Esophageal cancer is one of the leading causes of cancer-related mortality because of poor prognosis. Long noncoding RNAs (lncRNAs) have been gradually demonstrated to play critical roles in cancer development. We identified a novel long noncoding RNA named linc00460 by microarray analysis using esophageal squamous cell carcinoma (ESCC) clinical samples, which has not been studied before. Our research indicated that linc00460 was overexpressed in the majority of tumor tissues and ESCC cell lines. Linc00460 expression was positively correlated with ESCC TNM stage, lymph node metastasis, and predicted poor prognosis. In vitro experiments showed that linc00460 depletion suppressed ESCC cell growth through regulating cell proliferation and cell cycle; in additional, linc00460 depletion accelerated ESCC cell apoptosis. We further revealed that linc00460 overexpression was manipulated by transcriptional co-activator CBP/P300 through histone acetylation. Given the high expression and important biological functions of linc00460, we suggest that linc00460 works as an oncogene and might be a valuable prognostic biomarker for ESCC diagnosis and treatment.

Introduction

Esophageal cancer is the 8th most commonly diagnosed cancer type and the 6th leading cause of cancer-related death, with an estimated 456,000 new cases each year worldwide [1,2]. The esophageal cancer morbidity of men is 3-fold higher than women, and mostly occurred in rural areas [3]. Alcohol addiction and tobacco abuse are proved to be risk factors for esophageal cancer [4] with other carcinogens, such as HPV infection [5]. Esophageal squamous cell carcinoma (ESCC) is the major histological type versus esophageal adenocarcinoma, accounting for 80% of esophageal cancer cases in South-Eastern and Central Asia [6]. As >50% of esophageal cancers are unresectable or with local invasion and organ metastasis at the time of diagnosis, only 15–25% of the patients can survive over 5 years despite of the improvements of surgery or other comprehensive treatments [7]. Therefore, investigating the mechanism underlying esophageal cancer initiation and development is critical for exploiting new diagnosis markers and therapeutic targets [8].

Long noncoding RNAs (lncRNAs) are defined as novel RNAs >200 nt in length, occupied at least 80% of human genome with no protein coding potential [9]. These ncRNAs are usually transcribed by RNA polymerase II, also spliced and mostly polyadenylated, analogues to protein coding genes [10]. Researchers have recognized many functional lncRNAs participating in tumorigenesis, due to their irregular expression and specific expression patterns in various tumor types [1113]. These lncRNAs have been demonstrated to modulate cancer cell behaviors including cancer progression, cell metastasis, and drug resistance. Some of the first identified lncRNAs such as HOX transcript antisense intergenic RNA (HOTAIR) [14], MALAT1 [15], and H19 [16] are highly expressed in multiple tumor tissues and play regulatory roles in chromatin remodeling through histone modification, DNA methylation, or function as competing endogenous RNAs through interacting with microRNAs [17]. Previous studies have identified that some lncRNAs expressed in ESCC disorderly, affecting cancer development and prognosis [18]. However, the delicate molecular basis of lncRNA function relies on deeper research and advances in scientific technology.

In the present study, we identified many differentially expressed lncRNAs by microarray analysis using five paired ESCC clinical tissues. Microarray results indicated that a newly identified lncRNA named long intergenic nonprotein coding RNA 460, abbreviated to linc00460, had a relatively great alteration between cancer tissues and normal tissues. As a novel identified lncRNA, the expression and function of linc00460 in ESCC were unknown. Our research indicated that linc00460 might function as a novel oncogene and might be a valuable prognostic biomarker for ESCC diagnosis and treatment.

Materials and methods

Patients and clinical tissues

ESCC tissues and corresponding adjacent normal tissues were collected from patients underwent esophagectomy in Southwest Hospital (the Third Military Medical University, Chongqing China) between 2006 and 2014. Our research project was approved by constituted Ethics Committee of the university and it conformed to the provisions of the Declaration of Helsinki. All patients enrolled were informed and consent. None of them received chemo- or radiotherapy or other preoperative treatments before surgery. The clinical tissues were stored at liquid nitrogen immediately after surgery. Patients were staged according to the American Joint Committee on Cancer (AJCC) by at least two pathologists. Our research observed the Declaration of Helsinki and was approved by the Human Ethics Committee of the Third Military Medical University.

Microarray screening and bioinformatics analysis

The microarray profiling was performed using five paired ESCC tissues and normal tissues, the clinical tissues were obtained from five ESCC male patients who were clinically diagnosed and pathologically confirmed, and none of them received chemo- or radiotherapy or other preoperative treatments before surgery. RNA extraction and sequential microarray hybridization were conducted by Kangchen Company (Shanghai, China), the detected human genome transcripts were from the Human lncRNA microarray v2.0 (8×60 K, arraystar, U.S.A.). Hierarchical cluster analysis was performed using Cluster software to obtain differential expressed lncRNAs and mRNAs. The results are available in Gene Expression Omnibus (GEO) with the serial number GSE89102.

Cell culture

The human ESCC cell lines EC109, KYSE150, and KYSE450 were purchased from the Cell Bank of the Chinese Academy of Science (Shanghai, China), the human normal esophageal epithelial cell line Het-1A was purchased from American Type Culture Collection (Maryland, U.S.A.). The other cancer cell lines used in the present study were preserved by our laboratory for years. EC109, KYSE150, KYSE450, and Het-1A cell lines were verified by STR genotype method at Key Laboratory of Birth Defects and Reproductive Health (Chongqing, China) (Supplementary Figure S1). All cells were cultured in RPMI-1640 medium (Hyclone, U.S.A.), containing 10% newborn bovine serum; Het-1A was cultured in BEGM medium from Lonza/Clonetics Corporation; all cells were maintained in a humidified incubator at 37°C containing 5% CO2.

RNA extraction and qRT-PCR assay

Total RNA was isolated from either clinical tissues or cultured cells using the Trizol reagent (Takara, Japan) according to the manufacturer’s instructions. The RNA concentration and quality were measured by NanoDrop ND-1000 spectrophotometer. First strand cDNA was synthesized from 200 ng of total RNA using the PrimeScript RT reagent Kit (Takara, Japan). SYBR Premix Ex Taq (Takara, Japan) was used for Quantitative real-time PCR assay on the CFX Connect Real-Time System (Bio-Rad, U.S.A.). The primers used for qPCR were listed in Supplementary Table S1. The relative expression of linc00460 was calculated from the formula 2−ΔΔCt and normalized with GAPDH.

