described during mouse liver regeneration, leading to the identification of lncRNA-LALR1 as a regulator of liver regeneration. LncRNA-LALR1 promoted hepatocyte proliferation by facilitating cyclin D1 expression through the activation of Wnt/β-catenin signaling. This study may provide a novel mechanism and potential therapeutic target for liver failure and liver transplantation.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References
  8. Supporting Information
Chromatin Immunoprecipitation

Chromatin immunoprecipitation (ChIP) was performed using an EZ ChIP Chromatin Immunoprecipitation Kit (Millipore, Bedford, MA) according to the manufacturer's instructions. Briefly, cross-linked chromatin was sonicated into 200-bp to 1000-bp fragments. The chromatin was immunoprecipitated using anti-CTCF (Cell Signaling Technology, Beverly, MA) and anti-RNA Pol II antibodies. Normal mouse immunoglobulin G (IgG) was used as a negative control. Quantitative polymerase chain reaction (PCR) was conducted using SYBR Green Mix (Takara Bio, Otsu, Japan). The primer sequences are listed in Supporting Table 1.

RNA Immunoprecipitation

We performed RNA immunoprecipitation (RIP) experiments using a Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore) according to the manufacturer's instructions. The CTCF antibodies were used for RIP (Cell Signaling Technology). The coprecipitated RNAs were detected by reverse transcription PCR and quantitative PCR. The primer sequences are listed in Supporting Table 1. Total RNAs (input controls) and isotype controls were assayed simultaneously to demonstrate that the detected signals were the result of RNAs specifically binding to CTCF (n = 3 for each experiment).

For a description of other materials and methods used in this study, see the Supporting Information.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References
  8. Supporting Information
Genome-Wide LncRNA Changes During Liver Regeneration

To determine the overall impact of lncRNAs on liver regeneration, we analyzed the expression profiles of lncRNAs and protein-coding RNAs in mouse livers at 0, 1.5, 12, and 24 hours after 2/3 PH using microarray analysis. Among all of the mouse liver samples, the intensity of one mouse at 24 hours (D11) was an outlier and was discarded from the subsequent bioinformatics analysis. A differential expression profile at each timepoint was obtained by comparing the microarray signal value with that obtained at 0 hour, which showed that ∼1,231 lncRNAs and 3,141 protein-coding RNAs were differentially expressed (Supporting Table 2). Hierarchical clustering showed systematic variations in the expression of differentially expressed lncRNAs and protein-coding RNAs in mouse livers at various timepoints (Fig. 1A,B). To understand the behavior of these differentially expressed lncRNAs, we explored how the patterns of gene expression change over a period of time because biologically related gene groups can share the same patterns of change. In total, 11 significant profiles (Supporting Fig. S1A) were obtained by Series Test of Cluster (STC) analysis and the most significant profile (Profile #17, Fig. S1B) including 117 lncRNAs is shown with the genes in detail (Supporting Table 3). Taking the protein-coding RNAs of this profile as input, the GO analysis results were determined and are listed in Fig. S2. KEGG pathways analysis (Fig. 1C) revealed many enrichment-related pathways, including the Wnt/β-catenin signaling pathway, in this profile. The KEGG pathways analysis of the other 10 significant profiles indicated that lncRNAs may participate in various signaling pathways (Fig. S3). The related gene coexpression networks extracted from the significant pathways of Profile #17 are shown in Fig. 1D, which indicates that 18 lncRNAs and 4 protein-coding genes were identified as relevant (Supporting Table 4). Considering that β-catenin (1st) is a component of the Wnt/β-catenin pathway and that the Wnt/β-catenin pathway was the most significantly different pathway, we selected the Wnt/β-catenin signaling pathway and the 18 lncRNAs for further study.

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Figure 1. LncRNA expression profile changes during liver regeneration. Hierarchical clustering analysis of 1,231 lncRNAs (A) and 3141 protein-coding RNAs (B) that were differentially expressed (P < 0.045 and false discovery rate [FDR] < 0.05) in mouse livers at 0, 1.5, 12, and 24 hours after 2/3 PH; three mice were analyzed for each timepoint. The clustering tree for lncRNAs and protein-coding RNAs is shown at the top. The expression values are represented in shades of red and green, indicating expression above and below the median expression value across all of the samples (log scale 2, from −1.21 to +1.21), respectively. (C) Pathway analysis shows the significant pathways of differentially expressed coding genes from the most significant profile (Profile #17) (P < 0.05). (D) A portion of the coexpression network of the differentially expressed lncRNAs in the most significant profile (Profile #17) and the protein-coding genes representing the significant pathways. The coexpression network included connections among 22 genes. The 18 lncRNAs were selected according to k-core threshold (k-core = 12), and four mRNAs were selected according to k-core and degree thresholds (k-core = 11 and degrees ≥ 28). A green node represents a protein-coding gene and a purple node represents an lncRNA. (E) The expression levels of lncRNA-uc008aun, lncRNA-uc008ofr, and lncRNA-uc007ppd were examined using qRT-PCR in CCL-9.1 cells that were treated with different HGF concentrations. Error bars represent ±SEM. *P < 0.05 and **P < 0.01.

