Increasing evidence demonstrates that long non-coding RNAs (lncRNA) play a vital role in the progression of tumors, containing esophageal squamous cell carcinoma (ESCC). LINC00239 was reported as an oncogene in diverse kinds of cancers, whereas its specific role is still unclear in ESCC. In this study, we detected the expression and functional role of LINC00239 in ESCC specimens and cells, and investigated the molecular mechanisms of it. LINC00239 was highly expressed in ESCC tissues and cells, and was related to poor prognosis of patients with ESCC. The proliferation, metastasis, and invasion ability as well as epithelial–mesenchymal transition (EMT) process were all enhanced in LINC00239-overexpressed ESCC cells. LINC00239 was upregulated in TGF-β1–treated ESCC cells. Furthermore, LINC00239 was found to bind directly to the transcription factor c-Myc promoter–binding protein-1 (MBP-1). MBP-1 was detected to inhibit the transcription of c-Myc in ESCC. Moreover, LINC00239 could activate c-Myc transcription through influencing MBP-1–binding ability to c-Myc promoter. These data suggest that LINC00239 may act as an oncogene to promote the transcription of c-Myc by competitively combining with MBP-1 in ESCC, and may serve as a potential target for antitumor therapy in ESCC.

Implications:

LINC00239 may function as an oncogenic lncRNA in ESCC through the LINC00239/MBP-1/c-Myc axis to activate EMT process.

Esophageal cancer ranks the seventh and eighth in the primary cancer morbidity and mortality, respectively, and its prognosis is poor (1, 2). Because of the delayed diagnosis, early metastasis, and the lack of efficient treatments, the five-year survival rate of esophageal cancer is only 25% to 30% (3). The main pathological type of esophageal cancer in China is esophageal squamous cell carcinoma (ESCC). ESCC has the characteristics of familial aggregation, and has a high incidence in some rural areas around Taihang Mountains, such as in Hebei, Henan, and Shanxi provinces (4). Although great progress has been made in the etiology of tumors, the pathogenesis of ESCC has not been fully elucidated.

Long non-coding RNAs (lncRNA), longer than 200 base pairs, have no capacity for coding protein (5). An increasing number of lncRNAs have been demonstrated to play crucial roles in tumor biology as potential therapeutic targets or biomarkers. Recent studies have indicated that lncRNAs participated in the occurrence and development of cancers by acting as RNA decoy, microRNA sponge, RNP component, or molecular scaffold (6, 7). The differentially expressed lncRNAs derived from microarrays were widely reported to play vital roles in tumorigenesis. For instance, lncRNA LUCAT1 could act as a scaffold to promote ubiquitination and reduce the protein level of DNMT1 to induce apoptosis in ESCC cells; LINC00673 could arrest the cell cycle at the G1–S checkpoint through repressing CDKN2C in ESCC; lncRNA SNHG12 induced EMT progress in ESCC via post-transcriptional regulation of BMI1 and CTNNB1 (8–10). Therefore, investigation of the molecular functions and mechanisms of differentially expressed lncRNAs in cancer may help to seek potential therapy targets for cancer treatment.

Recently, we performed an RNA-seq array on TGF-β1–treated ESCC cells to screen the differentially expressed lncRNAs. According to the list of differentially expressed lncRNAs, LINC00239 was found to be highly expressed (log FC >3) in the TGF-β1–treated group. LINC00239 (NR026774.1) is located on human chromosome 14q32.31 and consists of 662 nucleotides. LINC00239 has been validated the oncogenic role in hepatocellular carcinoma (HCC) and acute myelogenous leukemia (AML), and has been found to promote tumor characteristics by activating the PI3K/Akt/mTOR pathway in AML (11, 12). However, the specific mechanisms and functional roles of LINC00239 in ESCC need to be clarified.

In this study, we detected the expression level and functional roles of LINC00239, investigated the mechanisms of LINC00239 in regulating EMT process, and analyzed the potential diagnostic and prognostic value of LINC00239 in ESCC.

Clinical specimens

Clinical tumor specimens and corresponding paired normal tissues were obtained from 123 patients with ESCC who experienced surgical treatment at the fourth hospital of Hebei Medical University during 2008–2012. The patients had not received chemotherapy and radiotherapy before operation. There were 88 males and 35 females, ages from 35 to 80-years-old, and the median age of them was 61. The cancer specimens and normal tissues were separately collected from tumor tissues and the tissues 3–5 cm away from the tumor body. The tumor and normal tissues were divided into two parts for hematoxylin and eosin (H&E) stains and total RNA extraction. All H&E slices were explored by three pathologists. Clinicopathological features and clinical data were collected from pathological diagnosis and hospital records. Family history of upper gastrointestinal cancer was defined as individuals having at least one first-degree or at least two second-degree relatives with esophageal/cardia/gastric cancers. The study was approved by the Ethics Committee of the Fourth Hospital, Hebei Medical University and written informed consents were signed by all patients.

Cell culture

ESCC cell lines KYSE30, TE1, ECA109, KYSE150, KYSE170, and YES2 were purchased from the China Center for Type Culture Collection (CCTCC) and were cultured in RPMI-1640 (Gibco) with 10% FBS (Invitrogen) at 37°C incubator filled with 5% CO2. When cells reached about 80% confluence, starved them overnight in serum-free medium, and then regular cultured and added 10 ng/mL of TGF-β1 (R&D Systems) for 21 days with an interval of 2 days. The cells were separately harvested on 0, 7, 14, and 21 days (13).

