Long Non-coding RNAs are Differentially Expressed in Hepatocellular Carcinoma Cell Lines with Differing Metastatic Potential

BACKGROUND
Metastasis is a major reason for poor prognosis in patients with cancer, including hepatocellular carcinoma (HCC). A salient feature is the ability of cancer cells to colonize different organs. Long non-coding RNAs (lncRNAs) play important roles in numerous cellular processes, including metastasis.


MATERIALS AND METHODS
In this study, the lncRNA expression profiles of two HCC cell lines, one with high potential for metastasis to the lung (HCCLM3) and the other to lymph nodes (HCCLYM-H2) were assessed using the Arraystar Human LncRNA Array v2.0, which contains 33,045 lncRNAs and 30,215 mRNAs. Coding-non-coding gene co-expression (CNC) networks were constructed and gene set enrichment analysis (GSEA) was performed to identify lncRNAs with potential functions in organ-specific metastasis. Levels of two representative lncRNAs and one representative mRNA, RP5-1014O16.1, lincRNA-TSPAN8 and TSPAN8, were further detected in HCC cell lines with differing metastasis potential by qRT-PCR.


RESULTS
Using microarray data, we identified 1,482 lncRNAs and 1,629 mRNAs that were differentially expressed (≥1.5 fold-change) between the two HCC cell lines. The most upregulated lncRNAs in H2 were RP11-672F9.1, RP5-1014O16.1, and RP11-501G6.1, while the most downregulated ones were lincRNA-TSPAN8, lincRNA-CALCA, C14orf132, NCRNA00173, and CR613944. The most upregulated mRNAs in H2 were C15orf48, PSG2, and PSG8, while the most downregulated ones were CALCB, CD81, CD24, TSPAN8, and SOST. Among them, lincRNA-TSPAN8 and TSPAN8 were found highly expressed in high lung metastatic potential HCC cells, while lowly expressed in no or low lung metastatic potential HCC cells. RP5- 1014O16.1 was highly expressed in high lymphatic metastatic potential HCC cell lines, while lowly expressed in no lymphatic metastatic potential HCC cell lines.


CONCLUSIONS
We provide the first detailed description of lncRNA expression profiles related to organ-specific metastasis in HCC. We demonstrated that a large number of lncRNAs may play important roles in driving HCC cells to metastasize to different sites; these lncRNAs may provide novel molecular biomarkers and offer a new basis for combating metastasis in HCC cases.


Introduction
Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related deaths worldwide (Jemal et al., 2011;Llovet et al., 2012).Prognosis for the HCC patient is dismal due to poor therapeutic response and a high probability of relapse after treatment (Hong et al., 2003).Metastasis is not only a sign of deterioration but also the main obstacle to ameliorating HCC's poor prognosis (Genda et al., 1999;Poon and Wong, 2000;Ye et al., 2003).Therefore, exploring the molecular mechanisms under lying metastasis is important for developing effective intervention methods and improving patient prognosis.
Metastasis is a complex process involving a series of sequential and interrelated steps, often termed the