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Next, quantitative real-time polymerase chain reaction (qRT-PCR) was performed to analyze the expression of the 18 lncRNAs (Supporting Table 5) in the mouse liver samples at the various timepoints. The expression of most lncRNAs was consistent with the microarray analysis except in the cases of lncRNA-uc007ukb, lncRNA-uc008fcf, and lncRNA-uc.77+. Due to the important role of hepatocyte growth factor (HGF) in liver regeneration after 2/3 PH,[16] we determined the expression levels of lncRNAs in CCL-9.1 cells (normal mouse liver cell line) that were treated with HGF at different concentrations (Fig. 1E; Fig. S1C). Among the 15 lncRNAs, the expression levels of lncRNA-uc008aun, lncRNA-uc008ofr, and lncRNA-uc007ppd were significantly increased by HGF treatment. Finally, lncRNA-uc008aun was selected for further analysis because it shares high nucleotide homology with the human sequence (Supporting Table 6), and it was designated lncRNA-LALR1.

Taken together, the microarray data also suggested that thousands of lncRNAs undergo dynamic changes during different stages of liver regeneration after 2/3 PH, and they may regulate multiple and diverse signaling pathways to restore the lost liver mass, reflecting their key role in regulating the regenerative process.

Newly Identified LncRNA Involved in Liver Regeneration

The 2/3 PH in C57BL/6 mice caused an increase in lncRNA-LALR1 expression that was detectable at 6 hours, peaked between 18 and 24 hours, and returned to almost normal levels by 72 hours after surgery (Fig. 2A). The timing of the lncRNA-LALR1 surge suggested that it might be involved in liver regeneration. We also analyzed lncRNA-LALR1 expression in purified hepatocytes from the mouse liver samples at various timepoints after depleting the nonparenchymal cells. The trend (Fig. S4E) was similar to that of the mouse liver samples at various timepoints after 2/3 PH (Fig. 2A). Next, in situ hybridization was performed to analyze lncRNA-LALR1 expression in the mouse liver samples at 0 and 18 hours after surgery (Fig. S4F). The transcript of lncRNA-LALR1 was mainly located in the nucleus and cytoplasm of hepatocytes and was up-regulated at 18 hours after surgery. These results suggest that lncRNA-LALR1 is specifically up-regulated in hepatocytes after 2/3 PH. The full-length sequence of lncRNA-LALR1 and the transcription start and end sites are presented in Fig. S4A. Next, we detected lncRNA-LALR1 in the BNL CL.2 cells (mouse embryo liver cell line) and mouse liver samples using northern blot analysis (Fig. 2B). Our results indicated that lncRNA-LALR1 was present and that the length of the lncRNA-LALR1 fragment was similar to that determined by RACE analysis. The transcript for lncRNA-LALR1 was located both in the nucleus and in the cytoplasm of BNL CL.2 cells, and the expression of lncRNA-LALR1 in the nucleus was higher than that in the cytoplasm (Fig. 2C). The qRT-PCR analysis revealed significantly higher lncRNA-LALR1 expression in BNL CL.2 cell than in CCL-9.1 cells (Fig. S4B). To investigate the biological functions of lncRNA-LALR1 in vitro, we constructed CCL-9.1 cells with stable overexpression of lncRNA-LALR1 and BNL CL.2 cells with stable down-regulation of lncRNA-LALR1 (Fig. S4C,D).

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Figure 2. LncRNA-LALR1 participates in liver regeneration. (A) The expression levels of lncRNA-LALR1 were determined by qRT-PCR at different timepoints after 2/3 PH. (B) Northern blot analysis of lncRNA-LALR1 shows the length of the lncRNA-LALR1 fragment. Molecular weight markers are indicated on the left. The major product is marked by an arrow on the right. (C) RNA was extracted from the nuclei and cytoplasm (total) or only nuclei (nuclear) of BNL CL.2 cells. One μg of RNA was used for the qRT-PCR analysis of lncRNA-LALR1, U2 snRNA (nuclear retained), and hypoxanthine phosphoribosyltransferase (HPRT) mRNAs (exported to cytoplasm). The values represent the median of three technical replicates. Error bars represent ±SEM. *P < 0.05.

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LncRNA-LALR1 Enhances Cell Cycle Progression and Hepatocyte Proliferation In Vitro

To investigate the role of lncRNA-LALR1 in hepatocyte proliferation, cell counting kit-8 assays, bromodeoxyuridine (BrdU) enzyme-linked immunosorbent assay (ELISA) assays, EdU immunofluorescence, and BrdU immunocytochemistry staining were performed in the lncRNA-LALR1-down-regulated BNL CL.2 cells and the lncRNA-LALR1-up-regulated CCL-9.1 cells. The cell counting kit-8 assays (Fig. 3A) and BrdU ELISA assays (Fig. 3B) indicated that cell proliferation was reduced by the knockdown of lncRNA-LALR1 in BNL CL.2 cells and enhanced by the overexpression of lncRNA-LALR1 in CCL-9.1 cells. As Fig. 3C,D shows, lncRNA-LALR1-up-regulated CCL-9.1 cells had higher numbers of BrdU and EdU-positive nuclei than the control cells, and the number of EdU-positive nuclei was lower in lncRNA-LALR1-down-regulated BNL CL.2 cells than in the control cells. Thus, these results indicate that lncRNA-LALR1 promotes hepatocyte proliferation.