RNA extraction and qRT-PCR assay

RNA from cells and tissues was isolated by TRizol reagent (SolarBio) according to the instruction. The first-strand cDNA synthesis Kit (Roche) was used to achieve cDNA followed the instructions. qRT-PCR was performed by Agilent Strata gene Mx3005P (Agilent) using GoTaq qPCR Master Mix (Promega). The β-actin was used as endogenous control. An optimized comparative Ct (2–ΔΔCt) value method was used to measure the relative expression level. The test was performed in duplicate for each sample. The sequences of primers were listed in Supplementary Table S1.

Construction of vectors and cell transfection

The shRNAs of LINC00239 or c-Myc promoter–binding protein-1 (MBP-1) and the over-expression plasmids of pcDNA3.1–00239, pcDNA3.1–00239-Mut, pcDNA3.1-MBP-1, and pcDNA3.1-c-Myc were, respectively, synthesized by Genepharma and GeneScript. The LINC00239 deletion fragments were PCR amplified with pcDNA3.1–00239 as templet and subcloned into pcDNA3.1+ vector. The sequences of the primers were listed in Supplementary Table S1. To construct the expression vectors for His-tagged MBP-1, cDNAs encoding MBP-1 or truncated versions of MBP-1 were synthesized by Generay and subcloned into the pET-28a vector. The promoter region (−2000 to 1bp) of c-Myc was synthesized and subcloned into pGL3-basic vector, named pGL3-c-Myc-WT. The mutants of MBP-1-binding-sites in pGL3-MYC-WT were generated by Q5 Site-Directed Mutagenesis Kit (NEB), named pGL3-MYC-WT-p1, pGL3-MYC-WT-p2, pGL3-MYC-WT-p3, and pGL3-MYC-Mut. The sequences of the primers used in vector mutation were listed in Supplementary Table S1.

The cells were seeded into 6-well plates and cultured until 80% confluence. Transfection was conducted using Lipofectamine 2000 (Invitrogen) following the protocol. The efficiency of transfection was determined by qRT-PCR and fluorescence microscopy after 24 hours of transfection.

Cell proliferation assay

The MTS assay (Promega) and plate colony formation assay were used to monitor the relative cell viability. For MTS assay, cells were harvested after 24 hours transfection and seeded in 96-well plate with 1 × 103 per well and cultured with 100 μL 10% fetal bovine serum RPMI-1640 medium. Then the optical density at 490 nm (OD490) was measured after incubation with 20 μL per well of MTS for 2 hours, and the intervals are 0, 24, 48, 72, and 96 hours. For colony formation assay, 1 × 103 cells per well after transfection for 24 hours were seeded in 6-well plates and cultured for 7 days. Then, the cells were washed with PBS buffer twice, and fixed in 4% paraformaldehyde for 10 minutes. The cells were further stained with crystal violet solution for 20 minutes at room temperature. The clones were counted only when the number of cells in it was more than 50 under the microscope (Leica).

Cell migration and invasion assays

The wound-healing assay was performed after the cells were transfected for 24 hours, and the cells were harvested and seeded in a 6-well plate for regular culture when the confluence reached about 80%. Then, the 200-μL tip was used to form scratches for each well and the medium was replaced with FBS-free RPMI-1640. Photos for each well scratch were taken at 0, 12, and 24 hours by microscope. Cell invasion assay was measured by Matrigel-coated chambers (Corning) with 8-μm pore membranes. The 1 × 106 transfected cells were collected and seeded in the upper chamber with 200-μL pure RPMI-1640 medium. The lower chamber was filled up with 600 μL 20% FBS medium. After 24 hours regular incubation at 37°C, the upside of upper chamber was cleaned and the underside was fixed with 4% paraformaldehyde and stained with crystal violet. The number of cells migrated was counted under the microscope and the invasive rate was measured.

Subcellular fractionation assay

The PARIS Kit Protein and RNA Isolation System (Invitrogen) was used to isolate the nuclear and cytoplasm of ESCC cell lines (ECA109, TE1, KYSE30, KYSE150, KYSE170, and YES2) according to the protocol. The subcellular localization of LINC00239 was detected by the qRT-PCR method.

RNA pull-down assays and mass spectrometry analysis

Biotin-labeled LINC00239, antisense-LINC00239, and LINC00239 deletion fragments were separately transcribed in vitro with T7 RiboMAX Express RNA Production System (Promega) using the linearized plasmids as templates. RNA pull-down assay was conducted with Pierce Magnetic RNA-Protein Pull-Down Kit (Thermo Fisher Scientific) following the manufacturer's instructions. After elution of lncRNA-interacting proteins, the protein mixes were separately for SDS-PAGE testing. Then, the gels were subjected to mass spectrometric analysis. Protein library experiments were performed by bio-company (APTBIO) and the proteins with Base Peak F >300 were selected (Supplementary Table S4).

RNA immunoprecipitation assay

Magna RNA immunoprecipitation (RIP) RNA-Binding Protein Immunoprecipitation Kit (Millipore) was used to detect the RNA binding to specific protein. In brief, the KYSE150 cells were collected (1 × 107) and lysed by lysis buffer. Then, the lysates were divided into two groups and were separately incubated with anti–MBP-1 (11204–1-AP, Proteintech) and anti-IgG (PP64B, Millipore) antibodies, which already incubated with protein A/G magna beads. The RNA was extracted for further qRT-PCR analysis.