SiRNA transfection

Small interfering RNAs (siRNAs) were designed and synthesized by Shanghai GenePharma (Shanghai, China). The siRNA sequences used in the present study were listed in Supplementary Table S2. The cells were seeded and cultured in six-well plate with density of 3 × 105/well overnight. Then, cells were transfected with siRNAs or negative control at a final concentration of 50 nM using Lipofectamin 2000 reagent (Invitrogen, Carlsbad, U.S.A.) according to the manufacturer’s instruction.

Cell growth and proliferation assay

ESCC cells transfected with siRNAs or negative control were seeded in 96-well plate (5 × 103/ well). Cell growth assay was performed using Cell Counting Kit-8 (CCK-8) (Dojindo Laboratory, Japan) every 24 h according to the protocol. The number of viable cells was quantified by the absorbance of 450 nm using the microplate reader.

After transfection for 48 h, cell proliferation assay was performed using Cell-LightTM Edu Apollo567 In Vitro Kit (Ribobio, China) with fluorescence microscope according to the protocol. The cell proliferation rate was calculated according to Edu incorporation rate. Each experiment group had three replicates.

Cell cycle and apoptosis analysis

After transfected with siRNA for 48 h, cells were harvested by trypsin, and then cells were fixed with 70% ethanol at 4°C overnight. Then fixed cells were incubated with RNase A for 30 min to completely degrade RNA, and then stained with propidium oxide for another 30 min in dark place using Cell Cycle Analysis Kit (Beyotime Biotechnology, China). The managed cells were detected by flow cytometer FACSCalibur (BD Bioscience, U.S.A.) and analyzed with Flowjo software.

For apoptosis analysis, transfected cells were collected by trypsin 48 h after transfection, and then stained with Annexin V-FITC and PI using Cell apoptosis Analysis Kit (Beyotime Biotechnology, China). Stained cells were detected by flow cytometer FACSCalibur (BD Bioscience, U.S.A.) and analyzed with Flowjo software.

Nucleus and cytoplasm isolation assay

Nucleus and cytoplasm isolation assay was performed using Nuclei Isolation Kit: Nuclei Ez Prep (Sigma, U.S.A.) according to manufacturer‘s instruction. Then we used Trizol reagent to extract RNA from isolated nucleus and cytoplasm fraction. Reverse transcription and PCR reaction were done as described before. GAPDH and U6 were used as important criteria of isolation quality. The primers of GAPDH and linc00460 used for PCR were same with qPCR primers described before; the primers of U6 were purchased from GeneCopoeiaTM (#HmiRQP9001, China).

Chromatin immunoprecipitation assay

Chromatin immunoprecipitation (ChIP) was performed using the SimpleChIP® Enzymatic Chromatin IP Kit (#9003, CST, U.S.A.) according to the manufacturer’s instruction. Cells were harvest at 80–90% confluence after siRNA transfection. Briefly, cells were cross-linked with 1% formaldehyde for 15 min, and then terminated with glycine before scrapped from culture dishes. Afterwards, the collected cells were digested with nuclease to break the cross-linked chromatin to 100–200 bp length. Then appropriate amount of chromatin was immunoprecipitated using anti-CBP (ab2832, Abcam, U.S.A.), anti-P300 (ab14984, Abcam, U.S.A), anti-histone H3A (acetyl K27) (ab4729, Abcam, U.S.A.), and anti-histone H3A (acetyl K18) (Cat.#17-10111, Millipore, U.S.A.); goat anti-Rabbit IgG and goat anti-Mouse IgG were used as negative control respectively. The immunoprecipitation reaction was performed overnight at 4°C with rotation. The next day, the precipitated chromatin were washed and eluted from the antibody/protein G magnetic beads, and then DNA purification was performed and analyzed by qPCR with specific primers listed in Table 1.

Table 1
Correlation between linc00460 expression and ESCC clinical parameters
  Low-expression High-expression P valuea 
Clinical parameters  N=33 N=32  
Age <60 years 23 19  
 >60 years 10 13  0.384 
Gender Male 24 27  
 Female 0.253  
Lymph node metastasis Negative 24 15  
 Positive 17 0.033* 
TNM stage I/II 28 17  
 III/IV 15  0.006**  
Histological differentiation Well/moderate 31 27  
 Poor 0.258 
 Smoking Yes 21 24  
 No 12 0.321 
 Drinking Yes 19 25  
 No 14 0.077  
  Low-expression High-expression P valuea 
Clinical parameters  N=33 N=32  
Age <60 years 23 19  
 >60 years 10 13  0.384 
Gender Male 24 27  
 Female 0.253  
Lymph node metastasis Negative 24 15  
 Positive 17 0.033* 
TNM stage I/II 28 17  
 III/IV 15  0.006**  
Histological differentiation Well/moderate 31 27  
 Poor 0.258 
 Smoking Yes 21 24  
 No 12 0.321 
 Drinking Yes 19 25  
 No 14 0.077  

aChi-squared test results

*P<0.05

**P<0.01

Statistical analysis

Experimental data are presented as mean ± SE from three independent experiments in triplicate. Statistical analysis was performed using the SPSS software package version 16.0. For comparison, paired or independent Student’s t-test, Chi-square test, or one-way ANOVA were chosen as appropriate. Kaplan–Meier method and log-rank test were used to delineated survive curve. All P values were two sided, and a P<0.05 was considered significant.

Results

Linc00460 is a novel long noncoding RNA with potential function in ESCC

In order to identify aberrantly expressed lncRNAs in human esophageal squamous cell carcinoma, we performed microarray analysis using five paired ESCC tissues and adjacent nontumor tissues. The results revealed that 2939 (12.4%) lncRNA transcripts were up-regulated (fold change>2, P<0.05), and 3517 (14.64%) lncRNA transcripts were down-regulated (fold change<0.5, P<0.05) in ESCC tissues compared with normal tissues. The microarray results were validated by qRT-PCR and performed high repeatability (Supplementary Figure S2). Of all differentially expressed lncRNAs, linc00460 was one of the mostly up-regulated lncRNAs in tumor tissues (fold change = 41.9, P=0.006), which aroused our attention.