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Figure 3. LncRNA-LALR1 regulates hepatocyte proliferation and the cell cycle in vitro. LncRNA-LALR1-down-regulated BNL CL.2 cells and lncRNA-LALR1-up-regulated CCL-9.1 cells were seeded into 96-well plates, and cell proliferation was assessed using the CCK-8 assay (A) and a BrdU ELISA assay (B). LncRNA-LALR1-down-regulated BNL CL.2 cells and lncRNA-LALR1-up-regulated CCL-9.1 cells were seeded onto coverslips and cell proliferation was assessed using EdU immunofluorescence staining (C) and BrdU immunocytochemistry staining (D); original magnification ×200. The graph on the right shows the percentage of EdU- and BrdU-positive nuclei. The data shown are the mean of three independent experiments. (E) Western blot analysis demonstrates the expression of cyclin B1 and cyclin E1 in lncRNA-LALR1-down-regulated BNL CL.2 cells at different release timepoints during the cell cycle. (F) An analysis of the cell cycle phase distribution by flow cytometry shows the cell cycle phases of lncRNA-LALR1-up-regulated CCL-9.1 cells and lncRNA-LALR1-down-regulated BNL CL.2 cells. BNL CL.2 cells that were transfected with the transfection agent but no siRNA were called Mock, and cells transfected with scramble-control siRNA were the negative control. CCL-9.1 cells that were transfected with the pcDNA3.1 plasmid without the construct were called pcDNA3.1. Error bars represent ±SEM. *P < 0.05 and **P < 0.01.

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We wondered whether lncRNA-LALR1 accelerates hepatocyte proliferation by promoting cell cycle progression. To gain further insight into the role of lncRNA-LALR1 in cell cycle progression, we analyzed the expression of cyclin E1 and cyclin B1 in lncRNA-LALR1-down-regulated BNL CL.2 cells. We found that the G0/G1 phase was prolonged and that cell cycle progression was delayed in lncRNA-LALR1-down-regulated BNL CL.2 cells (Fig. 3E). Moreover, fluorescence-activated cell sorting (FACS) analysis (Fig. 3F) demonstrated a reduction in the G0/G1 population in lncRNA-LALR1-up-regulated CCL-9.1 cells and an increase in the G0/G1 population in lncRNA-LALR1-down-regulated BNL CL.2 cells. These observations suggest that lncRNA-LALR1 might facilitate the cell cycle progression of hepatocytes. Thus, lncRNA-LALR1 enhanced hepatocyte proliferation by accelerating the cell cycle progression of hepatocytes.

LncRNA-LALR1 Accelerates Mouse Hepatocyte Proliferation and Cell Cycle Progression During Liver Regeneration

To investigate whether lncRNA-LALR1 affected hepatocyte proliferation in vivo, we knocked down lncRNA-LALR1 in the mouse liver by Ambion in Vivo lncRNA-LALR1 small interfering RNA (siRNA)s and overexpressed lncRNA-LALR1 by pcDNA3.1-LALR1 plasmid. The lncRNA-LALR1 siRNAs or pcDNA3.1-LALR1 plasmid was separately injected into mice by way of the tail vein and the timepoints of the injections are shown in Fig. S5A. To confirm the effect of the inhibition or overexpression of lncRNA-LALR1 in the mouse liver, we analyzed lncRNA-LALR1 expression using qRT-PCR, northern blot analysis, and in situ hybridization. The mice injected with lncRNA-LALR1 siRNAs indeed expressed low levels (Fig. S5B-E) of lncRNA-LALR1 (∼0.3-fold), and the mice injected with the pcDNA3.1-LALR1 plasmid expressed high levels (Fig. S6A) of lncRNA-LALR1 (∼25-fold). The liver/body weight ratio in the lncRNA-LALR1-down-regulated mice was significantly lower in comparison to the control mice at 36 and 72 hours after 2/3 PH (Fig. 4A). The continued presence of mitotic figures was observed in lncRNA-LALR1-up-regulated mouse livers at 36 hours after 2/3 PH (Fig. 4B). Next, in vivo hepatocyte proliferation was analyzed by immunohistochemistry for BrdU, Ki67, and proliferating cell nuclear antigens (PCNAs). Initially, we found that lncRNA-LALR1-down-regulated mice exhibited lower numbers of BrdU, Ki67, and PCNA-positive nuclei in hepatocytes compared to controls at 36 hours after 2/3 PH (Fig. 4C), which coincides with the peak of DNA synthesis in mice.[17] We also used immunohistochemistry analysis to detect BrdU, Ki67, and PCNA at 24, 72, 120, and 168 hours after 2/3 PH (Fig. 4C). There was a decrease in proliferation at 72 hours and no significant difference at 24, 120, and 168 hours. We also found that lncRNA-LALR1-up-regulated mice exhibited higher numbers of BrdU, Ki67, and PCNA-positive nuclei in hepatocytes compared to controls at 36 and 72 hours after 2/3 PH, and no significant difference was found at 24, 120, and 168 hours (Fig. S6C).

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Figure 4. LncRNA-LALR1 regulates mouse hepatocyte proliferation and the cell cycle after 2/3 PH. (A) The mice were injected with lncRNA-LALR1 siRNA or control siRNA by way of the tail vein before and after 2/3 PH. The liver mass to body weight ratio was then calculated at each timepoint. (B) Hematoxylin and eosin staining shows prominent mitotic figures (denoted by arrows) in mice injected with pcDNA3.1-lncRNA-LALR1 at 36 hours after 2/3 PH; original magnification ×200. (C) Immunohistochemistry analysis for Ki67, BrdU, and PCNA shows the differences in hepatocyte proliferation between mice injected with lncRNA-LALR1 siRNA and those injected with control siRNA; original magnification ×200. Quantification of Ki67, BrdU, and PCNA positive cells at 24, 36, 72, 120, and 168 hours after 2/3 PH is on the right. The data shown are the mean of three independent experiments. (D) qRT-PCR analysis reveals the differences in the expression levels of cyclin D1, E1, A2, and B1 between mice injected with lncRNA-LALR1 siRNA and those injected with control siRNA at 24, 36, 72, and 120 hours after 2/3 PH. Error bars represent ±SEM. *P < 0.05 and **P < 0.01.

