Long non-coding RNA LINC00152 regulates cell proliferation and migration by epigenetically repressing LRIG1 expression in cholangiocarcinoma

Background: Recently, long non-coding RNAs (lncRNAs) have been veried to have signicant regulatory roles in multiple human cancer processes. Long non-coding RNA LINC00152, located on chromosome 2p11.2, was identied as an oncogenic lncRNA in various cancers. However, the biological function and molecular mechanism of LINC00152 in cholangiocarcinoma (CCA) are still unknown. Methods: Bioinformatic analysis was performed to determine LINC00152 expression levels in the CCA and normal tissues by using raw microarray data downloaded from Gene Expression Omnibus (GSE76297) and The Cancer Genome Atlas (TCGA). Quantitative reverse transcription PCR (qRT-PCR) was used to validate LINC00152 expression in the CCA tissues compared with that in the paired normal tissues. CCK8, colony formation, Edu assays, transwell assays, ow cytometry, and in vivo tumor formation assays were performed to investigate the biological function of LINC00152 on CCA cell phenotypes. RNA-seq was carried out to identify the downstream target gene which was further examined by qRT-PCR, western bolt and rescue experiments. RNA immunoprecipitation (RIP) and Chromatin immunoprecipitation (ChIP) assays were performed to reveal the factors involved in the mechanism of LINC00152 functions in CCA. Results: LINC00152 is signicantly upregulated in cholangiocarcinoma. LINC00152 regulated the proliferation and migration of cholangiocarcinoma cells both in vitro and in vivo. RNA-seq revealed that LINC00152 knockdown preferentially affected genes linked with cell proliferation, cell differentiation and cell adhesion. Furthermore, mechanistic investigation validated that LINC00152 could bind EZH2 and modulate the histone methylation of promoter of leucine rich repeats and immunoglobulin like domains 1 (LRIG1), thereby affecting cholangiocarcinoma cells growth and migration. Conclusion: Taken together, these results demonstrated the signicant roles of LINC00152 in homeobox Expression levels of by analysis of TCGA and GSE76297 data. (C) The decreased expression of LRIG1 was detected in 17 pairs of CCA tissues by qRT-PCR. The ΔCt value was determined by subtracting the GAPDH Ct value from the LRIG1 Ct value. A smaller ΔCt value indicates higher expression. (D-F) CCK8 assays (D), colony formation (E) and transwell assays (F) were implemented to analyze cells transfected with vector/pcDNA-LINC00152/pcDNA-LRIG1or co-transfected with pcDNA- LINC00152 and pcDNA-LRIG1. The results showed that overexpression of LINC00152 could promote cell proliferation and migration, and upregulated LRIG1 could reverse LINC00152-mediated growth and migration promotion.

and normal tissues by using raw microarray data downloaded from Gene Expression Omnibus (GSE76297) and The Cancer Genome Atlas (TCGA). Quantitative reverse transcription PCR (qRT-PCR) was used to validate LINC00152 expression in the CCA tissues compared with that in the paired normal tissues. CCK8, colony formation, Edu assays, transwell assays, ow cytometry, and in vivo tumor formation assays were performed to investigate the biological function of LINC00152 on CCA cell phenotypes. RNA-seq was carried out to identify the downstream target gene which was further examined by qRT-PCR, western bolt and rescue experiments. RNA immunoprecipitation (RIP) and Chromatin immunoprecipitation (ChIP) assays were performed to reveal the factors involved in the mechanism of LINC00152 functions in CCA. Background Cholangiocarcinoma (CCA) is one of the most invasive and lethal cancers derived from ductal epithelial cells lining the intrahepatic and extrahepatic biliary ducts [1,2]. CCA is a devastating malignancy with an abysmal 5-year overall survival rate of less than 10% [3]. In recent years, molecular targeted therapy with powerful pertinence shows wide application prospects, which may control the malignant progression [4]. Therefore, it is of paramount importance to understand the carcinogenesis and progression mechanisms underlying CCA, which make for nding novel diagnosis markers and therapeutic targets.