RESEARCH ARTICLE
Long Non-coding RNAs are Differentially Expressed in Hepatocellular Carcinoma Cell Lines with Differing Metastatic Potential Ting-Ting Fang, Xiao-Jing Sun, Jie Chen, Yan Zhao, Rui-Xia Sun, Ning Ren*, Bin-Bin Liu* invasion-metastasis cascade (Talmadge and Fidler, 2010).A salient feature of metastasis is the ability of cancer cells to colonize specific organ sites (Hanahan and Weinberg, 2011).For instance, HCC cells metastasize to the lung more frequently than the lymph nodes.It is well recognized that the probability of metastatic seeding and growth is determined by both intrinsic genetic properties of the cancer cells involved and characteristics of the stromal microenvironment that surrounds them (Lu and Kang, 2007;Nguyen et al., 2009).Functional genomic analysis of HCC cell lines with different organ-specific metastatic potential may provide new opportunities for identifying diagnostic biomarkers and developing therapies to combat HCC metastasis.
Large-scale genomic studies have demonstrated that the mammalian genome encodes many thousands of non-coding transcripts, both short (<200 nucleotides (nt) in length; sRNAs) and long (>200 nt; lncRNAs) (Kapranov et al., 2002;Bertone et al., 2004;Guttman et al., 2009;Cabili et al., 2011).miRNAs (microRNAs), the best-studied class of sRNAs, regulate their mRNA targets post-transcriptionally (Bartel, 2009).LncRNA transcripts were first revealed after large-scale sequencing of cDNA libraries in mouse (Okazaki et al., 2002).While only a small fraction of lncRNAs have been characterized in detail, it is clear that mRNA-like lncRNAs may act through a range of molecular mechanisms to function in epigenetic pathways and infrastructure developmental processes.LncRNAs have been reported to interact with chromatin modifications (Guttman et al., 2011;Guttman and Rinn, 2012), serve as precursors for the generation of sRNAs (Fejes-Toth et al., 2009;Guttman and Rinn, 2012), and play regulatory roles in the expression and activity of genes and the localization of the proteins they encode (Wilusz et al., 2009).In addition, lncRNAs function in numerous cellular processes, ranging from embryonic stem cell pluripotency to cell cycle regulation to disease, including cancer (Ponting et al., 2009;Wapinski and Chang, 2011).Recently, hundreds of lncRNAs have been discovered, as those "dark matters of the genome" are selectively over-or underexpressed in different tumors, but the cellular mechanisms of lncRNAs in tumors are still poorly understood (Qiu et al., 2013).Deregulation of lncRNAs is associated with the occurrence of various tumors and has potential significance for cancer diagnosis.Some lncRNAs are already considered biomarkers for specific cancers and outcomes.Examples include lncRNA DD3 for prostate cancer (Tinzl and Horvath, 2004;Wang et al., 2014), long intergenic non-coding RNA (lincRNA) HOTAIR for primary breast cancer (Gupta et al., 2010;Zhang et al., 2014), and serum lncRNA HULC for HCC (Panzitt et al., 2007;Xie et al., 2013).The expression profile of lncRNAs in HCC and their biological function in metastasis remain poorly understood.Better understanding of the roles that lncRNAs play will advance our understanding of cell regulatory and disease mechanisms.
In the present study, we compared the expression profiles of HCC cell lines with a similar genetic background but different potential for lung or lymph node metastasis.We found that expression signatures comprising lncRNAs and protein-coding mRNAs were significantly associated with organ-specific HCC metastasis.Our results indicate that lncRNA expression profiles may represent new molecular biomarkers for HCC metastasis.

HCC Cell lines and animals
Nine HCC cell lines (Hep3B, HepG2, SMMC-7721, MHCC-97L, MHCC-97H, HCCLM3, HCCLM6, HCCLYM-H, HCCLYM-H2) used in this study.MHCC-97L, MHCC-97H, HCCLM3, HCCLM6 were all established by the authors' institute.Hep3B, HepG2, SMMC-7721 were obtained from the Cell Bank of Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences.The HCCLM3 (LM3) and HCCLM6 cell lines were derived from the same parental cell line MHCC-97H.LM3 metastasizes to the lung, while HCCLM6 can metastasize to multiple organs in a mouse model (Li et al., 2001;Li et al., 2003).Hep3B was cultured in MEM (GibocloBRL, USA) supplemented with 10% fetal bovine serum (GibocloBRL, USA ), and other cells were cultured in high glucose DMEM (GibocloBRL, USA) supplemented with 10% fetal bovine serum (GibocloBRL, USA).By subcloning HCCLM6, we established another cell line, HCCLYM-H (unpublished data), which frequently metastasizes to the lymph nodes.We used limited dilution method (Dexter et al., 1978) and in vivo consecutive selection (Sun et al., 1996) to optimize the HCCLYM-H cell line.HCCLYM-H cells were seeded into the footpads of nude mice (BALB/C-nu/ nu, male, 4 weeks old, 18-20 g) to assess tumor growth.Athymic nude mice were obtained from Shanghai Institute of Material Medicine and maintained in a pathogen-free environment.Animal care and experimental protocols were performed in accordance with the guidelines established by the Shanghai Medical Experimental Animal Care Commission.Ethical approval was obtained from the Zhongshan Hospital Research Ethics Committee.In addition, liver orthotopic transplantation was performed in nude mice, and 5 weeks later, metastasis to the lungs and lymph nodes were detected by pathological examination.In this way, we established a cell line named HCCLYM-H2 (H2), which showed stable and high metastatic potential specific to the lymph nodes.