Luciferase reporter assay

In KYSE150 cells, pGL3-MYC-WT-p1, pGL3-MYC-WT-p2, pGL3-MYC-WT-p3, pGL3-MYC-WT, and pGL3-MYC-Mut were separately co-transfected with pRL-TK and pcDNA3.1-MBP-1/pcDNA3.1-NC or pcDNA3.1–00239 using Lipofectamine 2000. The firefly and Renilla luciferase activity were measured by Synergy HT (Biotek) with the Dual Luciferase Reporter Assay System kit (Promega) after 48 hours of transfection.

ChIP assay

Cells were collected after transfected for 48 hours, and then ChIP assays were conducted by the ChIP Assay Kit (Upstate) following the protocol. The anti–MBP-1 (11204–1-AP, Proteintech) and anti-IgG (PP64B, Millipore) antibodies were used to immune-precipitate the chromatin complexes, and the samples were analyzed by the qPCR method. The primers for ChIP-qPCR were shown in Supplementary Table S1.

Western blot assay

Western blots (WB) were performed as previously described (14). The primary antibodies were exhibited as follows: anti–β-actin (AC026, ABclonal), anti–E-cadherin (E-AB-35932, Elabscience), anti–N-cadherin (E-AB-64011, Elabscience), anti-Vimentin (bs-0756R, Bioss), anti–c-Myc (E-AB-62131, Elabscience), anti–MBP-1 (11204–1-AP, Proteintech), anti–His-Tag (E-AB-20009, Elabscience), and anti-SNAI1 (E-AB-32931, Elabscience).

Statistical analysis

Results were shown as mean ± SD from at least three independent experiments. Two groups comparison was assessed by the Student t test. The χ2 test was conducted to compare the pathological features and clinical data. Spearman correlation was also used to assess the correlation between two genes. The survival curves were assessed with log-rank test and calculated using Kaplan–Meier analysis. Cox regression analysis was performed to assess the independent factors that could affect survival and recurrence. Bivariate correlations between variables in tissues were calculated by Spearman correlation analysis. All statistical tests were two-sided and the P value of <0.05 was considered statistically significant. *, P < 0.05; **, P < 0.01; and ***, P < 0.001, respectively.

LINC00239 is overexpressed in ESCC and correlated with poor prognosis

To characterize the expression level of LINC00239 in different kinds of cancers, we searched its expression level at StarBase (15) and GEPIA (16) websites that are based on TCGA and GTEx datasets. High expression level of LINC00239 was observed in multiple cancers, including esophageal cancer (Supplementary Fig. S1A–S1C). Higher expression level of LINC00239 in ESCC tissues compared with corresponding normal tissues was detected in clinical specimens (Fig. 1A). The expression level of LINC00239 in ESCC cell lines was also higher than that in the control group (Fig. 1B). Among the 123 ESCC cases, 88 cases (71.5%) manifested that the expression level of LINC00239 in tumor tissues was higher than that in corresponding normal tissues. The TNM stage, depth of invasion, and lymph node metastasis were associated with the relatively high expression level of LINC00239 (the expression level of LINC00239 was 200% higher in tumor specimens than that in corresponding normal tissues; Supplementary Table S2). In addition, univariate analysis revealed that TNM stage, depth of invasion, lymph node metastasis, and the expression level of LINC00239 were correlated with patients' with ESCC overall survival (Supplementary Table S3 and Fig. 1C). Multivariate Cox regression analysis revealed that the lymph node metastasis and expression level of LINC00239 were independent prognostic indicators of patients with ESCC (Supplementary Table S3).

Figure 1.

LINC00239 is overexpressed in ESCC and correlated with poor prognosis. A, The qRT-PCR method was used to detect the relative expression of LINC00239 in 123 paired samples of ESCC. The result was expressed as ΔCt, and each specimen has three duplicate wells in qRT-PCR assay to obtain mean the Ct value. B, The expression level of LINC00239 in ESCC cells (KYSE30, TE1, ECA109, KYSE150, KYSE170, and YES2) compared with Pool measured by qRT-PCR experiment (the control group Pool was 10 randomly selected and well mixed corresponding normal tissues' cDNA). C, Overall survival of patients with ESCC was assessed by Kaplan–Meier analysis with low or high expression of LINC00239, Log-rank P = 0.002739. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 1.

LINC00239 is overexpressed in ESCC and correlated with poor prognosis. A, The qRT-PCR method was used to detect the relative expression of LINC00239 in 123 paired samples of ESCC. The result was expressed as ΔCt, and each specimen has three duplicate wells in qRT-PCR assay to obtain mean the Ct value. B, The expression level of LINC00239 in ESCC cells (KYSE30, TE1, ECA109, KYSE150, KYSE170, and YES2) compared with Pool measured by qRT-PCR experiment (the control group Pool was 10 randomly selected and well mixed corresponding normal tissues' cDNA). C, Overall survival of patients with ESCC was assessed by Kaplan–Meier analysis with low or high expression of LINC00239, Log-rank P = 0.002739. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

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LINC00239 regulates ESCC cells proliferation, migration, and invasion

According to the expression level of LINC00239 in six ESCC cell lines, KYSE150, KYSE170, TE1, and ECA109 cell lines were adopted to conduct knockdown and overexpression analysis. The sh-00239–1/2/3/4 and pcDNA3.1–00239 vectors were synthesized to knockdown and overexpress LINC00239 in KYSE150, KYSE170, TE1, and ECA109 cells (Fig. 2A and B; Supplementary Fig. S2A and S2B). To investigate whether LINC00039 participated in the progression of ESCC, cell functional experiments were performed. The downregulation of LINC00239 could alleviate proliferation, migration, and invasion capability in KYSE150, KYSE170, TE1, and ECA109 cells, whereas upregulation of LINC00239 provided opposite results in the above cell lines (Fig. 2C–F; Supplementary Fig. S2C–S2F).