According to RefSeq database, linc00460 is located at chromosome 13 in human genome, the transcript length is 935 bp (NR_034119, GenBank), consisting of three exons (Figure 1A). Using ORFfinder from NCBI we failed to predict a protein sequence longer than 80 amino acids (Figure 1B), strongly suggesting that linc00460 lacked protein coding capacity. Bioinformatic analysis indicated that linc00460 was transcribed from a gene desert region, suggesting that linc00460 belonged to long intergenic noncoding RNA (lincRNA). The conservation of linc00460 genomic region among primate genomes indicated its importance in evolution (Figure 1A). In addition, linc00460 had been detected to be dysregulated in a multitude of physiology and pathology processes according to GEO Profiles database. For instance, linc00460 expression was significantly lower in Neural tube defect patient than normal (GDS2470) (Supplementary Figure S3A); linc00460 expression was decreased when knockdown LSD1 in neuroblastoma cell lines (GDS5281) (Supplementary Figure S3B); MTX-sensitive HT29 colon adenocarcinoma cell line presented higher linc00460 expression than MTX-resistant HT29 colon adenocarcinoma cell line (GDS3330) (Supplementary Figure S3C). These suggested that linc00460 might function in organ development and tumorigenesis. Thus, we considered linc00460 might function in ESCC carcinogenesis and further detected the expression and function in ESCC tissues.

Linc00460 is a novel long noncoding RNA with potential function in ESCC

Figure 1
Linc00460 is a novel long noncoding RNA with potential function in ESCC

(A) The genomic construction of linc00460 coding region according to UCSC dataset (February 2009, GRCH37/hg19). (B) Results of ORFfinder software to predict protein sequence of linc00460. (C) Linc00460 subcellular localization analyzed by nucleus and cytoplasm isolation assay. Electrophoresis analysis of PCR products synthesized from nucleus and cytoplasm fraction of KYSE150 and KYSE450. N represents for nucleus, C represents for cytoplasm. (D) Percentage bar chart represents for linc00460 distribution between nucleus and cytoplasm in KYSE150 and KYSE450. Data were presented as mean ± SE from three independent experiments.

Figure 1
Linc00460 is a novel long noncoding RNA with potential function in ESCC

(A) The genomic construction of linc00460 coding region according to UCSC dataset (February 2009, GRCH37/hg19). (B) Results of ORFfinder software to predict protein sequence of linc00460. (C) Linc00460 subcellular localization analyzed by nucleus and cytoplasm isolation assay. Electrophoresis analysis of PCR products synthesized from nucleus and cytoplasm fraction of KYSE150 and KYSE450. N represents for nucleus, C represents for cytoplasm. (D) Percentage bar chart represents for linc00460 distribution between nucleus and cytoplasm in KYSE150 and KYSE450. Data were presented as mean ± SE from three independent experiments.

To determine the subcellular localization of linc00460 in ESCC cells, we also performed nucleus and cytoplasm isolation assay. The results in KYSE150 and KYSE450 cells showed that linc00460 was located both in nucleus and cytoplasm, mainly in cytoplasm (Figure 1C and D).

Linc00460 is overexpressed in ESCC tissues and correlated with ESCC clinical characteristics

To further confirm linc00460 expression pattern in ESCC, we performed qRT-PCR using ESCC clinical tissues and normal tissues. We found that linc00460 expression was higher in 95.4% (62/65) of the ESCC tissues compared with adjacent normal tissues (Figure 2A). Linc00460 expression was positively correlated with ESCC primary tumor invasion depth (Figure 2B). Patients with lymph node metastasis and later TNM stage had higher linc00460 expression level (Figure 2C and D). The worse differentiation status of ESCC was correlated with high linc00460 expression (Figure 2E). The positive rate of linc00460 overexpression in ESCC was remarkably, indicating that it could be used as a biomarker for ESCC molecular diagnosis clinically.

Linc00460 expression is higher in ESCC tissues and correlated with ESCC clinical characteristics

Figure 2
Linc00460 expression is higher in ESCC tissues and correlated with ESCC clinical characteristics

(A) Fold change of linc00460 expression of ESCC tissues to adjacent normal tissues normalized by log2. The red column indicates linc00460 expression was higher in ESCC tissues, while the blue column indicates the opposite. (B) Linc00460 expression was positively correlated with ESCC primary tumor invasion depth. The statistical significance was calculated by independent-samples Student’s t-test. (C) ESCC patients with lymph node metastasis had higher linc00460 expression. The statistical significance was calculated by independent-samples Student’s t-test. (D) The expression of linc00460 was significantly up-regulated in higher TNM stage of ESCC tissues. The statistical significance was calculated by one-way ANOVA analysis. (E) The worse differentiation status of ESCC was correlated with high linc00460 expression. The statistical significance was calculated by one-way ANOVA analysis. (F) Kaplan–Meier analysis of overall survive rate of 42 patients indicated that higher linc00460 expression exhibited poorer overall survive. P value was calculated by log-rank test.

Figure 2
Linc00460 expression is higher in ESCC tissues and correlated with ESCC clinical characteristics

(A) Fold change of linc00460 expression of ESCC tissues to adjacent normal tissues normalized by log2. The red column indicates linc00460 expression was higher in ESCC tissues, while the blue column indicates the opposite. (B) Linc00460 expression was positively correlated with ESCC primary tumor invasion depth. The statistical significance was calculated by independent-samples Student’s t-test. (C) ESCC patients with lymph node metastasis had higher linc00460 expression. The statistical significance was calculated by independent-samples Student’s t-test. (D) The expression of linc00460 was significantly up-regulated in higher TNM stage of ESCC tissues. The statistical significance was calculated by one-way ANOVA analysis. (E) The worse differentiation status of ESCC was correlated with high linc00460 expression. The statistical significance was calculated by one-way ANOVA analysis. (F) Kaplan–Meier analysis of overall survive rate of 42 patients indicated that higher linc00460 expression exhibited poorer overall survive. P value was calculated by log-rank test.