RNA extraction and RNA quality control
Total RNA was extracted from HCC cell lines LM3 and H2 using Trizol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol.RNA clean up including a DNase I digestion step performed with RNeasy spin columns (Qiagen, Germany).RNA quantity and quality were assessed using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, USA).RNA integrity was measured by the relative abundance of 28S/18S ribosomal subunits, verified through denaturing agarose gel electrophoresis.

Microarray experiment and data analysis
Gene expression measurements were performed using the Arraystar Human LncRNA Microarray v2.0 (8660 K, Arraystar, Rockville, MD) which is designed for the global profiling of human LncRNAs and proteincoding transcripts.33, 045 LncRNAs and 30, 215 coding transcripts can be detected by the second-generation LncRNA microarray.The LncRNAs are carefully collected from the most authoritative databases such as RefSeq, UCSC Knowngenes, Ensembl and many related literatures.We measured three samples for each cell line, for a total of six samples.Sample labeling and array hybridization were performed according to the Agilent One-Color Microarray-Based Gene Expression Analysis protocol (Agilent Technologies, Santa Clara, CA).The hybridized arrays were washed and fixed, then processed slides were scanned using the DNA Microarray Scanner (Agilent Technologies).Agilent Feature Extraction software (version 11.0.1.1)was used to analyze array DOI:http://dx.doi.org/10.7314/APJCP.2014.15.23.10513Long non-coding RNAs are Differentially Expressed in Hepatocellular Carcinoma Cell Lines with Different Metastatic Potential images.Quantile normalization and subsequent data processing were performed using the GeneSpring GX v11.5.1 software package (Agilent Technologies, Santa Clara, CA).LncRNAs and mRNAs in which at least one out of the six samples had flags in Present or Marginal for the "All Targets Value" was chosen for further data analysis.We identified lncRNAs and mRNAs that were differentially expressed between the two groups using volcano plotfiltering.P-values were calculated using the paired t-test.The threshold set for up and downregulated genes was a fold change≥1.5 and a P-value≤0.05.The microarray data discussed in this article have been deposited in National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) and are accessible through (GEO) Series accession number GSE61015 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE61015).Hierarchical clustering was performed using Agilent GeneSpring GX software (version 11.5.1).

Construction of the co-expression network
Coding non-coding gene co-expression (CNC) networks were constructed according to the normalized signal intensity of differentially expressed genes.We determined the network adjacency between two genes, X and Y, which was defined as the power of the Pearson correlation between the corresponding gene expression profiles, Kx and Ky.In this way, we obtained the gene adjacency matrix, M (X, Y) (Prieto et al., 2008).The adjacency matrix, M (X, Y), was visualized as a graph, and the topological properties of this graph were examined.To make a visual representation, only the strongest correlations (≥0.99) were drawn.In co-expression networks, each gene corresponds to a node and two genes are connected by an edge.Within the network, a degree is defined as the number of directly linked neighbors; this is the most important measure of the centrality of a gene within a network and determines its relative importance (Barabasi, 2004).We chose 132 lncRNAs in H2 and 173 in LM3 which with the highest level of co-expression to construct the H2 and the LM3 co-expression network respectively.And ten lncRNAs (CR613944, KLHL23, TPRXL, AX747582, AX746887, NCRNA00173, BC058547, RP11-672F9.1, SMEK3P, AX747284) which have the highest level of co-expression were chose to construct the subnetwork.CR613944, KLHL23, TPRXL, AX747582, AX746887 and NCRNA00173 are upregulated in LM3 compared with H2.BC058547, RP11-672F9.1, SMEK3P and AX747284 are up-regulated in H2 compared with LM3.

Gene set enrichment analyses
Functional associations were computed using Gene Set Enrichment Analysis (GSEA), performed with Gene Spring GX software (version 11.5.1).Correlations between the expression levels of each lncRNA locus and selected protein-coding loci were calculated, similar to the method described by Guttman et al (Guttman et al., 2009).For each lncRNA locus, a list of protein-coding loci ranked based on correlation was constructed and subjected to GSEA (Mootha et al., 2003;Subramanian et al., 2005).
Gene sets were filtered using a false-discovery rate (FDR) threshold of 0.05.An association matrix between lncRNA loci and gene ontology (GO) terms was constructed, using a FDR threshold of 0.01.Rows (lncRNA loci) and columns (GO terms) were clustered (k-means, 10 clusters), resulting in distinct subsets of lncRNAs associated with functional GO terms.To determine the enrichment level of positively associated GO terms for each cluster with respect to other clusters, positively correlated GO terms were ranked according to a binomial test.