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On the basis of the previous finding that most hepatocytes enter S phase by 36 hours after 2/3 PH in adult male C57BL/6 mice,[18] we speculated that lncRNA-LALR1 might play a role in the regulation of cell cycle events preceding S phase because its expression level peaked between 18 and 24 hours during liver regeneration (Fig. 2A). To gain further insight into the cell cycle of proliferating hepatocytes, we analyzed the expression of various cyclins. We found that at 24, 36, and 72 hours after 2/3 PH, lncRNA-LALR1-down-regulated mice have lower expression of cyclin D1, E1, and A2, which are known to play a role in the G1 to S transition of hepatocytes during regeneration. Additionally, the expression of cyclin B1 was decreased at 72 hours (Fig. 4D). Furthermore, we did not find any difference in the expression levels of cyclin D1, E1, A2, and B1 at 120 hours. In addition, lower protein levels of these cyclins were found in lncRNA-LALR1-down-regulated mice at 24, 36, and 72 hours after 2/3 PH (Fig. S5F). We also found that at 24, 36, and 72 hours after 2/3 PH, the lncRNA-LALR1-up-regulated mice had a higher expression of cyclin D1, E1, and A2. In addition, cyclin B1 expression was increased at 72 hours (Fig. S6D). Furthermore, we did not find any difference in the expression of cyclin D1, E1, A2, and B1 at 120 hours. Thus, all the results suggested that lncRNA-LALR1 enhanced liver regeneration in mice mainly at an early phase after surgery by accelerating cell cycle progression.

To assess the repair of liver injury, we detected serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) at 24, 72, and 168 hours after 2/3 PH. More serious liver damage was documented in the control mice than in the lncRNA-LALR1-up-regulated mice (Fig. S6B).

Increased Expression of LncRNA-LALR1 Is Induced by Hepatocyte Growth Factor

Because lncRNA-LALR1 increased at an early phase after 2/3 PH and HGF played a critical role in liver regeneration following partial hepatectomy,[3, 16] we wondered whether the increase in lncRNA-LALR1 expression was driven by HGF. The expression levels of lncRNA-LALR1 increased with the elevated concentrations of HGF (Fig. 5A). In addition, a correlation was observed between HGF and lncRNA-LALR1 in mouse livers at different timepoints (Fig. 5B). To examine the influence of HGF on lncRNA-LALR1 expression, we cloned the promoter of lncRNA-LALR1 (a region spanning −1,988 bp to +166 bp nucleotide relative to transcription site) into a pGL3 basic firefly luciferase reporter. As Fig. 5C indicates, HGF significantly increased the luciferase activity of this construct. The pGL3 basic firefly luciferase reporter was used as a negative control. These results demonstrated that HGF increased the expression of lncRNA-LALR1 by enhancing the activity of the lncRNA-LALR1 promoter.

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Figure 5. HGF increases the expression of lncRNA-LALR1. (A) The expression levels of lncRNA-LALR1 were determined using qRT-PCR in CCL-9.1 cells that were treated with different HGF concentrations. (B) The correlation of the expression levels of HGF and lncRNA-LALR1 in mouse liver samples at different timepoints after 2/3 PH was confirmed by qRT-PCR. (C) Dual luciferase assay in CCL-9.1 cells transfected with firefly luciferase constructs containing the lncRNA-LALR1 promoter or nothing (negative control) with different concentrations of HGF. The values are presented as the mean ± standard error of the ratio of firefly luciferase activity to renilla luciferase activity and are representative of at least three independent experiments. Error bars represent ±SEM. *P < 0.05 and **P < 0.01.

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LncRNA-LALR1 Activates the Wnt/β-Catenin Pathway in Mouse Hepatocytes by Suppressing Axin1

According to the results of the coexpression network and pathway analysis, lncRNA-LALR1 is associated with the Wnt/β-catenin pathway during liver regeneration. We measured the luciferase activity of TOPFlash/FOPFlash and examined the expression levels of Wnt/β-catenin pathway target genes to evaluate the activation of Wnt/β-catenin pathway in lncRNA-LALR1-down-regulated BNL CL.2 cells and lncRNA-LALR1-up-regulated CCL-9.1 cells. The TOP/FOP ratio was higher in lncRNA-LALR1-up-regulated CCL-9.1 cells and lower in lncRNA-LALR1-down-regulated BNL CL.2 cells when compared to the respective control cells (Fig. 6A). As Fig. S7A shows, the expression levels of cyclin D1, Axin2, and TCF7 were increased with the overexpression of lncRNA-LALR1 and decreased by the knockdown of lncRNA-LALR1.

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Figure 6. LncRNA-LALR1 activates the Wnt/β-catenin pathway in mouse hepatocytes. (A) TOPflash or FOPflash plasmids were transfected into lncRNA-LALR1-up-regulated CCL-9.1 cells and lncRNA-LALR1-down-regulated BNL CL.2 cells. Luciferase activity for TOPflash/FOPflash was determined after 48 hours. These results show data from at least three independent experiments. (B) Western blot analysis shows changes in Wnt/β-catenin pathway components in lncRNA-LALR1-down-regulated BNL CL.2 cells and lncRNA-LALR1-up-regulated CCL-9.1 cells. (C) qRT-PCR analysis reveals the expression levels of β-catenin degradation components in lncRNA-LALR1-up-regulated CCL-9.1 cells and lncRNA-LALR1-down-regulated BNL CL.2 cells. (D) Immunofluorescence staining demonstrates the expression level and nuclear translocation (denoted by arrows) of β-catenin (green) in lncRNA-LALR1-up-regulated CCL-9.1 cells and control cells; original magnification ×400. (E) Western blot analysis shows the protein levels of total, nuclear, and cytoplasm β-catenin in lncRNA-LALR1-up-regulated CCL-9.1 cells and control cells. (F) Western blot analysis shows changes in Wnt/β-catenin pathway components in lncRNA-LALR1-down-regulated mouse livers at 36 hours after 2/3 PH. BNL CL.2 cells that were transfected with the scramble-control siRNA were the negative control. CCL-9.1 cells that were transfected with pcDNA3.1 plasmid without the construct were called pcDNA3.1. Error bars represent ±SEM. *P < 0.05 and **P < 0.01.