To further explore the relationship between linc00460 expression and ESCC clinical characteristics, we divided the 65 ESCC patients into two groups according to linc00460 expression: the linc00460 high-expression group (n=32, fold change > average) and the linc00460 low-expression group (n=33, fold change < average). Statistical analysis suggested that linc00460 high-expression group presented more lymph node metastasis and later TNM stage than linc00460 low-expression group. However, there was no difference of histological differentiation, smoking, and drinking status between these two groups (Table 1). To investigate the relationship between linc00460 expression and ESCC prognosis, we delineated survive curve using Kaplan–Meier method. The results showed that linc00460 expression was in inverse proportion to ESCC survive rate (Figure 2F). All the above results indicated that up-regulation of linc00460 was a common effect in the carcinogenesis of ESCC and predicted poor prognosis.

Linc00460 promotes ESCC cell growth and apoptosis in vitro

In order to explicit the meaning of linc00460 overexpression, we next investigated its biological function in ESCC. First, we measured linc00460 expression in three human ESCC cell lines (EC109, KYSE150, and KYSE450) and one human normal esophageal epithelial cell line (Het-1A), we found that the linc00460 expression in KYSE150 and KYSE450 was significantly higher than that in EC109 and Het-1A, especially the expression in KYSE150 (Figure 3A). To our knowledge, EC109 and KYSE450 were established from ESCC surgical specimen with well-differentiated histology, whereas KYSE150 was established from poor differentiated ESCC carcinoma [19]. So, the particularly high expression of linc00460 in KYSE150 was in accordance with the expression pattern in ESCC tissues. Additionally, we measured linc00460 expression in multiple cancer cell lines, the results indicated that linc00460 was overexpressed in many digestive system cancers (Supplementary Figure S4).

Linc00460 promotes ESCC cells growth in vitro

Figure 3
Linc00460 promotes ESCC cells growth in vitro

(A) Linc00460 expression in three ESCC cell lines (EC109, KYSE150, and KYSE450) and a normal esophageal epithelial cell line Het-1A. Data were presented as expression fold-change relative to Het-1A. (B) Linc00460 expression of KYSE150 and KYSE450 after transfection of three different siRNAs targeting linc00460 and negative control. Si2 was used in the present study. (C and D) CCK-8 assay indicated that knockdown of linc00460 expression decreased cell growth in KYSE150 and KYSE450. (E and F) EDU incorporation assay indicated that linc00460 knockdown decreased cell proliferation in KYSE150 and KYSE450. Data were presented as mean ± SE from three independent experiments in triplicate; *P<0.05, **P<0.01, and ***P<0.001.

Figure 3
Linc00460 promotes ESCC cells growth in vitro

(A) Linc00460 expression in three ESCC cell lines (EC109, KYSE150, and KYSE450) and a normal esophageal epithelial cell line Het-1A. Data were presented as expression fold-change relative to Het-1A. (B) Linc00460 expression of KYSE150 and KYSE450 after transfection of three different siRNAs targeting linc00460 and negative control. Si2 was used in the present study. (C and D) CCK-8 assay indicated that knockdown of linc00460 expression decreased cell growth in KYSE150 and KYSE450. (E and F) EDU incorporation assay indicated that linc00460 knockdown decreased cell proliferation in KYSE150 and KYSE450. Data were presented as mean ± SE from three independent experiments in triplicate; *P<0.05, **P<0.01, and ***P<0.001.

To investigate the biological functions of linc00460 on ESCC in vitro, three different siRNAs were used to knockdown linc00460 expression in KYSE150 and KYSE450 that had higher level of linc00460. The interference efficiency was tested 48 h after transfection, as shown in Figure 3B, the si2 presented the best interference efficiency, which was used in the following experiments. The CCK-8 assay showed that the vital cell number of KYSE150 and KYSE450 transfected with linc00460 siRNA was less than the control group (Figure 3C and D), indicated that linc00460 promoted ESCC cell growth. Since both cell proliferation and apoptosis can contribute to the effect on cell growth, we further studied the function of linc00460 on the two aspects.

EDU proliferation assay showed that the incorporation rate of KYSE150 and KYSE450 decreased after transfected with linc00460 siRNA (Figure 3E and F), demonstrating that depletion of linc00460 damaged ESCC cell proliferation. Then we performed flow cytometer experiment to test cell cycle and cell apoptosis. The results showed that in KYSE150, linc00460 depletion resulted in the increase in G0/G1-phase distribution and decrease in G2/M-phase distribution (Figure 4A); however, in KYSE450, linc00460 depletion only resulted in decrease of S-phase distribution (Figure 4A). Moreover, cell apoptosis rates in KYSE150 and KYSE450 were increased after linc00460 siRNA transfection (Figure 4B). These results indicated that cell growth induced by linc00460 was probably due to both cell proliferation promotion and apoptosis inhibition.

Linc00460 knockdown affects ESCC cell cycle and cell apoptosis

Figure 4
Linc00460 knockdown affects ESCC cell cycle and cell apoptosis

(A) Representative images of cell cycle distribution of siRNA transfected KYSE150 and KYSE450, the bar diagram indicated the percentage of cells distributed in G0/G1, S, and G2/M phases. (B) Representative images of cell apoptosis of siRNA transfected KYSE150 and KYSE450, the bar diagram indicated the overall apoptosis rates. Data were presented as mean ± SE from three independent experiments in triplicate; *P<0.05, **P<0.01, and ***P<0.001.

Figure 4
Linc00460 knockdown affects ESCC cell cycle and cell apoptosis

(A) Representative images of cell cycle distribution of siRNA transfected KYSE150 and KYSE450, the bar diagram indicated the percentage of cells distributed in G0/G1, S, and G2/M phases. (B) Representative images of cell apoptosis of siRNA transfected KYSE150 and KYSE450, the bar diagram indicated the overall apoptosis rates. Data were presented as mean ± SE from three independent experiments in triplicate; *P<0.05, **P<0.01, and ***P<0.001.