' G A A G C A A G C C T A T G A
, s e n s e : 5'CATCTCTCATTGACTTATCTGGTAGC3', antisense: 5'ACGTCCCCCTAAG GTTTGGT3'; mRNA-CALCB, sense: 5'TCTGTTGTTTTTCATAGGCTTGCT3', a n t i s e n s e : 5

' A C T TA G AT T T G A A A A C A G C T C C T A G G A 3 ' ; G A P D H
, s e n s e : 5'TGACTTCAACAGCGACACCCA3', antisense: 5'CACCCTGTTGCTGTAGCCAAA3'. The level of each transcript was normalized by the level of GAPDH and represented as fold change using the 2-ΔCt method.

Establishment of lymph node-specific metastasis in the HCCLYM-H cell line
We established the HCCLYM-H2 cell line, which has high lymphatic metastatic potential (100%) and low lung metastatic potential (20%).Figure 1A shows the size of subcutaneous/orthotopic implantation tumors.Figure 1B shows lymph nodes metastases.

LncRNA and mRNA expression profiles in both cell lines
Hierarchical clustering was used to identify lncRNAs and protein-coding mRNAs that were differentially expressed between H2 and LM3 (Figure 3D and E).The expression profiles were shown by calculating log fold change H2/LM3.A total of 1, 629 mRNAs and 1, 483 lncRNAs exhibited significant differential expression (P≤0.05,≥1.5-fold change) between the two cell lines; these included antisense lncRNAs, bidirectional lncRNAs, transcripts with overlap to RefSeq exons, and transcripts mapping to intronic and intergenic regions.More differentially expressed lncRNAs were detected in LM3 than H2.Three hundred and six lncRNAs were upregulated in H2, and 1, 177 lncRNAs were downregulated (Table 1).The numbers of expressed intronic and intergenic lncRNAs in H2 (42 and 151, respectively) were comparable to those in LM3 (209 and 545, respectively).RP11-672F9.1 was the lncRNA that showed the greatest increase in expression in H2 (log2 fold change = 9.543524).Long intergenic noncoding RNAs (lincRNAs) TSPAN8 (log2 fold change = 65.7) and CALCA (log2 fold change = 57.2) showed the greatest decrease in expression of all lncRNAs (Figure 2).Interestingly, the fold changes in expression of TSPAN8   and CALCB mRNAs (48.6 and 74.1, respectively) were as high as those for the lincRNAs, according to the microarray data.
Of 1, 629 differentially expressed mRNAs, 717 mRNAs were upregulated in H2 while 912 were downregulated (Table 2).GO analysis showed that differentially expressed mRNAs were related to cell adhesion, cell migration, apoptosis, and regulation of transcription.Pathway analysis revealed that the functions of the differentially expressed mRNAs included primary substance metabolism, tumorigenesis, inflammatory factors associated with signaling pathways.

LncRNA classification and subgroup analysis
Our contained subgroups of lncRNAs, such as Rinn lincRNAs (Guttman et al., 2009;Khalil et al., 2009), enhancer-like lncRNAs, and HOX lncRNAs.Microarray results suggested that 2, 163 lincRNA transcripts could be detected in both cell lines, and 43 of them were differentially expressed.Among the differentially expressed lincRNA transcripts, 15 were upregulated and 28 were downregulated in H2 (Table 3).The coding genes near the differentially expressed lincRNAs are also shown in Table 3. Differentially expressed enhancer-like lncRNAs and nearby coding genes (distance < 300 kb) are shown in Table 4.These 12 differentially expressed enhancer-like lncRNAs included 1 lncRNA upregulated in H2 and 11 downregulated and regulated nearby coding mRNAs in cis.Moreover, 407 HOX lncRNAs from four HOX loci were detected.

Gene set enrichment analysis
To define the functions of the differentially expressed lncRNAs and their co-expressed mRNAs, we performed GSEA.When analyzing both lncRNA and mRNA expression levels, the six samples (three from H2 and three from LM3) could be grouped into two broad classes (Figure 3D and E).These co-expression patterns corresponded to the two HCC cell lines.These results suggest that the cell lines'different lncRNA expression profiles may be related to their distinct patterns of organspecific metastasis.