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We further investigated the expression levels of Wnt/β-catenin pathway components in lncRNA-LALR1-up-regulated CCL-9.1 cells and lncRNA-LALR1-down-regulated BNL CL.2 cells. First, we analyzed the expression levels of β-catenin degradation components, including Axin1, GSK-3β, and APC. There was a decrease in the protein and messenger RNA (mRNA) levels of Axin1 as a result of the overexpression of lncRNA-LALR1, and knockdown of lncRNA-LALR1 resulted in an increase in Axin1. However, no significant difference was observed in the protein and mRNA levels of GSK-3β and APC (Fig. 6B,C). Our results also showed that overexpression of lncRNA-LALR1 resulted in a decrease in the level of phosphorylated β-catenin protein (inactive) and that the level of nonphosphorylated β-catenin protein (active) increased (Fig. 6B). As Fig. 6D shows, active β-catenin was translocated to the nucleus, and total β-catenin staining increased because of the overexpression of lncRNA-LALR1. Western blot analysis (Fig. 6E) of β-catenin demonstrated that there was no significant difference in the cytoplasm protein level of β-catenin. However, the nuclear protein level of β-catenin significantly increased with the overexpression of lncRNA-LALR1. Next, we analyzed several target genes of the Wnt/β-catenin pathway that are associated with liver regeneration. The expression of c-myc and cyclin D1 increased with the overexpression of lncRNA-LALR1 and declined after knockdown of lncRNA-LALR1 (Fig. 6B). We also investigated the changes in the protein levels of Wnt/β-catenin pathway components in lncRNA-LALR1-down-regulated mouse liver at 36 hours after 2/3 PH. The trend was similar to that in lncRNA-LALR1-down-regulated BNL CL.2 cells (Fig. 6F).

We transfected pcDNA3.1-Axin1 into lncRNA-LALR1-up-regulated CCL-9.1 cells and control cells, and performed a TOP/FOP ratio analysis (Fig. S7B), western blots of Wnt/β-catenin pathway components (Fig. S7C), BrdU ELISA assays (Fig. S7D), EdU immunofluorescence (Fig. S7E), and FACS analysis (Fig. S7F). These analyses indicated that overexpression of Axin1 attenuated the function of lncRNA-LALR1 which activated the Wnt/β-catenin pathway and facilitated hepatocyte proliferation and cell cycle progression.

To address the physiological relevance of the mechanism of Axin1 regulation by lncRNA-LALR1, we analyzed the change in Axin1 mRNA and protein expression during liver regeneration. The 2/3 PH in C57BL/6 mice caused a decrease in Axin1 expression that was detectable at 12 hours, lowest between 24 and 36 hours, and began to return at 48 hours after surgery (Fig. S8A,B). The expression changes in Axin1 suggest that Axin1 might be inhibited by lncRNA-LALR1 during liver regeneration.

Taken together, these data showed that lncRNA-LALR1 activated the Wnt/β-catenin pathway in hepatocytes. LncRNA-LALR1 decreased the expression of Axin1, and the stability of the β-catenin destruction complex receded, which led to the decline in the levels of phosphorylated β-catenin (inactive); active β-catenin could no longer stay bound and was released. This monomeric form of β-catenin binds to proteins such as T-cell factor-4 (TCF-4) and lymphoid enhancement factor (LEF) and translocates to the nucleus to control the transcription of target genes, including c-myc and cyclin D1. Finally, lncRNA-LALR1 facilitated mouse cell cycle progression and hepatocyte proliferation (Fig. 7).

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Figure 7. Schematic representation of lncRNA-LALR1 mediated enhanced proliferation of hepatocytes.

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LncRNA-LALR1 Inhibits the Expression of Axin1 by Recruiting CTCF to the Promoter Region