Figure 2.

LINC00239 regulates ESCC cells proliferation, migration, and invasion. A and B, The knockdown and overexpression efficiency of sh-00239 and pcDNA3.1–00239 transfection in KYSE170 and KYSE150 cell lines determined by the qRT-PCR method; the bar was represented as mean ±SD of three duplicate wells. C and D, Effect of LINC00239 knockdown or overexpression on KYSE170 and KYSE150 cells proliferation ability determined by MTS and colony formation assays, respectively (data represented as mean ± SD of three biological replicate experiments). E and F, Wound-healing and Transwell assays were conducted, respectively, to measure the effect of knockdown or overexpression of LINC00239 on KYSE170 and KYSE150 cells migration and invasion ability (data represented as mean ± SD of three biological replicate experiments). Data represented as mean ± SD of three biological replicate experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 2.

LINC00239 regulates ESCC cells proliferation, migration, and invasion. A and B, The knockdown and overexpression efficiency of sh-00239 and pcDNA3.1–00239 transfection in KYSE170 and KYSE150 cell lines determined by the qRT-PCR method; the bar was represented as mean ±SD of three duplicate wells. C and D, Effect of LINC00239 knockdown or overexpression on KYSE170 and KYSE150 cells proliferation ability determined by MTS and colony formation assays, respectively (data represented as mean ± SD of three biological replicate experiments). E and F, Wound-healing and Transwell assays were conducted, respectively, to measure the effect of knockdown or overexpression of LINC00239 on KYSE170 and KYSE150 cells migration and invasion ability (data represented as mean ± SD of three biological replicate experiments). Data represented as mean ± SD of three biological replicate experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

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LINC00239 is regulated by TGF-β1 and participates in EMT process

Because LINC00239 was found to be highly expressed in TGF-β1–treated ECA109 cells by microarray, and TGF-β1 was reported to activate EMT process in cancers (17, 18), we further detected the role of LINC00239 in EMT process. The expression alteration of EMT-related markers (downregulation of E-cadherin, upregulation of N-cadherin, Vimentin, β-catenin, TWIST1, SMAD2, and SMAD3) was confirmed in TGF-β1–treated ECA109 cells (Supplementary Fig. S3A). Because we selected KYSE150 (with the lowest expression level of LINC00239 in six cell lines) and KYSE170 (with the highest expression level of LINC00239 in six cell lines) cells for subsequent experiments, we also treated KYSE150 and KYSE170 cells with TGF-β1. The expression alteration of EMT related markers was also confirmed in TGF-β1–treated KYSE150 and KYSE170 cells (Fig. 3A and B). LINC00239 was detected to be highly expressed in TGF-β1–treated KYSE150, KYSE170, and ECA109 cells (Fig. 3C and D; Supplementary Fig. S3B).

Figure 3.

LINC00239 is regulated by TGF-β1 and participates in EMT process. A and B, The expression of EMT markers (E-cadherin, N-cadherin, Vimentin, β-catenin, TWIST1, SMAD2, and SMAD3) in TGF-β1–treated (0, 7, 14, and 21 days) KYSE150 and KYSE170 cells compared with non-treated cells, and detected by qRT-PCR assay with three duplicate wells. C and D, The expression of LINC00239 in TGF-β1–treated (0, 7, 14, and 21 days) KYSE150 and KYSE170 cells compared with non-treated KYSE150 and KYSE170 cells, and detected by the qRT-PCR method. E–H, The variations of EMT markers (E-cadherin, N-cadherin, Vimentin, and SNAI1) were measured by qRT-PCR and Western blot methods in LINC00239 knockdown and overexpression cells. The data were represented as mean ± SD with three replicant wells of qRT-PCR assays. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 3.

LINC00239 is regulated by TGF-β1 and participates in EMT process. A and B, The expression of EMT markers (E-cadherin, N-cadherin, Vimentin, β-catenin, TWIST1, SMAD2, and SMAD3) in TGF-β1–treated (0, 7, 14, and 21 days) KYSE150 and KYSE170 cells compared with non-treated cells, and detected by qRT-PCR assay with three duplicate wells. C and D, The expression of LINC00239 in TGF-β1–treated (0, 7, 14, and 21 days) KYSE150 and KYSE170 cells compared with non-treated KYSE150 and KYSE170 cells, and detected by the qRT-PCR method. E–H, The variations of EMT markers (E-cadherin, N-cadherin, Vimentin, and SNAI1) were measured by qRT-PCR and Western blot methods in LINC00239 knockdown and overexpression cells. The data were represented as mean ± SD with three replicant wells of qRT-PCR assays. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

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In addition, the roles of LINC00239 on the expression level of EMT markers, including E-cadherin, N-cadherin, Vimentin, and SNAI1, were further measured. The descent of E-cadherin and the ascent of N-cadherin, Vimentin, and SNAI1 were found in LINC00239-overexpressed KYSE150 and KYSE170 cells, whereas the lessened expression level of N-cadherin, Vimentin, and SNAI1 and the enhanced expression level of E-cadherin were detected in LINC00239 knockdown KYSE170 and KYSE150 cells (Fig. 3E–H), suggesting that LINC00239 may affect the EMT process of ESCC cells.