Results
Over the past decade, with the development of human genome sequencing and the Encyclopedia of DNA Elements (ENCODE) project, ENCODE annotation have revealed that over 90% human genome is transcribed, while less than 2% could be subsequently translated, which reveals that most genome generates many thousands of non-coding RNAs (ncRNAs) transcriptions [5][6][7]. Long non-coding RNAs (lncRNAs), members of ncRNA, are classically characterized as regulatory RNA members longer than 200 nt without detectable open reading frame (ORF) to encode protein [8,9]. Recently, more and more studies have revealed that lncRNAs participate in various biological processes, including cellular proliferation [10], migration [11] and differentiation [12], etc. Notably, the dysregulation of lncRNAs have been linked with a broad-spectrum of human cancers, including CCA [13,14].
As modulators in epigenetic process, lncRNAs could regulated gene expression in chromatin modi cation, transcriptional, as well as post-transcriptional processing [15,16]. For example, the H3K4me3 and H3K27Ac-activated lncRNA HOXCAS3 could lead transcriptional activation of the downstream target genes through binding to transcriptional activator YBX1 in gastric cancer [17]. Moreover, lncRNA-ATB have the capacity to upregulate zinc-nger E-box-binding homeobox 1 (ZEB1) and ZEB2 by competitively binding the miR-200 family in hepatocellular carcinoma [18].
In this study, we found that LINC00152 was signi cantly upregulated in CCA tissues compared with normal tissues in CCA. In addition, the contributions of LINC00152 to CCA development were investigated in vitro and in vivo. RNA-seq analysis showed that downregulated LINC00152 preferentially affected the expression of proliferation and migration-related genes. More importantly, LINC00152 could directly bind to EZH2 to suppress the leucine rich repeats and immunoglobulin like domains 1 (LRIG1) expression, thereby promoting CCA tumorigenesis.

Materials And Methods
Tissue gathering and Ethics statement A total of 17 cholangiocarcinoma patients analyzed in this study had undergone surgeries at the Second A liated Hospital of Nanjing Medical University (China). All tissue samples were instantly snap-frozen in liquid nitrogen until extraction of RNA. This study was approved by the Research Ethics Committee of Nanjing Medical University (Nanjing, Jiangsu, People's Republic of China). Written informed consent was obtained from all participants.

RNA extraction and qRT-PCR analysis
All RNA was isolated from specimens or cultured cells with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) in accordance with manufacturer's instructions. Then, RNA (1 µg) was reverse transcribed to cDNA through a Reverse Transcription Kit (Takara, Dalian, China). For Real-time PCR analyses, we used SYBR colony formation test, the transfected cells were placed in 6-well plates with suitable media containing 10% FBS. After 2 weeks, colonies were xed with methanol and stained with 0.1% crystal violet (Sigma).
The numbers of visibly stained colonies were counted to determine the colony formation. Edu assays were performed using the Edu Cell Proliferation Assay Kit (Ribobio, Guangzhou, China) following the manufacturer's instructions. Then, the percentages of Edu-positive cells were examined in the sample.
Experiments were independently repeated three times in triplicate.

Cell migration assays
For migration assays, 3 × 10 4 transfected cells in media with 1% FBS were placed into the upper chamber of the insert (Millipore, Billerica, MA, USA), while the medium in lower chamber contained 10% FBS. After 24 h of incubation, cotton wools were used to remove the cells remaining on the upper chamber. The cells migrated through the membrane were xed with methanol, stained with 0.1% crystal violet, and imaged and counted with an IX71 inverted microscope (Olympus, Tokyo, Japan). All wells were assessed three times.
Western blot assay and antibodies Cells protein lysates were divided using 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) transferred to 0.22 µm NC membranes (Sigma) and incubated with speci c antibodies. The ECL chromogenic substrates were quantifed by densitometry (Quantity One software; Bio-Rad). An anti-GAPDH antibody was employed as a control. AntiEZH2 was from Millipore (Billerica, MA, USA) and Anti-LRIG1 was purchased from GeneTex (Irvine, USA). Chromatin immunoprecipitation assays Chromatin immunoprecipitation (ChIP) assays were conducted using EZ-CHIP KIT following the manufacturer's instructions (Millipore, USA). EZH2 and H3 trimethyl Lys 27 (H3K27me3) antibodies were from Millipore and Abcam, respectively. The ChIP primer sequences were shown in Supplementary Table S1. Quanti cation of precipitated chromatin DNA was analyzed by qPCR. ChIP data were calculated as a proportion relative to the input DNA using the following equation: 2 [Input Ct− Target Ct] × 0.1 × 100.
RNA immunoprecipitation assays RNA immunoprecipitation (RIP) assays were conducted using a Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore, Billerica, MA, USA) in accordance with the manufacturer's protocols. The EZH2 antibody used in RIP was obtained from Millipore (Billerica, MA, USA).
Whole transcriptome deep sequencing Total RNA from LINC00152 knockdown and control HuCCT1 cells were separated and quanti ed. The concentration of each sample was measured with NanoDrop 2000 (Thermo Scienti c, USA). The quality was evaluated with the Agilent2200 (Agilent, USA). The sequencing library of each RNA specimen was prepared using Ion Proton Total RNA-Seq Kit v2 (Life technologies, USA). Data of six samples are available in Supplementary Table S2 and Table S3.