We wondered whether the mechanism of lncRNA-LALR1 activates the Wnt/β-catenin pathway by suppressing Axin1. We performed a computational screen (http://jaspar.genereg.net; CTCFBSDB2.0[19]) and found a CTCF binding site within the AXIN1 promoter region (−1,892 bp upstream of the transcription start site of AXIN1). Recent studies have reported that the transcription factor CTCF can bind to the promoter region of target genes and inhibit their expression.[20] There was no significant difference in the CTCF mRNA and protein levels in lncRNA-LALR1-up-regulated CCL-9.1 cells compared to those in the control cells (data not shown). To determine whether lncRNA-LALR1 could change the binding of CTCF to the AXIN1 promoter region, we performed ChIP analysis in lncRNA-LALR1-up-regulated CCL-9.1 cells and lncRNA-LALR1-down-regulated BNL CL.2 cells. We observed that overexpression of lncRNA-LALR1 increased the binding of CTCF at the AXIN1 promoter region in CCL-9.1 cells, and the binding declined in lncRNA-LALR1-down-regulated BNL CL.2 cells (Fig. 8A). These results confirmed that lncRNA-LALR1 could increase the binding of CTCF to the AXIN1 promoter region in hepatocytes. In addition, we tested whether lncRNA-LALR1 could associate with CTCF. We performed RIP with an antibody against CTCF from extracts of BNL CL.2 cells and CCL-9.1 cells. We observed significant enrichment of lncRNA-LALR1 with the CTCF antibody compared with the nonspecific IgG control antibody (Fig. 8B). Next, we performed an in vitro RNA pulldown to validate the association between lncRNA-LALR1 and CTCF in BNL CL.2 cells and CCL-9.1 cells. This analysis confirmed that lncRNA-LALR1 physically associated with CTCF in vitro (Fig. 8C). Together, the RIP and RNA pulldown results demonstrate a specific association between CTCF and lncRNA-LALR1. The expression level of Axin1 was not statistically different in lncRNA-LALR1-down-regulated BNL CL.2 cells and lncRNA-LALR1-up-regulated CCL-9.1 cells that were transfected with CTCF siRNA (Fig. 8D), and the same result was found for the Axin1 protein level (Fig. 8E). These results indicate that lncRNA-LALR1 inhibited the expression of Axin1 by way of transcription factor CTCF. Taken together, our results demonstrate that lncRNA-LALR1 can specifically associate with transcription factor CTCF and recruit CTCF to the AXIN1 promoter region to inhibit its expression.

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Figure 8. LncRNA-LALR1 inhibits the expression of Axin1 by way of transcription factor CTCF. (A) ChIP analyses of lncRNA-LALR1-up-regulated CCL-9.1 cells and lncRNA-LALR1-down-regulated BNL CL.2 cells were conducted with the AXIN1 promoter region using the CTCF antibodies. Enrichment was determined relative to the input controls. The data are the mean ±SEM of three independent biological replicates. (B) RIP experiments were performed using the CTCF antibody for immunoprecipitation (IP) and a primer to detect lncRNA-LALR1. RIP enrichment was determined relative to the input controls (n = 3). (C) RNA pulldown assay performed in CCL-9.1 and BNL CL.2 cells. Biotinylated lncRNA-LALR1 or antisense RNA was incubated with cell extracts, targeted with streptavidin beads, and washed, and the associated proteins were resolved on a gel. Western blot analysis detected the specific association of CTCF and lncRNA-LALR1 (n = 3). The expression of Axin1 was examined using qRT-PCR (D) and western blot (E) in lncRNA-LALR1-down-regulated BNL CL.2 cells and lncRNA-LALR1-up-regulated CCL-9.1 cells that were transfected with CTCF siRNA or the scramble-control siRNA (CTCF Negative control). BNL CL.2 cells that were transfected with the scramble-control siRNA were the negative control. CCL-9.1 cells that were transfected with pcDNA3.1 plasmid without the construct were called pcDNA3.1. Error bars represent ±SEM. *P < 0.05 and **P < 0.01.

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Human Ortholog RNA of LALR1 Is Expressed in Human Liver Tissues

Sequence homology analysis revealed that the murine LALR1 lncRNA most likely has a human ortholog RNA, referred to as hLALR1, which is located on human chromosome 16 (Supporting Table 6). We identified the 5′ and 3′ transcription start and termination sites of the hLALR1 transcript by RACE analysis, and the sequences of the full-length hLALR1 are presented in Fig. S9A. Analysis of the sequences using ORF Finder failed to predict a protein of more than 52 amino acids (Fig. S9B). Moreover, it did not contain a valid Kozak sequence, suggesting the unlikelihood of translation. Thus, the hLALR1 transcript is consistent with an lncRNA.

We next measured the expression of lncRNA-hLALR1 in three human liver tissues and QSG 7701 cells by northern blot analysis (Fig. S9C). Our results indicated that lncRNA-hLALR1 could be expressed in human liver tissues and human liver cells, and the length of the lncRNA-hLALR1 fragment was similar to that determined by RACE analysis. Furthermore, qRT-PCR analysis of two human liver cell lines (Fig. S9D) and 20 human liver tissues (Fig. S9E) revealed that the lncRNA-hLALR1 expression level was between the level for the two well-known lncRNA-MVIH and HOTAIR.

Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References
  8. Supporting Information

Although various cytokine,[3] growth factors,[4] and miRNAs[5] have been shown to regulate the genes that orchestrate proliferation in liver regeneration, little information exists on the lncRNAs that regulate liver regeneration. To identify lncRNAs that regulate the regenerative capabilities of hepatocytes, we performed a comprehensive lncRNA expression profiling analysis during different phases of mouse liver regeneration. Genome-wide changes in lncRNA expression were documented during liver regeneration after 2/3 PH, leading to the identification of lncRNA-LALR1, which accelerated hepatocyte proliferation during liver regeneration. LncRNA-H19 was also involved in hepatocyte proliferation in the rat and mouse.[15] These results led us to propose that lncRNAs are critical regulators of hepatocyte proliferation during liver regeneration.

HGF plays an important role in liver regeneration following PH.[3, 16] HGF activates a receptor tyrosine kinase c-Met, which stimulates diverse signaling pathways including Ras, mitogen-activated protein kinase (MAPK),[21] and certain transcription factors, such as STAT3 [22] and c-jun.[23] Our results showed that HGF increased the expression of lncRNA-LALR1, while the exact mechanism was not determined (see Supporting Discussion for further discussion on the mechanism of HGF).