Expression of lincRNA-TSPAN8 and TSPAN8 were evaluated in HCC cells with different lung metastatic potiential by qRT-PCR
Since lincRNA-TSPAN8 was found the most upregulated lncRNA in LM3 (Table 1), to evaluate the biological functions of lincRNA-TSPAN8, we first examined the expression of the gene in a variety of cell lines with different lung metastatic potential, including Hep3B, HepG2, SMMC-7721, MHCC-97L, MHCC-97H and LM3 by qRT-PCR.As shown in Figure 4C, comparing with no or low lung metastatic potiential cell lines (Hep3B, HepG2, SMMC-7721 and MHCC-97L), lincRNA-TSPAN8 was found highly expressed in high lung metastatic potiential cell lines (MHCC-97H and LM3).And the trend of the corresponding mRNA TSPAN8 expression in these HCC cells was the same as lincRNA-TSPAN8 (Figure 4C).

Expression of RP5-1014O16.1 was evaluated in HCC cells with different lymphatic metastatic potential by qRT-PCR
RP5-1014O16.1 was the second most upregulated lncRNA in H2 (Table 1).It is a new-found lncRNA, which is located on chrX: 149758021-149758295.To obtain the expression information about RP5-1014O16.1 in HCC, we detected its level in several different lymphatic metastatic potential HCC cell lines.We found that comparing with no lymphatic metastatic potential HCC cell lines (Hep3B, HepG2, SMMC-7721), RP5-1014O16.1 was highly expressed in high lymphatic metastatic potential HCC cell lines (HCCLM6, HCCLYM-H, H2) (Figure 4D).