Statistical analysis
Statistical analyses were performed with GraphPad Prism5 (GraphPad Software, La Jolla, USA). Student's t-test, #2 test or Wilcoxon test were used to estimate the signi cance of the distinctions between groups. The dys-regulated genes from GEO and TCGA datasets were obtained by limma R package and edger R package, respectively. All resulting data were recounted as mean ± SD. Two-sided Pvalues were calculated, and less than 0.05 was considered for statistical signi cance.

Result
LINC00152 is upregulated in the human CCA tissues To identify the dysregulated lncRNAs involved in CCA, we investigated the CCA and normal tissue RNA-Seq data of the public gene pro ling datasets from Gene Expression Omnibus (GEO) database (GSE76297; 91 pairs of cancer tissue specimens and normal tissue specimens, as well as a single normal tissue specimen without pairing; we discarded the single normal tissue specimen data and analyzed the 91 paired data synthetically) [33] and The Cancer Genome Atlas (TCGA) (9 normal and 36 cancer specimens) (Fig. 1A, B and C). LncRNA LINC00152 which is consistently highly upregulated in these datasets caught our attention. Accordant with these results, remarkable increasing expression levels of LINC00152 from normal tissues to carcinoma tissues were con rmed in a cohort of the 17 paired CCA and non-tumor tissues (Fig. 1D).

Knockdown of LINC00152 inhibits CCA cell proliferation and migration
The siRNA-mediated knockdown and plasmid-mediated overexpression were used to manipulate the expression of LINC00152 exogenously, both in HuCCT1 and RBE cell lines, to evaluate the biological functions of LINC00152 in CCA (Supplementary Figure S1A and S1B).
The CCK-8 assay revealed that LINC00152 silencing signi cantly decreased cell viability in HuCCT1 and RBE cell lines, while the overexpression of LINC00152 could promote the cell viability by contrast ( Fig. 2A). Similarly, the colony formation capacity of the CCA cells was also greatly impaired with LINC00152 knockdown and enhanced after overexpression of LINC00152 (Fig. 2B). Furthermore, EdU staining assays demonstrated that LINC00152 had a signi cant impact on CCA cell proliferation as well (Fig. 2C). Then, using a transwell assay, we found that HuCCT1 and RBE cells migration were observably impaired or promoted after the knockdown or overexpression of LINC00152, respectively (Fig. 2D). Taken together, these results indicated that LINC00152 could accelerate the proliferation and migration phenotype of CCA cells.

LINC00152 knockdown induces cell cycle arrest and cell apoptosis
To determine whether LINC00152 is involved in regulating CCA cell apoptosis, ow cytometry was performed. Compared with the control group, LINC00152 silencing evidently improved the proportion of apoptotic CCA cells (Fig. 3A). We further examined if the change of cell cycle progression accounted for the in uence of LINC00152 silencing on cell viability. LINC00152 downregulation made an increased cell cycle arrest at the G1/G0 phase (Figs. 3B). These results suggested that LINC00152 downregulation could inhibit the cell proliferation and migration by in uencing the apoptosis and cell cycle progression of CCA cells.