Wnt/β-catenin signaling is an evolutionarily well-conserved pathway that has been shown to be crucial in regulating cell proliferation in liver regeneration.[24] The mechanisms of Wnt/β-catenin signaling activation are likely enriched and diversified, including variations in the Wnt/β-catenin signaling components[25] (see Supporting Discussion for further discussion on the role of lncRNA-LALR1 activates Wnt/β-catenin signaling).

In summary, we have reported an extensive genome-wide expression profile of lncRNAs during different phases of mouse liver regeneration after 2/3 PH. The overall changes in lncRNA expression were described during mouse liver regeneration, leading to the identification of lncRNA-LALR1 as a regulator of liver regeneration. Our results reveal that lncRNA-LALR1 accelerates hepatocyte proliferation during liver regeneration by activating Wnt/β-catenin signaling. We detected thousands of differentially expressed lncRNAs in our microarray analysis after 2/3 PH. Other novel regulators and molecular mechanisms of liver regeneration will require further study.

Acknowledgment

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References
  8. Supporting Information

We thank Jin-feng Huang (Department of Medical Genetics, Second Military Medical University) for experimental assistance, Sheng-xian Yuan (Third Department of Hepatic Surgery, Eastern Hepatobiliary Surgery Hospital) for human liver samples support, and GMINIX Co. for bioinformatics support.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References
  8. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References
  8. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
hep26361-sup-0001-suppfig1.tif3205KFigure S1. Series test of cluster (STC) analysis of differentially expressed lncRNAs and protein-coding RNAs. (A) These lncRNAs and protein-coding RNAs were grouped into 26 sets and classified as down-regulated, unchanged and up-regulated at 0, 1.5, 12 and 24 hours after 2/3 PH. Among them, 11 significantly different profiles of lncRNAs and protein-coding RNAs were identified (P-value<0.05), and the most significantly different profile (Profile # 17) is shown in Figure B. (C) The expression levels of the 12 selected lncRNAs were examined using qRT-PCR in CCL-9.1 cells that were treated with different HGF concentrations. Error bars represent ±SEM.
hep26361-sup-0002-suppfig2.tif5118KFigure S2. Gene Ontology (GO)-based annotation analysis was used to perform functional enrichment analysis using the DAVID tools. Fold enrichment of the differentially expressed coding genes from the most significant profile (Profile # 17) is measured by the bar length. The number of genes refers to the number of differentially expressed genes described by that annotation and is also expressed as a percentage of the total genes described for that term. The P-value represents the significance of the enrichment. Only annotations with a significant P-value of < 0.05 are shown.
hep26361-sup-0003-suppfig3.tif4136KFigure S3. Pathway analysis shows the significant pathways for the differentially expressed coding genes from the 10 significant profiles (Profile # 1, 2, 3, 10, 11, 12, 15, 16, 21 and 24) (P<0.05).
hep26361-sup-0004-suppfig4.tif4681KFigure S4. Information about lncRNA-LALR1. (A) Agarose gel electrophoresis of PCR products from the 5'-RACE and 3'-RACE procedure (left). The sequencing of second-round PCR products reveals the boundary between the universal anchor primer and lncRNA-LALR1 sequences (right). The thymine marked by a red arrow indicates a putative transcriptional start and end site. Nucleotide sequence of the full-length lncRNA-LALR1 gene is given below. (B) The expression levels of lncRNA-LALR1 in different mouse hepatocyte cell lines were detected by qRT-PCR. The values are shown with respect to the expression level of lncRNA-LALR1 in CCL-9.1 cells set to value of 1. (C) The expression levels of lncRNA-LALR1 in CCL-9.1 cell clones that were stably transfected with pcDNA3.1 encoding lncRNA-LALR1 cDNA or a pcDNA3.1 plasmid control were detected by qRT-PCR. (D) The expression levels of lncRNA-LALR1 in BNL CL.2 cells that were stably transfected with a plasmid encoding shRNA against lncRNA-LALR1 or a negative control were quantified by qRT-PCR. (E) LncRNA-LALR1 expression was determined by qRT-PCR in purified hepatocytes from the mouse liver samples at various time points after depleting the non-parenchymal cells. (F) Mouse liver samples at 0 and 18 hours after surgery were subjected to QD-FISH and observed under ultraviolet light excitation using a fluorescence microscope. Original magnifications: 400×. BNL CL.2 cells that were transfected with the scramble-control siRNA served as the Negative control. CCL-9.1 cells that were transfected with pcDNA3.1 plasmid without the construct were called pcDNA3.1. Error bars represent ±SEM. *P<0.05.
hep26361-sup-0005-suppfig5.tif3545KFigure S5. (A) Schematic representation of the experimental design. (B) Northern blot analysis shows the differences in the expression levels of lncRNA-LALR1 in the livers of mice injected with lncRNA-LALR1 siRNA compared to the livers of mice injected with control siRNA at 0 hours after 2/3 PH. qRT-PCR analysis reveals the differences in the expression levels of lncRNA-LALR1 in the livers of mice injected with lncRNA-LALR1 siRNA compared to the livers of mice injected with control siRNA before (C) and after (D) 2/3 PH. (E) QD-FISH of lncRNA-LALR1 reveals the differences in the expression levels of lncRNA-LALR1 in the livers of mice injected with lncRNA-LALR1 siRNA compared to the livers of mice injected with control siRNA at 0 hours after surgery. Original magnification: 400×. (F) Western blot analysis reveals the differences in the expression levels of cyclin D1, E1, A2 and B1 between mice injected with lncRNA-LALR1 siRNA and those injected with control siRNA at 24, 36, 72 and 120 hours after 2/3 PH. Error bars represent ±SEM.