Discussion
Recent studies have shown that many thousands of lncRNAs are encoded in the human genome.They serve as transcriptional and post-transcriptional regulators and as guides for chromatin-modifying complexes, and many affect various cellular and developmental pathways (Mercer et al., 2009;Wilusz et al., 2009;Taft et al., 2010).It is not surprising that the dysregulation of lncRNAs appears to be a significant feature of many complex human diseases, especially cancer (Ren et al., 2013).For example, the expression of lncRNA BC200 is upregulated invarious human tumors, such as breast, lung, parotid gland, ovary, cervix, and tongue cancers, but it is undetectable incorresponding normal tissue (Chen et al., 1997;Iacoangeli et al., 2004).Similarly, H19, which is located within a cluster of imprinted genes on human chromosome 11 in p15.5, is expressed in many types of cancers, such as gastric, breast, liver, and esophageal, at significantly higher levels than in corresponding normal tissue (Hibi et al., 1996;Berteaux et al., 2008;He et al., 2014;Zhang et al., 2014).Other lncRNAs are expressed in normal tissues and act as tumor suppressor genes.One example, MEG3, is expressed in normal tissues of the adrenal gland, pancreas, ovary, brain tissue, and pituitary; it is rarely expressed in pituitary tumors or human cancer cell lines.Moreover, ectopic expression of this gene inhibits the growth of human cancer cell lines, including HeLa, MCF-7 and H4, indicating that MEG3 may represent a novel tumor suppressor (Zhang et al., 2003).The loss of GAS5 expression have been found in many types of tumors including melanoma, prostate cancers and breast but the concrete mechanism still needs further research (Smedley et al., 2000;Nupponen and Carpten, 2001;Mourtada-Maarabouni et al., 2009).In addition, the expression of GAS5 in Renal Cell Carcinoma specimens was obviously lower than that in adjacent normal tissues, and GAS5 can arrest cell cycling, induce cell apoptosis, also suppress cell migration and invasion (Qiao et al., 2013).These results suggest that GAS5 may have potential value to become a tumor marker for several tumors. .Previous studies showed that TUG1 over-express in bladder cancer and is connected to some characteristics of tumor cells, such as proliferation , apoptosis and so on (Khalil et al., 2009).Additionally, TUG1 can inhibit cancer cell proliferation and promote apoptosis in osteosarcoma (Zhang et al., 2013).TUG1 may act as a new diagnostic marker and therapeutic target of these tumors.
It is believed that lncRNAs play an important regulatory role in cancer progression.Some research suggests that lncRNAs are associated with cancer metastasis and prognosis.According to Gupta et al. (2010), an lncRNA called HOTAIR can be overexpressed nearly two-thousand-fold in breast cancer metastases.High HOTAIR expression levels are significantly associated with breast tumor metastasis and a low survival rate.HOTAIR regulates metastatic progression by recruiting the PRC2 complex to specific, genome-wide target genes; this, in turn, leads to H3K27 methylation and epigenetic silencing of metastasis suppressor genes such as JAM2 and PCDH1 (Simon and Kingston, 2009).It has been known for some time that another lncRNA, MLATA1, is associated with lung cancer metastasis and poor prognosis (Ji et al., 2003).Recent studies show that MLATA1 is also overexpressed in breast cancer, prostate tumors, rectal carcinoma, HCC, and cervical cancer (Lin et al., 2007;Guo et al., 2010).In addition, a number of reports have demonstrated that MLATA1 overexpression is linked to cancer metastasis (Yamada et al., 2006).Recent studies have also revealed that lncRNAs exhibit different patterns of expression in different types of tumors.Some lncRNAs are very sensitive and specific markers of tumors; one example includes DD3 (also known as PCA3) in prostate tumors.Because its expression appears to be restricted to the prostate and it is highly overexpressed in prostate cancer cells, DD3 is a promising marker for the early diagnosis of prostate cancer (Bussemakers et al., 1999;de Kok et al., 2002).Similarly, OCC1 encodes two noncoding regulatory RNAs and its expression is restricted to human colon carcinoma cells (Pibouin et al., 2002).Finally, the HOST genes are rarely expressed in normal tissues or non-ovarian cancers, but they are frequently expressed in ovarian cancer-derived cell lines and primary tumors.Therefore, the HOST genes have been proposed to be specific biomarkers and their study may lead to novel strategies for ovarian cancer diagnosis and therapy (Rangel et al., 2003).Multiple studies have addressed the ectopic expression of lncRNAs in HCC.For example, the highly upregulated in liver cancer (HULC) gene encodes an mRNA-like non-coding RNA (ncRNA) that is highly upregulated in HCC tissue (Panzitt et al., 2007;Matouk et al., 2009).HULC may downregulate miR-372 and induce phosphorylation of the cAMP responsive element binding protein1 (CREB1) in liver cancer (Wang et al., 2010).In addition, Yang et, al's studies (Yang et al., 2011) indicate that the lncRNA HEIH promotes tumor progression.The expression level of lncRNA-HEIH in hepatitis Bvirus (HBV)-related HCC is associated with recurrence and is an independent prognostic factor for survival.Measuring lncRNA-HEIH levels may help predict HCC patient prognosis.According to another study, levels of the lncRNA uc.338 are elevated in HCC cells and may be a promising marker for this type of cancer.Aberrant expression of its transcript, TUC338, is found in transformed hepatocytes, and its functional role in modulating growth may make it a desirable therapeutic target for selected HCC cases (Braconi et al., 2011).Collectively, these studies lead us to propose that lncRNAs may serve as key regulatory hubs in HCC progression.Until now, however, there have been few reports on the role of lncRNAs in the organ-specific metastasis of HCC.
In this study, we established the HCC cell line H2, which has high potential to metastasize to the lymph nodes.It and cell line LM3, which has high potential to metastasize to the lung, are similar in terms of genetic background, but very different in terms of metastatic potential.By comparing their expression profiles and validating a significant portion of differentially expressed lncRNAs using qRT-PCR, we were able to identify a small number of lncRNAs related to organ-specific metastasis in HCC.There have been several reports of aberrant lncRNA expression in various types of human cancers, including HCC (Zhu et al., 2012).However, to the best of our knowledge, this is the first time that lncRNA expression profiles have been studied with regard to organ-specific metastasis in HCC.
