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Introduction

There are >1.6 million new cases of breast cancer diagnosed each year, making it the most common malignancy among women worldwide (1). Breast cancer metastasis is a life-threatening occurrence that constitutes the primary cause of breast cancer-related deaths (2). Previous studies have aimed to identify the molecular processes that cause breast cancer metastasis and to search for new targets that can stop its progression (3,4). The specific underlying mechanisms driving cell migration and invasion remain largely unknown, despite the crucial role they serve in the metastatic development of breast cancer.

According to previous studies, long non-coding RNAs (lncRNAs) serve an important role in human cancer (5,6). Numerous types of human cancer cells exhibit dysregulated expression of these ncRNA molecules, which are ~200 nucleotides in length and are transcribed from the corresponding gene locus (7,8). LncRNAs exert biological functions through the epigenetic modulation of transcriptional and post-transcriptional regulation in physiological and pathological activities. Notably, lncRNAs are essential regulators in human cancer and have a substantial association with tumor prognosis (9,10).

The lncRNA differentiation antagonizing non-protein coding RNA (DANCR) was initially reported to be associated with osteoclastogenesis and osteoblast differentiation in osteoporosis (11). It has also been reported that DANCR is overexpressed in neoplastic tissues and functions as an oncogenic lncRNA by facilitating the development of tumors (12). Previous research has reported that lncRNAs are involved in the epigenetic modification of multiple diseases, including both transcriptional and post-transcriptional regulation (5,6). In the present study, the association between DANCR and E2F transcription factor 1 (E2F1) expression is assessed. However, to the best of our knowledge, there has been no research to date reporting the mechanism underlying the relationship between DANCR and E2F1 in cancer; therefore, the present study aimed to elucidate the oncogenic function of DANCR in cancer.

Materials and methods

The Cancer Genome Atlas (TCGA) data access and analysis

DANCR and other gene expression profiles and clinicopathological factors were downloaded from TCGA (Data Release v21.0; December 10, 2019) (https://tcga-data.nci.nih.gov/). To analyze the expression levels of DANCR, the data were dichotomized using the median expression as the cut-off point, defining ‘high’ as expression levels at or above the median and ‘low’ as expression levels below the median. The log-rank test was used to assess differences in survival between different groups of patients.

Human DNA methylation profiles were determined experimentally using the Illumina Infinium Human Methylation 450 platform (Illumina, Inc.). β-values were obtained from Johns Hopkins University and TCGA Genome Characterization Center of the University of Southern California (California, USA). DNA methylation and β-values of each array probe were measured across all samples using the BeadStudio (version 3.2; Illumina, Inc.) software. The β-values varied from 0–1, which represented the ratio of the methylated bead type to the combined locus intensity. Consequently, increased β-values indicated increased DNA methylation levels, while decreased β-values indicated decreased DNA methylation levels. These values were treated as continuous variables. Additionally, information about histone modifications of the DANCR locus was retrieved from the ENCODE database (ENCODE 3 Nov 2018) (https://www.encodeproject.org/).

Transcription factor (TF) prediction and guilt-by-association analysis

The JASPAR database (https://Jaspar.bind.Ku.dk/) was used to predict TFs and the chromatin immunoprecipitation (ChIP)-seq data obtained from ENCODE served as the basis for analysis.

According to previous investigations, the guilt-by-association methodology was adopted to discern genes that exhibited a positive correlation with DANCR (13–16). The pairwise Pearson correlation was applied to measure the correlation between DANCR expression and that of other genes. As a result, solely the genes that exhibited a favorable association with a correlation coefficient of R≥0.3 and attained statistical significance at a level of P<0.05 were chosen. Subsequently, DAVID Functional Annotation Bioinformatics Microarray Analysis (https://david.ncifcrf.gov/tools.jsp) was performed based on Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and Gene Ontology (GO) terms. P<0.05 and a gene count threshold of 4 were used to identify significant GO terms and KEGG pathways.

Human tissue specimens and ethics statement

The present study acquired five paired samples of breast cancer tissues and their respective adjacent noncancerous tissues (mean patient age, 58±13 years) as well as information on patient sex, age, patient number and molecular subtypes from The First Hospital of Harbin Medical University (Harbin, China). Patients with DANCR information were eligible for the present study, which met the ethical standards of The Declaration of Helsinki.

For RNA isolation, tissue samples were collected from patients with breast cancer and healthy controls (n=5/group) and immediately cryopreserved at −80°C after resection. Before surgery, written informed consent was obtained from all patients. Patients with a history of adjuvant chemotherapy, immunotherapy, radiotherapy, tumor recurrence, bilateral tumor, metastatic disease or other previous tumors were excluded from the present study. The Ethics Committee of the First Affiliated Hospital of Harbin Medical University (Harbin, China) granted ethical approval after obtaining written informed consent from patients (ethical approval no. 202438).