LINC00239 combines with MBP-1

To explore the potential molecular mechanisms of LINC00239 in ESCC progression, we detected the subcellular localization of LINC00239. LINC00239 was detected to be primarily localized in the nucleus rather than in the cytoplasm of ESCC cells (Fig. 4A). To investigate the proteins that LINC00239 could bind with, RNA pull-down assay and mass spectrometric analysis were carried out in KYSE170 cells that with the highest expression level of LINC00239 among six ESSC cell lines. Sixteen proteins were detected to bind with LINC00239 (Supplementary Fig. S4A and S4B and Supplementary Table S4). As demonstrated previously, LINC00239 was able to regulate the proliferation, migration, and invasion ability of ESCC cells, then MBP-1 was noticed from the proteins list according to its inhibiting effect on the transcription of c-Myc, an oncogenic gene related to cell proliferation and EMT process (Fig. 4B; refs. 19, 20). It should be noted that both MBP-1 and ENO1 protein contain the same mass spectrometric identification sequences; however, the location of the protein band shown in Fig. 4B demonstrated that LINC00239 may bind with MBP-1 (37kDa) instead of ENO1 (48kDa). RNA pull-down and RIP analysis further verified the binding ability of LINC00239 to MBP-1 (Fig. 4C–E). The nucleus lncRNAs regulate the expression of genes by interacting with DNA, chromatin-modifying complexes, and various transcriptional regulators (21). As MBP-1 was reported to act as a transcriptional factor (22), we inferred that LINC00239 might participate in EMT process through interacting with MBP-1 in ESCC.

Figure 4.

LINC00239 combines with c-Myc promoter–binding protein 1 (MBP-1). A, The subcellular localization of LINC00239 in ESCC cell lines (KYSE30, TE1, ECA109, KYSE150, KYSE170, and YES2) with the U6 and GAPDH as the internal reference of nucleus RNA and cytoplasm RNA. The bar was represented as mean ± SD of qRT-PCR three duplicate wells. B, Biotinylated LINC00239 and LINC00239-AS RNA pull-down assay and Coomassie brilliant blue staining gel indicated that LINC00239 could bind to MBP-1 protein; the 37kDa arrow direct the band of MBP-1 and 48kDa arrow direct the band of ENO1. C, Western blot assay conducting with MBP-1 antibody and RNA pull-down products indicated that LINC00239 bound to MBP-1 protein. D and E, RIP assays showed the association of MBP-1 with LINC00239. Immunoprecipitation with antibody IgG was served as the negative control. F, Deletion mapping of the MBP-1–binding domain in LINC00239. Top diagrams, full-length LINC00239 (exception of Poly-A tail) and the deletion fragments. Middle diagram, the transcribed LINC00239 fragments. Bottom diagram, immunoblot analysis for MBP-1 in protein samples pulled down by the different LINC00239 constructs. G, Immunoblot of His-tagged MBP-1 retrieved by biotinylated LINC00239; the structure of MBP-1 is shown below. H and I, Western blot and qRT-PCR assays appraised the expression of MBP-1 in LINC00239 knockdown and overexpression KYSE170 and KYSE150 cells. J, The expression of MBP-1 in ESCC cells (KYSE30, TE1, ECA109, KYSE150, KYSE170, and YES2) compared with the expression of Pool measured by qRT-PCR assays (the control group Pool was 10 randomly selected and well mixed corresponding normal tissues' cDNA). K, The overexpression and knockdown efficiency of MBP-1 transfection in KYSE150 and KYSE170 cell lines determined by the qRT-PCR method. L, The expression level of LINC00239 was measured in MBP-1 overexpression and knockdown KYSE150 and KYSE170 cells. Data are shown as mean ± SD of three replicate experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 4.

LINC00239 combines with c-Myc promoter–binding protein 1 (MBP-1). A, The subcellular localization of LINC00239 in ESCC cell lines (KYSE30, TE1, ECA109, KYSE150, KYSE170, and YES2) with the U6 and GAPDH as the internal reference of nucleus RNA and cytoplasm RNA. The bar was represented as mean ± SD of qRT-PCR three duplicate wells. B, Biotinylated LINC00239 and LINC00239-AS RNA pull-down assay and Coomassie brilliant blue staining gel indicated that LINC00239 could bind to MBP-1 protein; the 37kDa arrow direct the band of MBP-1 and 48kDa arrow direct the band of ENO1. C, Western blot assay conducting with MBP-1 antibody and RNA pull-down products indicated that LINC00239 bound to MBP-1 protein. D and E, RIP assays showed the association of MBP-1 with LINC00239. Immunoprecipitation with antibody IgG was served as the negative control. F, Deletion mapping of the MBP-1–binding domain in LINC00239. Top diagrams, full-length LINC00239 (exception of Poly-A tail) and the deletion fragments. Middle diagram, the transcribed LINC00239 fragments. Bottom diagram, immunoblot analysis for MBP-1 in protein samples pulled down by the different LINC00239 constructs. G, Immunoblot of His-tagged MBP-1 retrieved by biotinylated LINC00239; the structure of MBP-1 is shown below. H and I, Western blot and qRT-PCR assays appraised the expression of MBP-1 in LINC00239 knockdown and overexpression KYSE170 and KYSE150 cells. J, The expression of MBP-1 in ESCC cells (KYSE30, TE1, ECA109, KYSE150, KYSE170, and YES2) compared with the expression of Pool measured by qRT-PCR assays (the control group Pool was 10 randomly selected and well mixed corresponding normal tissues' cDNA). K, The overexpression and knockdown efficiency of MBP-1 transfection in KYSE150 and KYSE170 cell lines determined by the qRT-PCR method. L, The expression level of LINC00239 was measured in MBP-1 overexpression and knockdown KYSE150 and KYSE170 cells. Data are shown as mean ± SD of three replicate experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