Based on microarray data, we detected thousands of expressed lncRNAs in both cell lines, thousands of which were differentially expressed.We found that 306 lncRNAs were upregulated and 1177 downregulated in the H2 cell line, and the function of a large number of these RNAs is unknown.They may be involved in the occurrence, progression, and organ-specific metastasis of HCC.Hence, our work contributes new potential biomarkers for HCC metastasis.It also highlights the importance of investigating the biological relevance of lncRNAs to fully understand the molecular basis of organ-specific HCC metastasis.Furthermore, this study may help identify more effective therapeutic targets and facilitate the development of new personalized therapeutic strategies.
We constructed CNC networks to identify the mRNAs associated with 10 lncRNAs then performed GSEA to define their functions.The ten lncRNAs have the highest co-expression.The results revealed that four intergenic lncRNAs, TPRXL, AX747582, AX746887, and SMEK3P, were all co-expressed with TFF1 mRNA, which is associated with gastrointestinal tumors (Uchino et al., 2000).The gene for lncRNA RP11-672F9.1, upregulated in H2, was co-expressed with 10 mRNAs.One of these mRNAs, H2AFY2, is located near lncRNA RP11-672F9.1, which is reported to predict lung cancer recurrence (Sporn et al., 2009).The gene for lncRNA CR613944, downregulated in H2, was co-expressed with 22 mRNAs, 12 of which were related to cell adhesion, cell migration, and various types of cancer.Among the co-expressed genes, RASAL1 was also located on chromosome 12, relatively close to CR613944.This gene has been reported to contribute to colon tumor progression and gastric tumorigenesis (Ohta et al., 2009;Seto et al., 2011).Two lincRNAs, CR613944 and AX747582, were downregulated in H2 and co-expressed with WISP2 mRNA, which is translated into an important regulator involved in tumor cell invasion and metastasis (Fritah et al., 2008).Whether the progression and organ-specific metastasis of HCC are regulated by these lncRNAs warrants further study.
It has been shown that the transcription of lncRNAs can affect the expression of nearby coding genes (Da Sacco et al., 2012).LncRNAs can recruit chromatinmodifying enzymes to regulate the expression of genes, either in cis (near the site of lncRNA production) or in trans (when the genes involved are distant) through a phenomenon called transvection (Da Sacco et al., 2012).In our study, the lncRNAs that displayed the greatest decrease in expression in H2 were the lincRNAs TSPAN8 and CALCA; the nearby genes encoding TSPAN8 and CALCB mRNA were also differentially expressed in the H2 and LM3 cell lines and the fold change in their expression was equally high (48.58408 and 74.100555, respectively).These results indicate that the TSPAN8 and CALCA lncRNAs may increase the expression of nearby coding transcripts in cis.The possibility that these lncRNAs contribute to the different metastatic potential of the cell lines via cis regulatory functions requires further investigation.
The subgroup of lncRNAs contains Rinn lincRNAs, enhancer-like lncRNAs, and HOX lncRNAs.The subgroup analysis of lncRNAs consists of Rinn lincRNAs profiling, enhancer-like lncRNAs profiling, and HOX lncRNAs profiling, lincRNAs nearby coding gene data and enhancer LncRNAs nearby coding gene data.It may also help clarify the relationship between lncRNAs and the organ-specific metastasis of HCC.A large number of HOX lncRNAs, clustered around four chromosomal loci, termed HOXA through HOXD, are essential for specifying the positional identities of cells (Rinn et al., 2007).This subgroup of lncRNAs is expressed in a temporal and spatial pattern and could play a significant role in HOX regulation (Kmita and Duboule, 2003;Lemons and McGinnis, 2006).The HOTAIR lincRNA is one of the best-studied HOX lincRNAs, initially discovered as a repressor of the HOXD genes that is expressed from the HOXC lincRNA locus.HOTAIR lincRNA is a potential biomarker for metastasis and high probability of death in breast cancer patients (Gupta et al., 2010).Recently, another set of lncRNAs were identified as gene expression enhancers in multiple human cell lines, such as fibroblasts, keratinocytes, and HeLa cells.It was reported that the depletion of some of these lncRNAs led to decreased expression of neighboring protein-coding genes, such as the master regulator of hematopoiesis, SCL, Snai1, and Snai2 (Orom et al., 2010) (Orom et al., 2010) (Orom et al., 2010) (Orom et al., 2010).
Six lncRNAs and two mRNAs were selected for further validation by qRT-PCR, and the results were consistent with the microarray data.To obtain more information about these genes expression in HCC, we investigated their levels in some different metastatic potentials HCC cell lines.We found that the expression level of RP5-1014O16.1 in high lymphatic metastatic potentials HCC cell lines is evidently higher than that in no lymphatic metastatic potentials HCC cell lines.The data indicate that RP5-1014O16.1 is possible promote the lymphatic metastasis of HCC.RP5-1014O16.1 is a new-found lncRNA, which is located on chrX:149758021-149758295.The mechanisms of RP5-1014O16.1 promoting tumor metastasis need further research.In addition, the expression levels of lincRNA-TSPAN8 and the corresponding mRNA TSPAN8 in high lung metastatic potentials HCC cell lines were found to be evidently higher than that in low or no lung metastatic potentials HCC cell lines.Protein Tspan8 is encoded by TSPAN8, also known as TM4SF3 (transmembrane 4 superfamily 3), CO-029, D6.1A (in rats), a member of tetraspanin family, has been reported as a cancer associated gene in many types of tumors, like tumors of the gastrointestinal tractgastric, colorectal, pancreatic and liver tumors (Kanetaka et al., 2001;Zoller, 2006).Tspan8 overexpression has been reported to be correlated with intrahepatic spread of HCC (Kanetaka et al., 2003).The tumor growth-promoting and metastasis-promoting activity of the Tspan8 is possible due to its capacity to induce angiogenesis and cancer cell motility (Gesierich et al., 2006;Yue et al., 2013).Taken together, these results show that lincRNA-TSPAN8 DOI:http://dx.doi.org/10.7314/APJCP.2014.15.23.10513Long non-coding RNAs are Differentially Expressed in Hepatocellular Carcinoma Cell Lines with Different Metastatic Potential and TSPAN8 might play an important role in the lung metastasis of HCC.We speculate that lincRNA-TSPAN8 promotes the lymphatic metastasis of HCC possible via regulating TSPAN8 expression.The concrete mechanisms need further study.
In summary, the present work compares the expression profiles of two HCC cell lines with different metastatic potential.The results may contribute to more efficient diagnosis, aid in the identification of therapeutic targets, and facilitate the development of new, personalized therapeutic strategies.Future studies may reveal whether the progression and organ-specific metastasis of HCC is regulated by these lncRNAs or by other targets.