LINC00152 downregulation suppresses CCA cell proliferation in vivo
HuCCT1 cells stably expressing sh-LINC00152 or control vector were subcutaneously inoculated into nude mice to further con rm whether the LINC00152 in uences tumorigenesis of CCA in vivo. After 17 days, the tumors established in the LINC00152 silencing group were substantially smaller than those in the control group (Figs. 4A-C). Correspondingly, at the nal experiment, the mean tumor weight in the sh-LINC00152 group was markedly lighter than that in the control vector group (Fig. 4D). These ndings indicated that LINC00152 silencing could inhibit CCA growth in vivo.
Downstream Targets of LINC00152 in CCA To probe the LINC00152-associated potential target genes in CCA cells, we used an RNA transcriptome sequencing in control or siRNAs against LINC00152. A total of 261 genes were signi cantly upregulated or downregulated in HuCCT1 cells as a consequence of LINC00152 knockdown (fold change ≥ 1.5) (Fig. 5A, Supplementary Table S2 and S3). The thorough assessment of gene ontology analysis indicated that the most distinct overrepresented biological processes included pathways related to cell proliferation, cell adhesion and cell apoptosis (Fig. 5B). To prioritize most LINC00152-associated genes, more attention was paid for the overexpressed genes after silencing LINC00152. Many renowned proliferation and migration-associated genes (e.g. ACTG2, TNFAIP3, LRIG1, ADAM19, NFKB2, CMTM3, NFKBIE, et al) were included. Their regulation was veri ed by qRT-PCR after LINC00152 silencing in HuCCT1 and RBE cells (Fig. 5C). Noticeably, among these transcripts, LRIG1 was signi cantly elevated after LINC00152 knockdown, which is locating at chromosomes 3p14 and could act as a tumor-suppressive gene in various cancers, including bladder, lung, renal, squamous cell, breast, brain, glioma, and colorectal cancers (Figs. 5C) [34][35][36][37][38][39][40][41]. Western blotting assay further demonstrated that LINC00152 knockdown signi cantly increased the LRIG1 protein levels in CCA, con rming that LRIG1 was a bona target of LINC00152 (Figs. 5D).

LINC00152 epigenetically silences LRIG1 transcription through binding to EZH2
A growing number of studies have reported that a large number of lncRNAs have the cooperative function with chromatin modi ers, suggesting an important role for lncRNAs in the epigenetic regulation of gene expression [42]. Remarkably, polycomb repressive complex 2 (PRC2), the methyltransferase constituted with EZH2, SUZ12 and embryonic ectoderm development (EED), has been reported as the catalyst in the trimethylation of lysine residue 27 of histone 3 (H3K27me3) and mediates transcriptional silencing [43,44]. Previous study showed that approximately 20% of human lncRNAs are bound by PRC2 to exert their regulatory functions [45,46]. Notably, LINC00152 has been reported to bind to EZH2 which is the core component of PRC2 to regulate target gene expression epigenetically in hepatocellular carcinoma, renal cell carcinoma, lung adenocarcinoma and gastric cancer [20, 26, [47][48][49]. But little is known whether LINC00152 could interact with EZH2 in CCA. The promoter region hypermethylation has been reported to resulted in LRIG1 transcriptional inactivation in colorectal and cervical cancer [50,51]. Furthermore, previous studies have reported that DNA methylation and histone methylation could function synergistically in various cancers [52][53][54][55]. Thus, we hypothesized that LINC00152 might modulate LRIG1 expression by binding to EZH2 and then mediating H3K27me3 in the promoter regions of LRIG1 to facilitate CCA progress. Indeed, su cient enrichment of endogenous LINC00152 was validated in the anti-EZH2 RNA immunoprecipitation (RIP) fraction associated with the input in comparison with the nonspeci c IgG control fraction (Fig. 6A), which proved the bond of EZH2 and LINC00152. Additionally, the qRT-PCR and western blots results showed that knockdown of LINC00152 have no effect on the expression of EZH2 (Fig. 6B and C) which proved that LINC00152 could bind to EZH2 but have no effect on EZH2 expression. Moreover, EZH2 activation was reported to be closely related to cancer progression in various human malignant tumors, including CCA [56][57][58]. We inhibited the expression of EZH2 with e cient siRNAs (Fig. 6D). The results con rmed that knockdown of EZH2 could signi cantly inhibit cell proliferation and migration (Fig. 6E, F and G). Next, to investigate whether EZH2 could act as a negative regulator of transcription via H3K27me3, we further performed the following assays. The qRT-PCR and Western Blot results showed that the mRNA and protein levels of LRIG1 exhibited an increase in the EZH2-knockdown HuCCT1 and RBE cell lines ( Fig. 6H and I). Then we conducted chromatin immunoprecipitation (ChIP) assays to analyze the enrichment of EZH2 and H3K27me3 in the promoter region of LRIG1. As shown in Fig. 6J, knockdown of LINC00152 markedly reduced the EZH2 enrichment and H3K27me3 levels in the promoter region of LRIG1 genes. Above results validated that LINC00152 could motivate CCA cells proliferation and migration partially through downregulating LRIG1 by binding to EZH2.