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To investigate the specific region of LINC00239 interacts with MBP-1, we performed RNA pull-down assays with truncated LINC00239. The deletion mapping analysis identified a 222-nt region (213–434nt) was required for LINC00239 interaction with MBP-1 (Fig. 4F). Furthermore, protein domain mapping experiments using truncated MBP-1 was conducted, and the 103–223 region of MBP-1 was proved to interact with LINC00239 (Fig. 4G). In addition, upregulation of LINC00239 in KYSE150 and KYSE170 cells, and downregulation of LINC00239 in KYSE170 and KYSE150 cells could not affect the expression level of MBP-1 both in mRNA and protein level (Fig. 4H and I). We also detected the expression level of MBP-1 in ESCC cell lines, and found higher expression level of MBP-1 in ECA109 and KYSE170 cells and the lowest expression level of MBP-1 in KYSE150 cells (Fig. 4J). Then, MBP-1 was knocked down in KYSE170 and KYSE150 cells by transfecting sh-MBP-1–1/2, and was overexpressed in KYSE150 and KYSE170 cells by transfecting pcDNA3.1-MBP-1 (Fig. 4K). Knockdown or overexpression of MBP-1 could not influence the expression level of LINC00239 (Fig. 4L).

MBP-1 regulates the expression of c-myc by binding its promoter region

MBP-1 was reported to inhibit the transcription of c-Myc through binding with the promoter region of c-Myc as a transcriptional factor, and to influence growth, progression, and metastasis of cancers, including breast cancer, gastric cancer, and non–small cell lung cancer (22–26). However, the interaction between MBP-1 and c-Myc in ESCC has not been verified. We found that the expression level of MBP-1 was negatively correlated with c-Myc in ESCC clinical tissues (N = 123; Fig. 5A). The inhibition of c-Myc was observed in MBP-1–upregulated KYSE150 and KYSE170 cells, and elevation of c-Myc was detected in MBP-1–knockdown KYSE170 and KYSE150 cells (Fig. 5B). The web tool AnimalTFDB3.0 was used to find the motif sequence of MBP-1, and three specific binding sites in the c-Myc promoter region were discovered (Fig. 5C). We performed luciferase reporter assays in KYSE150 cell lines that with the lowest MBP-1 expression level among six ESCC cell lines, and the results demonstrated that the luciferase activity of MYC-WT-P1/P2/P3 were impaired by overexpression of MBP-1, whereas MYC-Mut had no response to the alteration of MBP-1 (Fig. 5D). ChIP assays were conducted in MBP-1–upregulated KYSE150 cells, and the binding ability of MBP-1 to c-Myc promoter was further verified (Fig. 5E).

Figure 5.

MBP-1 regulates the expression of c-Myc by binding its promoter region. A, Correlation analysis between MBP-1 and c-Myc in ESCC tissues by the Spearman's method (n = 123). B, The expression of c-Myc was measured by the qRT-PCR method in MBP-1 overexpression and knockdown KYSE150 and KYSE170 cells. C, The motif of MBP-1 was provided by AnimalTFDB3.0 website; the three specific binding sites between MBP-1 and c-Myc were shown. D, Luciferase assays were conducted to assess the luciferase activities of c-Myc promoter in response to MBP-1. E, ChIP assay revealed the direct interaction between MBP-1 and c-Myc promoter, with IgG as the negative control. The experiments are representative of three independent experiments with similar results. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 5.

MBP-1 regulates the expression of c-Myc by binding its promoter region. A, Correlation analysis between MBP-1 and c-Myc in ESCC tissues by the Spearman's method (n = 123). B, The expression of c-Myc was measured by the qRT-PCR method in MBP-1 overexpression and knockdown KYSE150 and KYSE170 cells. C, The motif of MBP-1 was provided by AnimalTFDB3.0 website; the three specific binding sites between MBP-1 and c-Myc were shown. D, Luciferase assays were conducted to assess the luciferase activities of c-Myc promoter in response to MBP-1. E, ChIP assay revealed the direct interaction between MBP-1 and c-Myc promoter, with IgG as the negative control. The experiments are representative of three independent experiments with similar results. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

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LINC00239 functions as an RNA decoy to regulate c-myc transcription by competitively binding with MBP-1 in ESCC

On the basis of above data, we suspected that LINC00239 might affect c-Myc transcription in ESCC by blocking the combination of MBP-1 to c-Myc promoter. A positive correlation between LINC00239 and c-Myc was observed in ESCC clinical tissues (Fig. 6A). We found that c-Myc was highly expressed in ESCC cell lines (Fig. 6B). Overexpression of LINC00239 ascended the mRNA and protein expression level of c-Myc in KYSE150 and KYSE170 cells, whereas knockdown of LINC00239 descended the mRNA and protein expression level of c-Myc in these cells (Fig. 6C and D). To further detect the relationship between LINC00239, MBP-1, and c-Myc, we produced LINC00239-Mut vector that was mutated at 213–434bp region of LINC00239. LINC00239 fortification augmented the expression of c-Myc and had a promotion on the activity of c-Myc transcription, whereas the overexpression of LINC00239-Mut could not influence the expression and transcription of c-Myc. The potentiation of upregulating LINC00239 on c-Myc expression and transcription could be attenuated by MBP-1 enhancement (Fig. 6E–H). ChIP assays further revealed that furtherance of LINC00239 could apparently decline the binding ability of MBP-1 to c-Myc promoter; however, overexpression of LINC00239-Mut could not influence the binding ability of MBP-1 to c-Myc promoter (Fig. 6I). Besides, the vector pcDNA3.1-c-Myc was built up and transfected in KSYE150 cells (Fig. 6J). The c-Myc protein was reported to activate the transcription of SNAI1 in multiple kinds of cancers (27, 28). The expression of SNAI1 was then measured by qRT-PCR and WB assays. Fortification of c-Myc enhanced the expression of SNAI1, and co-transfection of MBP-1 released the upregulation of SNAI1, whereas overexpression of LINC00239 partly reversed MBP-1 caused alleviation of SNAI1 (Fig. 6K and L). In all, LINC00239 boosted c-Myc transcription by blocking MBP-1 and further promoted the transcription of SNAI1.