Figure 1 .
Figure 1.Evaluation of the HCCLYM-H2 cell line's Ability to Initiate Tumor Growth and Metastasize to the Lymph Node in a Mouse Model.A) Tumor size in nude mice with subcutaneous (a & b) and orthotopic implantations(c) of HCCLYM-H2 cells.B) HCC metastases in regional lymph nodes: (a) subclavian, (b) axillary, (c) inguinal, (d) popliteal, (e) common iliac, and (f) para-aortic

Table 1 . LncRNAs Differentially Expressed in H2 vs LM3 Cell Lines
doi.org/10.7314/APJCP.2014.15.23.10513Long non-coding RNAs are Differentially Expressed in Hepatocellular Carcinoma Cell Lines with Different Metastatic Potential *LncRNAs are selected from the 306 upregulated in H2 according to greatest fold change and 1177 downregulated in H2 according to greatest fold change."up" means that the gene is upregulated in H2 compared with LM3, and "down" means that the gene is downregulated in H2 compared with LM3

Table 2 . mRNAs Differentially Expressed in H2 vs LM3 Cell Lines
DOI:http://dx.doi.org/10.7314/APJCP.2014.15.23.10513Long non-coding RNAs are Differentially Expressed in Hepatocellular Carcinoma Cell Lines with Different Metastatic Potential *mRNAs are selected from the 717 upregulated in H2 according to greatest fold change and the 912 downregulated in H2 according to greatest fold change."up" means that the gene is upregulated in H2 compared with LM3, and "down" means that the gene is downregulated in H2 compared with LM3

Table 3 . LincRNAs Differentially Expressed in H2 vs LM3 Cell Lines
*"down" means that the gene is downregulated in H2 compared with LM3, and "up" means that the gene is upregulated in H2 compared with LM3

Table 4 . Differentially Expressed Enhancer-Like lncRNAs and their Nearby mRNAs
:http://dx.doi.org/10.7314/APJCP.2014.15.23.10513Long non-coding RNAs are Differentially Expressed in Hepatocellular Carcinoma Cell Lines with Different Metastatic Potential *"down" means that the gene is downregulated in H2 compared with LM3, and "up" means that the gene is upregulated in H2 compared with LM3 DOI