CBP/P300 binding to linc00460 promoter activates linc00460 transcription through histone acetylation

Next, we asked why linc00460 was up-regulated in ESCC tissues. Bioinformatics analysis revealed that the promoter region of linc00460 was enriched of many histone modification signals, such as H3K4Me1, H3K4Me3, and H3K27Ac signals (Figure 1A); in addition, Transcription Factor ChIP-seq experiments performed by ENCODE project showed that many transcription factors and transcription co-activators could bind to linc00460 promoter, such as GATA2, CEBPB, P300, Fos, Jun etc., indicating that the linc00460 gene region was capable of transcription and could be regulated by chromatin modification (Figure 1A). CBP and P300 are closely related transcriptional co-activators and acetyltransferase enzymes in humans, which have been reported to activate gene expression through binding specific transcription factors to transcript machinery and chromatin modulation [20]. Considering that the promoter of linc00460 contains P300 binding signal (Figure 5A), we hypothesized that CBP and P300 might activate linc00460 transcription as co-activators by modifying histone acetylation in ESCC.

CBP/P300 binding to linc00460 promoter activates linc00460 transcription through histone acetylation

Figure 5
CBP/P300 binding to linc00460 promoter activates linc00460 transcription through histone acetylation

(A) Schematic diagram of linc00460 promoter. TSS represents transcription start site, represents P300 binding region. (B) CBP or P300 expression after transfection with siRNAs and negative control in KYSE150 and KYSE450 detected with qRT-PCR assay. (C) Linc00460 expression after transfection with CBP or P300 siRNAs and negative control in KYSE150 and KYSE450 detected with qRT-PCR assay. (D) CHIP-qPCR analysis of KYSE150 and KYSE450 using CBP and P300 antibody. (E) CHIP-qPCR analysis of KYSE150 and KYSE450 using H3K18Ac and H3K27Ac antibody; *P<0.05, **P<0.01, and ***P<0.001.

Figure 5
CBP/P300 binding to linc00460 promoter activates linc00460 transcription through histone acetylation

(A) Schematic diagram of linc00460 promoter. TSS represents transcription start site, represents P300 binding region. (B) CBP or P300 expression after transfection with siRNAs and negative control in KYSE150 and KYSE450 detected with qRT-PCR assay. (C) Linc00460 expression after transfection with CBP or P300 siRNAs and negative control in KYSE150 and KYSE450 detected with qRT-PCR assay. (D) CHIP-qPCR analysis of KYSE150 and KYSE450 using CBP and P300 antibody. (E) CHIP-qPCR analysis of KYSE150 and KYSE450 using H3K18Ac and H3K27Ac antibody; *P<0.05, **P<0.01, and ***P<0.001.

In order to verify our hypothesis, first, we knockdown CBP and P300 expression using siRNAs targeting three different sites respectively, the siRNAs with ideal interference efficacy were chosen for next experiments. The qRT-PCR results showed that both CBP and P300 depletion reduced linc00460 expression in KYSE150 and KYSE450 (Figure 5B and C). Then, we employed ChIP-qPCR assay to detect whether CBP and P300 bind to linc00460 promoter. The results showed that both individual CBP and P300 proteins could bind to linc00460 promoter (Figure 5D); meanwhile, we also detected high acetyl-Histone H3 (Lys18 and Lys27) enrichment signal in linc00460 promoter (Figure 5E). Therefore, we concluded that CBP/P300 binding to linc00460 promoter activates linc00460 transcription through histone H3 acetylation. The acetyl histone loosens the chromatin structure, this puffy conformation allows easier access for transcription machinery.

Disscusion

LncRNAs exists prevalently in human genome, characterization of lncRNAs function in cancer has been of ongoing interest [21]. In recent years, accumulating evidence prove that lncRNA dysregulation play important roles in ESCC carcinogenesis, cancer development, metastasis, and patient outcome. Our laboratory discovered that lncRNA MALAT1 and H19 expression were significantly higher in ESCC and were correlated with tumor TNM stage, lymph node metastasis [22,23], which was with accordance to other researches [24,25]. Besides, lncRNAs such as HOTAIR [2628], antisense noncoding RNA in the INK4 locus (ANRIL) [2931], and colon cancer associated transcript-1 (CCAT1) [3234] have been reported to be up-regulated in at least three types of digestive system cancers (DSCs) including ESCC; in additional, there are many other lncRNAs investigated in ESCC, such as linc-POU3F3 [35], AFAP1-AS1 [36], HNF1A-AS1 [37], HOTTIP [38] etc. Recently, scientists found that the circulating expression level of linc-POU3F3 in plasma showed reliable potential for ESCC diagnosis [39]; on the other hand, HOTAIR was the most commonly identified negative biomarker for ESCC prognosis [40]; moreover, Li et al. [41] established a three-lncRNA signature (ENST00000435885.1, XLOC_013014, and ENST00000547963.1) as a new independent biomarker for ESCC prognosis. However, the clinical application of lncRNAs as therapeutic targets in ESCC hasn’t been conducted.

In the present study, we identified a functional-unknown lncRNA named linc00460 through microarray analysis in ESCC tissues. We found that linc00460 was up-regulated in ESCC and was correlated with ESCC aggressiveness. Linc00460 exerted its oncogenic roles through regulating ESCC cell proliferation, cell cycle, and apoptosis. Our research revealed a remarkably high positive rate of linc00460 overexpression in ESCC clinical tissues, indicating that linc00460 can be a potential biomarker for ESCC molecular diagnosis. In view of the association of linc00460 expression with ESCC clinical characteristics, linc00460 could be a potential index for monitoring ESCC development and prognosis, which needs further study and analysis in a larger number of clinical samples.