Figure 6.

LINC00239 functions as an RNA decoy to regulate c-Myc transcription by competitively binding with MBP-1 in ESCC. A, Correlation analysis between LINC00239 and c-Myc in ESCC tissues by the Spearman's method (n = 123). B, The expression of c-Myc in ESCC cells lines (KYSE30, TE1, ECA109, KYSE150, KYSE170, and YES2) compared with control group Pool detected by the qRT-PCR method (the control group Pool was 10 randomly selected and well mixed corresponding normal tissues' cDNA). C and D, The expression of c-Myc was measured by qRT-PCR and western blot (WB) methods in LINC00239 overexpression and knockdown KYSE150 and KYSE170 cells. E and F, The expression of c-Myc was measured by qRT-PCR and WB methods in LINC00239, LINC00239-Mut or MBP-1 and LINC00239 co-transfected KYSE150 cells. G and H, The interplay between LINC00239, MBP-1, and c-Myc was confirmed by luciferase reporter assays. I, ChIP assays validated that LINC00239 fortified the promoter activity of c-Myc by blocking MBP-1; IgG was the normalized control. J, The overexpression efficiency of c-Myc transfection in KYSE150 cell lines determined by the qRT-PCR method. K and L, The qRT-PCR and WB assays were conducted to discover the effect of LINC00239, c-Myc, and MBP-1 on SNAI1. ACTB was used as the normalized control. M and N, MTS and colony formation assays were conducted to measure the effect of LINC00239/MBP-1/c-Myc axis on cell proliferation ability of KYSE150 cells (data represented as mean ± SD of three biological replicate experiments). O and P, Wound-healing assays and Transwell assays were used for evaluating the effect of the LINC00239/MBP-1/c-Myc axis on cell migration and invasion ability of KYSE150 cells (data represented as mean ± SD of three biological replicate experiments). Q, The possible mechanism of LINC00239 regulating ESCC progression. The experiments are representative of three independent experiments with similar results. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 6.

LINC00239 functions as an RNA decoy to regulate c-Myc transcription by competitively binding with MBP-1 in ESCC. A, Correlation analysis between LINC00239 and c-Myc in ESCC tissues by the Spearman's method (n = 123). B, The expression of c-Myc in ESCC cells lines (KYSE30, TE1, ECA109, KYSE150, KYSE170, and YES2) compared with control group Pool detected by the qRT-PCR method (the control group Pool was 10 randomly selected and well mixed corresponding normal tissues' cDNA). C and D, The expression of c-Myc was measured by qRT-PCR and western blot (WB) methods in LINC00239 overexpression and knockdown KYSE150 and KYSE170 cells. E and F, The expression of c-Myc was measured by qRT-PCR and WB methods in LINC00239, LINC00239-Mut or MBP-1 and LINC00239 co-transfected KYSE150 cells. G and H, The interplay between LINC00239, MBP-1, and c-Myc was confirmed by luciferase reporter assays. I, ChIP assays validated that LINC00239 fortified the promoter activity of c-Myc by blocking MBP-1; IgG was the normalized control. J, The overexpression efficiency of c-Myc transfection in KYSE150 cell lines determined by the qRT-PCR method. K and L, The qRT-PCR and WB assays were conducted to discover the effect of LINC00239, c-Myc, and MBP-1 on SNAI1. ACTB was used as the normalized control. M and N, MTS and colony formation assays were conducted to measure the effect of LINC00239/MBP-1/c-Myc axis on cell proliferation ability of KYSE150 cells (data represented as mean ± SD of three biological replicate experiments). O and P, Wound-healing assays and Transwell assays were used for evaluating the effect of the LINC00239/MBP-1/c-Myc axis on cell migration and invasion ability of KYSE150 cells (data represented as mean ± SD of three biological replicate experiments). Q, The possible mechanism of LINC00239 regulating ESCC progression. The experiments are representative of three independent experiments with similar results. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Close modal

LINC00239/MBP-1/c-myc axis induced EMT process in ESCC

Next, we performed cell-functional rescue assays. Upregulation of MBP-1 reversed the impacts of overexpressing c-Myc on cells proliferation, migration, and invasion ability, whereas c-Myc fortification combined with LINC00239 elevation partly normalized MBP-1 overexpression-induced phenomena (Fig. 6M–P). These results suggested that LINC00239 might induce the malignancy of ESCC through regulating c-Myc via MBP-1 (Fig. 6Q).