hep26361-sup-0006-suppfig6.tif5593KFigure S6. (A) qRT-PCR reveals the differences in the expression levels of lncRNA-LALR1 in the livers of mice injected with pcDNA3.1-lncRNA-LALR1 compared to the livers of mice injected with the pcDNA3.1 plasmid after 2/3 PH. (B) Blood biochemistry analysis showed liver injury after 2/3 PH in mice injected with the pcDNA3.1-lncRNA-LALR1 or pcDNA3.1 plasmid (AST/ALT, respectively). (C) Immunohistochemistry analysis for BrdU, Ki67 and PCNA shows the differences in hepatocyte proliferation between mice injected with pcDNA3.1-lncRNA-LALR1 and those injected with pcDNA3.1 plasmid; original magnification × 200. Quantification of BrdU, Ki67 and PCNA positive cells at 24, 36, 72, 120 and 168 hours after 2/3 PH is shown on the right. The data shown represent the mean values of three independent experiments. (D) qRT-PCR analysis reveals the differences in the expression levels of cyclin D1, E1, A2 and B1 between mice injected with pcDNA3.1-lncRNA-LALR1 and those injected with pcDNA3.1 plasmid at 24, 36, 72 and 120 hours after 2/3 PH. Error bars represent ±SEM. *P<0.05 and **P<0.01.
hep26361-sup-0007-suppfig7.tif2598KFigure S7. (A) qRT-PCR reveals the expression levels of the Wnt/β-Catenin pathway target genes, such as cyclin D1, Axin2 and TCF7, in lncRNA-LALR1-up-regulated CCL-9.1 cells and lncRNA-LALR1-down-regulated BNL CL.2 cells. (B) TOPflash/FOPflash luciferase activity assay of lncRNA-LALR1-up-regulated CCL-9.1 cells and control cells, which were cotransfected with the pcDNA3.1-Axin1 plasmid and luciferase constructs containing the TOPflash and FOPflash plasmids. (C) Western blot analysis shows changes in several representative Wnt/β-Catenin pathway components in lncRNA-LALR1-up-regulated CCL-9.1 cells and control cells, which were transfected with the pcDNA3.1-Axin1 plasmid. (D) LncRNA-LALR1-up-regulated CCL-9.1 cells and control cells, which were transfected with the pcDNA3.1-Axin1 plasmid, were seeded into 96-well plates, and cell proliferation was assessed using the BrdU ELISA assay. (E) LncRNA-LALR1-up-regulated CCL-9.1 cells and control cells that were transfected with the pcDNA3.1-Axin1 plasmid were seeded onto cover slips and incubated with EdU regent for 60 min. Then, cell proliferation was assessed using EdU immunofluorescence staining; original magnification × 200. (F) Representative histograms of FACS analysis shows the cell cycle phases of lncRNA-LALR1-up-regulated CCL-9.1 cells and control cells, which were transfected with the pcDNA3.1-Axin1 plasmid. BNL CL.2 cells that were transfected with the scramble-control siRNA were the Negative control. CCL-9.1 cells that were transfected with pcDNA3.1 plasmid without the construct were called pcDNA3.1. Error bars represent ±SEM.*P<0.05 and **P<0.01.
hep26361-sup-0008-suppfig8.tif2969KFigure S8. The expression levels of Axin1 were determined by qRT-PCR (A) and western blot analysis (B) at different time points after 2/3 PH. Error bars represent ±SEM. *P<0.05.
hep26361-sup-0009-suppfig9.tif2610KFigure S9. (A) Nucleotide sequence of the full-length cDNA of lncRNA-hLALR1. (B) Putative proteins possibly encoded by lncRNA-hLALR1 as predicted by the ORF Finder. The predicted proteins (gray) were subjected to a Blastp search and were consistent with noncoding. (C) Northern blot analysis of lncRNA-hLALR1 shows the expression and length of lncRNA-hLALR1 in three human liver tissues and QSG 7701 cells. Molecular weight markers are indicated on the left. The major product is marked by an arrow on the right. (D) The expression of lncRNA-hLALR1 is comparable to that of lncRNA-MVIH, HOTAIR and H19 in QSG 7701 cells and LO2 cells. The ΔCT values were used to measure gene expression, which was normalized to 18S rRNA expression. Error bars represent ±SEM. (E) qRT-PCR analysis reveals that lncRNA-hLALR1 expression is comparable to that of lncRNA-MVIH, HOTAIR and H19 in 20 human liver tissues. ΔCT values were used to measure gene expression, which was normalized to 18S rRNA expression. The horizontal lines in the box plots represent the median, the boxes represent the interquartile range, and the whiskers represent the 5th and 95th percentiles.
hep26361-sup-0010-supptab1.doc40KSupporting Table 1. Oligonucleotide Sequences
hep26361-sup-0011-supptab2.doc8921KSupporting Table 2. Differentially Expressed lncRNAs and mRNAs
hep26361-sup-0013-supptab3.doc1316KSupporting Table 3.Profile # 17 lncRNAs and mRNAs
hep26361-sup-0014-supptab4.doc89K

Supporting Table 4. Co-expression network

Supporting Table 5. Expression array data and qRT-PCR validation of 18 lncRNAs

Supporting Table 6. Sequence homology analysis

Supporting Table 7. Clinical Characteristics of the Patients who provided normal liver tissues.

hep26361-sup-0015-suppinfo1.doc43KSupporting Information
hep26361-sup-0016-suppinfo.doc65KSupporting Information

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