LncRNA overexpression is widely reported in different species and organs. The mechanisms underline could be genome variation [22,42], transcriptional activation by classical oncogenes [43], transcription factors regulation, chromatin modification epigenetically [36], and microRNAs [25]. Among these, CBP and P300 were reported to be important regulators. CBP (also called CREB-binding protein, CREBBP, or KAT3A) and P300 (also called EP300 or KAT3B) were first identified and investigated as members of E1A interacting proteins [44]. They have significant sequence homology and many overlapping functions, thus the two proteins are now referred as CBP/P300 [44,45]. CBP/P300 was traditionally recognized to be involved in the transcriptional activation of many protein coding genes [44,46,47], recently, CBP/P300 was reported to regulate lncRNA expression. It has been reported that HOTAIR was overexpressed in breast cancer partly because of the binding of the CBP/P300 to the promoter of HOTAIR [48]; also, it has been reported that CREB unregulated the expression of lncRNA HULC through binding to the core promoter of this lncRNA [49], whereas CBP/P300 can interact with CREB directly.

In the present study, we discovered that CBP/P300 binds to linc00460 promoter; meanwhile, we detected acetylation toward H3K18 and H3K27, which gives an epigenetic tag for transcriptional activation. This phenomenon suggested that the binding of CBP/P300 to linc00460 promoter modulates the closed, silenced chromatin to open, permissive chromatin. This chromatin architecture remolding facilitates transcription machinery access and activates transcription consequently. To our knowledge, CBP/P300 can be downstream effectors of many signaling pathways, such as NF-κB signaling pathway, Notch signaling pathway, hypoxia, and DNA damage [45,5052]. Thus, we may draw a picture that the above biological pathways alteration result in abnormal gene expression such as linc00460 through CBP/P300 function, and finally causes tumor formation and development.

In conclusion, we identified a novel lncRNA named linc00460, which acted as oncogene in ESCC; CBP/P300 up-regulated linc00460 expression through binding to linc00460 promoter and modulating chromatin architecture; linc00460 could be a candidate biomarker for ESCC diagnosis and treatment.

Funding

This work was supported by grants from National Natural Science Foundation of China [grant number 81372554]. The Southwest Hospital is affiliated with the Third Military Medical University.

Competing Interests

The authors declare no potential conflicts of interest and that all authors have contributed significantly.

Author Contribution

Yan Liang did the design, acquisition and analysis of data with the help of Yuanyuan Wu and Xuandan Chen; Shixin Zhang, Xingying Guan and Kang Yang collected the clinical samples; Kai Wang, Juan Li and Yun Bai supervised the experiments and revised the article for publication.