Cell culture

MCF7 (cat. no. HTB22), MDA-MB-231 (cat. no. CRM-HTB-26), MCF10A (cat. no. CRL-10317) and 293T (cat. no. ACS-4500) cell lines were obtained from the American Type Culture Collection. MCF7, MCF10A and 293T cell lines were maintained in DMEM (Gibco; Thermo Fisher Scientific, Inc.) and MDA-MB-231 was maintained in L15 (Gibco; Thermo Fisher Scientific, Inc.) for culture purposes. Before the experiments, the cells were tested to rule out the presence of Mycoplasma. Cells were cultured at 37°C in a humidified environment with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and 5% CO2.

Cell transfection

Small interfering (si)RNAs targeting DANCR, siDANCR negative control (NC; cat. no. siN0000001- 1–5), siRNAs targeting E2F1, siE2F1 NC, miRNA mimics, miRNA inhibitors, mimic NC (cat. no. miR1N0000001-1-5) and inhibitor NC (cat. no. miR2N0000001-1-5) were purchased from Guangzhou RiboBio Co, Ltd. The DANCR plasmid (pcDNA3.1-DANCR) was constructed by Shanghai GeneChem Co., Ltd. for DANCR overexpression. The experimental procedure involved seeding 1×105 cells into a 6-well plate and transfecting cells when they reached a confluence of 70–80%. Transfection was performed using JetPRIME (Polyplus-transfection SA) and different masses of nucleic acids as follows: DANCR overexpression plasmid (2,000 ng); miRNA inhibitor (50 nmol); siRNA (100 nmol); and miRNA mimic (100 nmol) for 24 h at 37°C and 5% CO2. The transfected cells were employed for subsequent experimentations at 24 h after transfection. The transfection efficiency was determined using reverse transcriptase-quantitative (RT-q) PCR and western blotting analysis at 24 and 48 h after the transfection, respectively. The sequences used were as follows: siDANCR#1 sense, 5′-CCAACUAUCCCUUCAGUUA-3′ and antisense, 5′-UAACUGAAGGGAUAGUUGG-3′; siDANCR#2 sense, 5′-GUGCUUCAUGUUCACCUUU-3′ and antisense, 5′-AAAGGUGAACAUGAAGCAC-3′; siE2F1 sense, 5′-GGGAGAAGUCACGCUAUGA-3′ and antisense, 5′-UCAUAGCGUGACUUCUCCC-3′; hsa-miR-34c-5p mimic sense, 5′-AGGCAGUGUAGUUAGCUGAUUGC-3′ and antisense, 5′-GCAAUCAGCUAACUACACUGCCU-3′; and hsa-miR-34c-5p inhibitor, 5′-GCAAUCAGCUAACUACACUGCCU-3′.

Lentiviral infection

DANCR-specific short-hairpin (sh)RNA-targeting coding sequences and non-targeting negative control sequences (Shanghai GeneChem Co., Ltd.) were cloned into GV112 vectors (Shanghai GeneChem Co., Ltd.) to produce DANCR knockdown vectors. The shDANCR sequence used in the experiment was as follows: 5′-GTGCTTCATGTTCACCTTT-3′. The NC of shDANCR sequence used in the experiment was as follows: 5′-TTCTCCGAACGTGTCACGT-3'. A 3rd generation system was used to package the lentivirus. To produce lentiviral particles, TransIT-LT1 (Mirus Bio, LLC) was used to co-transfect the expression vector with the packaging plasmid pHelper 1.0 (Shanghai GeneChem Co., Ltd.) and the envelope plasmid pHelper 2.0 (Shanghai GeneChem Co., Ltd.) into 293T cells at 37°C and 5% CO2 for 24 h. The supernatant was collected 48 h post-transfection and concentrated using ultracentrifugation at 75,500 × g and 4°C for 90 min and resuspended in an appropriate volume of OptiMEM (Gibco; Thermo Fisher Scientific, Inc.). MCF7 and MDA-MB-231 cells were seeded in 6-well plates at a density of 1×105 cells/well and cultured in DMEM with 10% FBS at 5% CO2 at 37°C and transfecting cells when they reached a confluence of 70–80%. The cells were transfected with 10 µg lentiviral plasmids, 7.5 µg packaging plasmid and 5 µg envelope plasmid at a multiplicity of infection of 10 using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) the following day when the cells were ~70% confluent. MCF7 and MDA-MB-231 cells were cultured at 37°C for 6 h followed by replacement of the medium. The cells were incubated at 37°C for 48 h and subsequently treated with puromycin (selection, 2 µg/ml; maintenance, 1 µg/ml; Calbiochem; Merck KGaA) at 37°C and 5% CO2 for 72 h to select transfected clones. Stable knockdown of DANCR was confirmed by RT-qPCR.

Proliferation assay

Cell proliferation was assessed using the 5-ethynyl-2′-deoxyuridine (EdU) assay (Beyotime Institute of Biotechnology), following the manufacturer's instructions. After a 4-h incubation with EdU, the cells were fixed in 4% formaldehyde for a duration of 30 min at room temperature. Then, a glycine solution (2 mg/ml) was applied for 5 min, followed by permeabilization using 0.5% Triton X-100 for 10 min at room temperature. A 1X reaction cocktail (from EdU kit) was then administered and the nuclei were stained with DAPI for 10 min at room temperature. The cells were then imaged under a fluorescence inverted microscope.