LRIG1 overexpression inhibits CCA cell proliferation and metastasis
Several studies have shown that LRIG1 could exert tumor-suppressive effects in various cancers [35,[59][60][61]. However, the function of LRIG1 in CCA has not been reported until now. To assess the expression of LRIG1 in CCA tissues, we analyzed the dataset TCGA and GSE76297, detecting the down-regulation of LRIG1 in CCA compared to that in normal tissue ( Fig. 7A and B). Indeed, by qRT-PCR, the measure of LRIG1 expression in 17 pairs of CCA tumor tissues and the adjacent tissues showed the similar results (Fig. 7C). Moreover, overexpression of LRIG1 could evidently suppress proliferation and migration of HuCCT1 and RBE cell lines. In addition, LRIG1 could also partially reverse the LINC00152-mediated growth promotion (Fig. 7D-F). Taken together, our study demonstrated that LINC00152 could bind to EZH2 and then epigenetically mediate H3K27me3 in the LRIG1 promoter regions to inhibit LRIG1 expression, thus promoting CCA progress.

Discussion
Recently, more and more studies have implied the important roles of lncRNAs in the diagnosis, prognostic and therapeutic evaluation of cancers. In our present study, publicly available data and qRT-PCR results both uncovered that LINC00152 expression in CCA tissues was observably higher than that in corresponding non-tumor tissues, which implied the important role of LINC00152 in CCA progression. LINC00152 was reported upregulated in various cancers, promoting cancer cell proliferation and migration [20-32]. Nevertheless, the possible role of LINC00152 in CCA is still undocumented.
As the results indicated in our study, LINC00152 could regulate the proliferation and migration of CCA cells both in vitro and in vivo. Although the oncogenic feature of LINC00152 have been clari ed in various cancers, the pathway and downstream genes correlated with LINC00152 in CCA remains uncertain. Therefore, we conducted RNA transcriptome sequencing and gene ontology analysis. The results implied that gene expression pro les were primarily associated with cell proliferation, adhesion and apoptosis and inducted us to estimate several target genes regulated by LINC00152. We observed a noticeable elevation of the suppressor LRIG1 expression in mRNA and protein levels and conducted further mechanism studies.
Previous studies have determined that a signi cant number of lncRNAs could function in cooperation with chromatin modifying enzymes to involved in promoting of epigenetic activation or silencing of gene expression [11,42,62]. Thereinto, LINC00152 was reported to have the capital to inactive target gene expression epigenetically through interaction with EZH2 in lung adenocarcinoma and gastric cancer [20,26]. EZH2, an well-known oncogenic gene in various human malignant tumors, was reported to be capable of interacting with lncRNAs to catalyze the trimethylation of lysine residues 27 of histone 3 (H3K27me3) in the promoter regions of the target gene, thereby mediating transcriptional silencing [44,63,64]. Further, our resulting data revealed that LINC00152 was capable to bind to EZH2 but have no effect on EZH2 expression, silencing target genes expression in CCA cells as well.
LRIG1 have been showed to function as the tumor suppressor in various types of cancers. However, the role of LRIG1 in CCA tumorigenesis remains unclear. It is reported that hypermethylation of the suppressor gene LRIG1 promoter region leads to the LRIG1 transcription suppression in colorectal cancer and cervical cancer [50,51]. Meanwhile, our results demonstrated that histone methylation (H3K27me3) induced by LINC00152 could also account for the LRIG1 downregulation in cancer. Furthermore, it was reported that histone methylation usually cooperate with DNA methylation to inactive target gene expression heritably [52,53,65]. As discovered by our ndings, LRIG1, the tumor suppressor, could be affected by LINC00152-EZH2 complex through promoter H3K27me3 in CCA.

Conclusion
In summary, our study showed that upregulated LINC00152 could facilitate CCA progress through silencing the target tumor suppressor gene LRIG1 transcription at the epigenetic level, which suggested that LINC00152 could provide a theoretical basis for clinical diagnosis and treatment strategies in CCA.

Consent for publication
All authors have agreed to publish this manuscript.

Availability of data and materials
The data generated or analyzed during this study are included in this article, or if absent are available from the corresponding author upon reasonable request.

Competing interests
The authors declare that they have no con ict of interest.       western blot after transfecting with si-LINC00152 into CCA cells. Error bars indicate means ± SD.*P < 0.05; **P < 0.01; ***P< 0.001. Colonies were counted and captured. (G) Transwell assays were used to investigate the migration of CCA cells after transfection. (H and I) Knockdown EZH2 triggered LRIG1 expression at the mRNA levels by qPCR and protein levels by western blot. (J) The enrichment of EZH2 and H3K27me3 in the promoter region of LRIG1 was identi ed via ChIP assays, and this enrichment was decreased after LINC00152 knockdown in the CCA cell line. Enrichment was quanti ed relative to the amount of input controls.