Abbreviations

     
  • CCK-8

    Cell Counting Kit-8

  •  
  • ChIP

    chromatin immunoprecipitation

  •  
  • ESCC

    esophageal squamous cell carcinoma

  •  
  • HOTAIR

    HOX transcript antisense intergenic RNA

  •  
  • LncRNA

    long noncoding RNA

References

References
1
Rubenstein
J.H.
and
Shaheen
N.J.
(
2015
)
Epidemiology, diagnosis, and management of esophageal adenocarcinoma
.
Gastroenterology
149
,
302
317
,
e301
2
Pennathur
A.
,
Gibson
M.K.
,
Jobe
B.A.
and
Luketich
J.D.
(
2013
)
Oesophageal carcinoma
.
Lancet
381
,
400
412
3
Ferlay
J.
,
Soerjomataram
I.
,
Dikshit
R.
,
Eser
S.
,
Mathers
C.
,
Rebelo
M.
et al. 
(
2015
)
Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012
.
Int. J. Cancer
136
,
E359
E386
4
Rustgi
A.K.
and
El-Serag
H.B.
(
2014
)
Esophageal carcinoma
.
N. Engl. J. Med.
371
,
2499
2509
5
Petrick
J.L.
,
Wyss
A.B.
,
Butler
A.M.
,
Cummings
C.
,
Sun
X.
,
Poole
C.
et al. 
(
2014
)
Prevalence of human papillomavirus among oesophageal squamous cell carcinoma cases: systematic review and meta-analysis
.
Br. J. Cancer
110
,
2369
2377
6
Arnold
M.
,
Soerjomataram
I.
,
Ferlay
J.
and
Forman
D.
(
2015
)
Global incidence of oesophageal cancer by histological subtype in 2012
.
Gut
64
,
381
387
7
Ohashi
S.
,
Miyamoto
S.
,
Kikuchi
O.
,
Goto
T.
,
Amanuma
Y.
and
Muto
M.
(
2015
)
Recent advances from basic and clinical studies of esophageal squamous cell carcinoma
.
Gastroenterology
149
,
1700
1715
8
Boniface
M.M.
,
Wani
S.B.
,
Schefter
T.E.
,
Koo
P.J.
,
Meguid
C.
,
Leong
S.
et al. 
(
2016
)
Multidisciplinary management for esophageal and gastric cancer
.
Cancer Manage. Res.
8
,
39
44
9
Chu
C.
,
Qu
K.
,
Zhong
F.L.
,
Artandi
S.E.
and
Chang
H.Y.
(
2011
)
Genomic maps of long noncoding RNA occupancy reveal principles of RNA-chromatin interactions
.
Mol. Cell
44
,
667
678
10
Ulitsky
I.
and
Bartel
D.P.
(
2013
)
lincRNAs: genomics, evolution, and mechanisms
.
Cell
154
,
26
46
11
Yang
P.
,
Xu
Z.P.
,
Chen
T.
and
He
Z.Y.
(
2016
)
Long noncoding RNA expression profile analysis of colorectal cancer and metastatic lymph node based on microarray data
.
Onco. Targets Ther.
9
,
2465
2478
12
Zhang
F.
,
Zhang
L.
and
Zhang
C.
(
2016
)
Long noncoding RNAs and tumorigenesis: genetic associations, molecular mechanisms, and therapeutic strategies
.
Tumour Biol.
37
,
163
175
13
Bartonicek
N.
,
Maag
J.L.
and
Dinger
M.E.
(
2016
)
Long noncoding RNAs in cancer: mechanisms of action and technological advancements
.
Mol. Cancer
15
,
43
14
Rinn
J.L.
,
Kertesz
M.
,
Wang
J.K.
,
Squazzo
S.L.
,
Xu
X.
,
Brugmann
S.A.
et al. 
(
2007
)
Functional demarcation of active and silent chromatin domains in human HOX loci by noncoding RNAs
.
Cell
129
,
1311
1323
15
Gutschner
T.
,
Hammerle
M.
and
Diederichs
S.
(
2013
)
MALAT1 – a paradigm for long noncoding RNA function in cancer
.
J. Mol. Med. (Berl.)
91
,
791
801
16
Ratajczak
M.Z.
,
Shin
D.M.
,
Schneider
G.
,
Ratajczak
J.
and
Kucia
M.
(
2013
)
Parental imprinting regulates insulin-like growth factor signaling: a Rosetta Stone for understanding the biology of pluripotent stem cells, aging and cancerogenesis
.
Leukemia
27
,
773
779
17
Thomson
D.W.
and
Dinger
M.E.
(
2016
)
Endogenous microRNA sponges: evidence and controversy
.
Nat. Rev. Genet.
17
,
272
283
18
Lin
C.Y.
and
Xu
H.M.
(
2015
)
Novel perspectives of long non-coding RNAs in esophageal carcinoma
.
Carcinogenesis
36
,
1255
1262
19
Shimada
Y.
,
Imamura
M.
,
Wagata
T.
,
Yamaguchi
N.
and
Tobe
T.
(
1992
)
Characterization of 21 newly established esophageal cancer cell lines
.
Cancer
69
,
277
284
20
Dancy
B.M.
and
Cole
P.A.
(
2015
)
Protein lysine acetylation by p300/CBP
.
Chem. Rev.
115
,
2419
2452
21
Li
L.
,
Feng
T.
,
Lian
Y.
,
Zhang
G.
,
Garen
A.
and
Song
X.
(
2009
)
Role of human noncoding RNAs in the control of tumorigenesis
.
Proc. Natl Acad. Sci. U.S.A.
106
,
12956
12961
22
Hu
L.
,
Wu
Y.
,
Tan
D.
,
Meng
H.
,
Wang
K.
,
Bai
Y.
et al. 
(
2015
)
Up-regulation of long noncoding RNA MALAT1 contributes to proliferation and metastasis in esophageal squamous cell carcinoma
.
J. Exp. Clin. Cancer Res.
34
,
7
23
Tan
D.
,
Wu
Y.
,
Hu
L.
,
He
P.
,
Xiong
G.
,
Bai
Y.
et al. 
(
2016
)
Long noncoding RNA H19 is up-regulated in esophageal squamous cell carcinoma and promotes cell proliferation and metastasis
.
Dis. Esophagus. 30, 1-9
,
30
,
1
9
24
Wang
W.
,
Zhu
Y.
,
Li
S.
,
Chen
X.
,
Jiang
G.
,
Shen
Z.
et al. 
(
2016
)
Long noncoding RNA MALAT1 promotes malignant development of esophageal squamous cell carcinoma by targeting beta-catenin via Ezh2
.
Oncotarget
7
,
25668
25682
25
Wang
X.
,
Li
M.
,
Wang
Z.
,
Han
S.
,
Tang
X.
,
Ge
Y.
et al. 
(
2015
)
Silencing of long noncoding RNA MALAT1 by miR-101 and miR-217 inhibits proliferation, migration, and invasion of esophageal squamous cell carcinoma cells
.
J. Biol. Chem.
290
,
3925
3935
26
Ge
X.S.
,
Ma
H.J.
,
Zheng
X.H.
,
Ruan
H.L.
,
Liao
X.Y.
,
Xue
W.Q.
et al. 
(
2013
)
HOTAIR, a prognostic factor in esophageal squamous cell carcinoma, inhibits WIF-1 expression and activates Wnt pathway
.
Cancer Sci.
104
,
1675
1682
27
Yang
X.D.
,
Xu
H.T.
,
Xu
X.H.
,
Ru
G.
,
Liu
W.
,
Zhu
J.J.
et al. 
(
2016
)
Knockdown of long non-coding RNA HOTAIR inhibits proliferation and invasiveness and improves radiosensitivity in colorectal cancer
.
Oncol. Rep.
35
,
479
487
28
Song
B.
,
Guan
Z.
,
Liu
F.
,
Sun
D.
,
Wang
K.
and
Qu
H.
(
2015
)
Long non-coding RNA HOTAIR promotes HLA-G expression via inhibiting miR-152 in gastric cancer cells
.
Biochem. Biophys. Res. Commun.