Esophageal cancer is characterized as one of the most frequent gastrointestinal tumors worldwide (4, 29). ESCC is the most common type of esophageal cancer in China, and is regulated by the dysregulation of gene networks containing both protein-coding genes and noncoding RNAs (30). Emerging quantity of lncRNAs is demonstrated to play important roles in regulating ESCC development (31). In our previous studies, we discovered the roles of lncRNA SEMA3B-AS1 and MEG3 in regulating EMT processes in ESCC, and the expression of them were all changed in TGF-β1–treated ESCC cell lines (32, 33). In the current study, we detected upregulation of LINC00239 in TGF-β1–treated ECA109 cells. High expression of LINC00239 promoted proliferation, migration, and invasion of ESCC cells, suggesting the oncogenic role of LINC00239 in ESCC. In addition, the association of LINC00239 expression with patients' with ESCC prognosis suggested its potential roles as prognostic biomarker in ESCC. LINC00239 was also reported as an oncogene in HCC and AML (11, 12). However, the functional roles and specific molecular mechanisms of LINC00239 need to be further clarified in ESCC.

LINC00239 was primarily located in nuclear. Nuclear lncRNAs may play different roles in regulating epigenetic, transcriptional, and post-transcriptional effect by binding with transcriptional factors or chromatin modifying complexes (34, 35). We revealed that LINC00239 could bind with MBP-1. Transcriptional factor MBP-1 was first identified from cDNA expression library of HeLa cell (36). MBP-1 protein consists of 341 amino acids in length and MBP-1 gene shares 97% similarity with the cDNA of ENO1. MBP-1 and ENO1 are both mapped to the same region of human chromosome 1, and MBP-1 protein is translated from the mRNA that is the isoform 2 of ENO1 gene. As a kind of enolase enzyme, ENO1 is mainly located and functioned in cytoplasm, whereas MBP-1 cannot exert its function as an enzyme, and is mainly located in nucleus and serves as a transcriptional factor (22). The molecular weight of MBP-1 protein is about 37kDa, and ENO1 is about 48kDa. RNA pull-down assay demonstrated a protein band at 37kDa, but not at 48kDa, suggesting the possible interaction of LINC00239 with MBP-1 not ENO1. MBP-1 has been observed to function as a tumor suppressor in various types of cancers. Overexpression of MBP-1 suppresses proliferation, migration, and invasion of different types of cancer cells, including breast, endometrial, and gastric cancers (37–39). However, the expression status and specific roles of MBP-1 in ESCC have not been clarified.

In the present study, MBP-1 was proved to inhibit transcriptional activity of c-Myc in ESCC cells by binding to the promoter region of c-Myc gene. C-Myc, a member of MYC family, is widely reported to be related with cancer cells growth and proliferation, and is regulated by TCF/LEF in cancer pathways to promote cancer cell stemness (40, 41). The role of MBP-1 in regulating transcription of c-Myc has been confirmed in a lot of tumor types, including cervical carcinoma, breast cancer, and leukemia (23, 42, 43). MBP-1 has been found to provide a negative regulatory role on c-Myc transcription via the 1–140 region of amino acid sequence (42). In this study, we found that LINC00239 could activate the expression of c-Myc by blocking the 103–223 region of MBP-1 amino acid sequence. For there is no specific DNA-binding domain reported about MBP-1, we assumed that the DNA-binding domain may exist in 103–140 region of MBP-1 based on our and previous study (41). Generally speaking, transcriptional factors may be regulated by lncRNAs through three main themes, including decoys, scaffold, and guides (44, 45). In this study, rescue assays demonstrated that the inhibitory effect of c-Myc caused by overexpression of MBP-1 could be released by overexpression of LINC00239, implying that LINC00239 may act as an RNA decoy to regulate EMT process through competitively binding with MBP-1 to regulate c-Myc transcription.

The epithelial–mesenchymal transition is a cellular transition process in which epithelial cells acquire mesenchymal-like properties that endow cancer cells with increased migratory and invasive behavior (46). EMT has been viewed as a binary process with distinct cell populations, epithelial and mesenchymal cells, and is often defined by the loss of epithelial marker E-cadherin and gain of mesenchymal marker Vimentin (47, 48). The transcription of E-cadherin can be controlled and affected by various transcriptional factors, such as ZEB1, ZEB2, TWIST1, and SNAI1 (49). SNAI1 was reported to repress the transcription of E-cadherin in breast cancer (50); meanwhile, SNAI1 could be transcriptionally activated by c-Myc in melanoma (28). In the present study, we demonstrated that LINC00239 could be regulated by TGF-β1 and promoted the expression of c-Myc and SNAI1. Combined with the results that co-transfection of MBP-1 and LINC00239 partly normalized the overexpression of MBP1-induced downregulation of SNAI1, we assumed that LINC00239 may regulate EMT process through the LINC00239/MBP-1/c-Myc/SNAI1/E-cadherin axis in ESCC. Further experiments are worthy to confirm the findings.

In summarize, LINC00239 may act as an oncogenic lncRNA in ESCC and promotes esophageal cancer cells proliferation, migration, and invasion. Furthermore, LINC00239 contributes to EMT process by activating c-Myc transcription through binding with MBP-1. LINC00239 may serve as a potential prognostic marker in predicting patients' with ESCC survival and may be a promising biomarker for ESCC treatment.

No disclosures were reported.

X. Liang: Conceptualization, resources, writing–original draft. J. Lu: Data curation, software. Z. Wu: Formal analysis. Y. Guo: Visualization, methodology. S. Shen: Investigation, visualization. J. Liang: Data curation, software. Z. Dong: Funding acquisition, validation. W. Guo: Supervision, funding acquisition, validation.

This study was supported by grants from the National Natural Science Foundation (no. 81772612) and the Natural Science Foundation of Hebei Province (H2020206363 and H2020206368).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/).

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Supplementary data