464
,
807
813
29
Zhang
E.B.
,
Kong
R.
,
Yin
D.D.
,
You
L.H.
,
Sun
M.
,
Han
L.
et al. 
(
2014
)
Long noncoding RNA ANRIL indicates a poor prognosis of gastric cancer and promotes tumor growth by epigenetically silencing of miR-99a/miR-449a
.
Oncotarget
5
,
2276
2292
30
Naemura
M.
,
Tsunoda
T.
,
Inoue
Y.
,
Okamoto
H.
,
Shirasawa
S.
and
Kotake
Y.
(
2016
)
ANRIL regulates the proliferation of human colorectal cancer cells in both two- and three-dimensional culture
.
Mol. Cell. Biochem.
412
,
141
146
31
Chen
D.
,
Zhang
Z.
,
Mao
C.
,
Zhou
Y.
,
Yu
L.
,
Yin
Y.
et al. 
(
2014
)
ANRIL inhibits p15INK4b through the TGFβ1 signaling pathway in human esophageal squamous cell carcinoma
.
Cell. Immunol.
289
,
91
96
32
Zhang
E.
,
Han
L.
,
Yin
D.
,
He
X.
,
Hong
L.
,
Si
X.
et al. 
(
2016
)
H3K27 acetylation activated-long non-coding RNA CCAT1 affects cell proliferation and migration by regulating SPRY4 and HOXB13 expression in esophageal squamous cell carcinoma
.
Nucleic Acids Res. 45, 3086-3101
33
Zhu
H.
,
Zhou
X.
,
Chang
H.
,
Li
H.
,
Liu
F.
,
Ma
C.
et al. 
(
2015
)
CCAT1 promotes hepatocellular carcinoma cell proliferation and invasion
.
Int. J. Clin. Exp. Pathol.
8
,
5427
5434
34
He
X.
,
Tan
X.
,
Wang
X.
,
Jin
H.
,
Liu
L.
,
Ma
L.
et al. 
(
2014
)
C-Myc-activated long noncoding RNA CCAT1 promotes colon cancer cell proliferation and invasion
.
Tumour Biol.
35
,
12181
12188
35
Li
W.
,
Zheng
J.
,
Deng
J.
,
You
Y.
,
Wu
H.
,
Li
N.
et al. 
(
2014
)
Increased levels of the long intergenic non-protein coding RNA POU3F3 promote DNA methylation in esophageal squamous cell carcinoma cells
.
Gastroenterology
146
,
1714
1726
,
e1715
36
Wu
W.
,
Bhagat
T.D.
,
Yang
X.
,
Song
J.H.
,
Cheng
Y.
,
Agarwal
R.
et al. 
(
2013
)
Hypomethylation of noncoding DNA regions and overexpression of the long noncoding RNA, AFAP1-AS1, in Barrett's esophagus and esophageal adenocarcinoma
.
Gastroenterology
144
,
956
966 e954
37
Yang
X.
,
Song
J.H.
,
Cheng
Y.
,
Wu
W.
,
Bhagat
T.
,
Yu
Y.
et al. 
(
2014
)
Long non-coding RNA HNF1A-AS1 regulates proliferation and migration in oesophageal adenocarcinoma cells
.
Gut
63
,
881
890
38
Chen
X.
,
Han
H.
,
Li
Y.
,
Zhang
Q.
,
Mo
K.
and
Chen
S.
(
2016
)
Upregulation of long noncoding RNA HOTTIP promotes metastasis of esophageal squamous cell carcinoma via induction of EMT
.
Oncotarget
7
,
84480
84485
39
Tong
Y.S.
,
Wang
X.W.
,
Zhou
X.L.
,
Liu
Z.H.
,
Yang
T.X.
,
Shi
W.H.
et al. 
(
2015
)
Identification of the long non-coding RNA POU3F3 in plasma as a novel biomarker for diagnosis of esophageal squamous cell carcinoma
.
Mol. Cancer
14
,
3
40
Li
X.
,
Wu
Z.
,
Mei
Q.
,
Li
X.
,
Guo
M.
,
Fu
X.
et al. 
(
2013
)
Long non-coding RNA HOTAIR, a driver of malignancy, predicts negative prognosis and exhibits oncogenic activity in oesophageal squamous cell carcinoma
.
Br. J. Cancer
109
,
2266
2278
41
Li
J.
,
Chen
Z.
,
Tian
L.
,
Zhou
C.
,
He
M.Y.
,
Gao
Y.
et al. 
(
2014
)
LncRNA profile study reveals a three-lncRNA signature associated with the survival of patients with oesophageal squamous cell carcinoma
.
Gut
63
,
1700
1710
42
Zhang
X.
,
Zhou
L.
,
Fu
G.
,
Sun
F.
,
Shi
J.
,
Wei
J.
et al. 
(
2014
)
The identification of an ESCC susceptibility SNP rs920778 that regulates the expression of lncRNA HOTAIR via a novel intronic enhancer
.
Carcinogenesis
35
,
2062
2067
43
Atmadibrata
B.
,
Liu
P.Y.
,
Sokolowski
N.
,
Zhang
L.
,
Wong
M.
,
Tee
A.E.
et al. 
(
2014
)
The novel long noncoding RNA linc00467 promotes cell survival but is down-regulated by N-Myc
.
PLoS One
9
,
e88112
44
Shiama
N.
(
1997
)
The p300/CBP family: integrating signals with transcription factors and chromatin
.
Trends Cell Biol.
7
,
230
236
45
Gerritsen
M.E.
,
Williams
A.J.
,
Neish
A.S.
,
Moore
S.
,
Shi
Y.
and
Collins
T.
(
1997
)
CREB-binding protein/p300 are transcriptional coactivators of p65
.
Proc. Natl. Acad. Sci. U.S.A.
94
,
2927
2932
46
Sauer
M.
,
Schuldner
M.
,
Hoffmann
N.
,
Cetintas
A.
,
Reiners
K.S.
,
Shatnyeva
O.
et al. 
(
2017
)
CBP/p300 acetyltransferases regulate the expression of NKG2D ligands on tumor cells
.
Oncogene
36
,
933
941
47
Cho
M.H.
,
Park
J.H.
,
Choi
H.J.
,
Park
M.K.
,
Won
H.Y.
,
Park
Y.J.
et al. 
(
2015
)
DOT1L cooperates with the c-Myc-p300 complex to epigenetically derepress CDH1 transcription factors in breast cancer progression
.
Nat. Commun.
6
,
7821
48
Bhan
A.
,
Hussain
I.
,
Ansari
K.I.
,
Kasiri
S.
,
Bashyal
A.
and
Mandal
S.S.
(
2013
)
Antisense transcript long noncoding RNA (lncRNA) HOTAIR is transcriptionally induced by estradiol
.
J. Mol. Biol.
425
,
3707
3722
49
Wang
J.
,
Liu
X.
,
Wu
H.
,
Ni
P.
,
Gu
Z.
,
Qiao
Y.
et al. 
(
2010
)
CREB up-regulates long non-coding RNA, HULC expression through interaction with microRNA-372 in liver cancer
.
Nucleic Acids Res.
38
,
5366
5383
50
Maekawa
Y.
,
Minato
Y.
,
Ishifune
C.
,
Kurihara
T.
,
Kitamura
A.
,
Kojima
H.
et al. 
(
2008
)
Notch2 integrates signaling by the transcription factors RBP-J and CREB1 to promote T cell cytotoxicity
.
Nat. Immunol.
9
,
1140
1147
51
Kung
A.L.
,
Zabludoff
S.D.
,
France
D.S.
,
Freedman
S.J.
,
Tanner
E.A.
,
Vieira
A.
et al. 
(
2004
)
Small molecule blockade of transcriptional coactivation of the hypoxia-inducible factor pathway
.
Cancer Cell
6
,
33
43
52
Tini
M.
,
Benecke
A.
,
Um
S.J.
,
Torchia
J.
,
Evans
R.M.
and
Chambon
P.
(
2002
)
Association of CBP/p300 acetylase and thymine DNA glycosylase links DNA repair and transcription
.
Mol. Cell
9
,
265
277
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