Wound healing assay

A wound healing assay was conducted to examine MCF7 and MDA-MB-231 cell migration. Initially, 1×105 cells were seeded in a 6-well plate and incubated until they reached ~100% confluence at 5% CO2 and 37°C. The cells were then scratched using a pipette tip to create wounds and the concentration of FBS in the cell culture medium was reduced from 10% to 1%. An image of the scratched area was immediately taken (0 h). The plates were then placed at 37°C and 5% CO2. After overnight (16-h) incubation, another image was taken of the same scratched area using light microscopy (magnification, ×10). The width of the scratch at 24 h was calculated as a percentage of the width at 0 h. The cell migration rate was analyzed by the edge-finding method using Image J 1.8.0 (National Institutes of Health).

Invasion assay

MCF7 (1×105) and MDA-MB-231 (1×105) cells were incubated in serum-free DMEM or RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc.) medium using the BRAND® Insert with Matrigel (cat. no. BR782806; MilliporeSigma; Merck KGaA) precoated for 4 h and 37°C. Serum-free medium was added to both upper chambers and medium containing 10% FBS was added to the lower cell chamber. After 24 h of incubation at 37°C, RPMI-1640 or DMEM containing 10% FBS was added to the lower chamber and non-invading cells were removed with a cotton swab. Cells that successfully traversed the membrane were immobilized in 100% methanol for 30 min and subsequently dehydrated by air drying. Furthermore, these cells were stained with a 0.5% crystal violet solution at room temperature for 30 min and counted manually after imaging using a light microscope.

RNA preparation and RT-qPCR

TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) was used to extract total RNA from cells and tissue. Subsequently, PrimeScript RT Reagent Kit (Takara Bio, Inc.) was used to reverse transcribe 0.5 µg total RNA. FastStart Universal SYBR Green Master Mix (Roche Applied Science) and gene-specific primers were used, with U6 or GAPDH used as internal controls. The ABI 7500 Fast Real-time PCR Detection System (Applied Biosystems; Thermo Fisher Scientific, Inc.) was used for RT-qPCR. To normalize the results, expression levels relative to GAPDH and U6 were assessed using the 2−ΔΔCq method (17). The miRNA stem-loop real-time PCR kit (Guangzhou RiboBio Co., Ltd.) was used to quantify miR-34c-5p and U6. The primer sequences were as follows: DANCR forward (F), 5′-CGGAGGTGGATTCTGTTAGGGACA-3′ and reverse (R), 5′-AGAGGGCTTCGGTGTAGCAAGT-3′; E2F1 F, 5′-GGACCTGGAAACTGACCATCAG-3′ and R, 5′-CAGTGAGGTCTCATAGCGTGAC-3′; U6 F, 5′-CTCGCTTCGGCAGCACAT-3′ and R, 5′-TTTGCGTGTCATCCTTGCG-3′; and GAPDH F, 5′-GTCTCCTCTGACTTCAACAGCG-3′ and R, 5′-ACCACCCTGTTGCTGTAGCCAA-3′. The thermocycling conditions were as follows: Initial denaturation at 95°C for 10 min; 40 cycles at 95°C for 15 sec and 60°C for 30 sec.

Western blotting analysis

Protease and phosphatase inhibitors (Beyotime Institute of Biotechnology) were added to the RIPA (Beyotime Institute of Biotechnology) to lyse the MCF7 and MDA-MB-231 cells. The protein concentrations were measured using a BCA Protein Assay Kit (Beyotime Institute of Biotechnology). Subsequently, equivalent quantities of protein (30 µg) was loaded per lane onto a 10% SDS gel, resolved using SDS-PAGE and translocated onto nitrocellulose membranes, followed by blocking with 5% milk in TBST (0.1% Tween) at room temperature for 2 h. Primary antibodies targeting E2F1 (1:1,000; cat. no. 3742; CST Biological Reagents Co., Ltd.) and β-actin (1:1,000; cat. no. TA09; OriGene Technologies, Inc.) were incubated with the membranes at 4°C overnight. Primary antibodies were then washed away with TBST (0.1% Tween), incubated with IRDye 800CW-conjugated secondary antibodies (1:10,000; cat. no. 92632210/92632211; LI-COR Biosciences) for 1 h at room temperature and visualized using the Odyssey® Imaging System and Image Studio (LI-COR Biosciences).

ChIP assay

The ChIP detection kit (cat. no. bes5001, Guangzhou Bersinbio Co., Ltd.) was used with slight modifications to the manufacturer's protocol (18). Cells were crosslinked using 1% formaldehyde and the reaction was terminated by adding glycine to a final concentration of 0.125 M. The E2F1 antibodies (1:100) were used to immunoprecipitate DNA from sonicated cell lysates, with IgG (BD Biosciences) as the negative control. To detect the binding sites of E2F1, the immunoprecipitated DNA was amplified by RT-qPCR, as aforementioned. Subsequently, 3% agarose gel electrophoresis was used to analyze the amplified fragments. Chromatin at a concentration of 10% was used as a control input before immunoprecipitation. The primer sequences were as follows: DANCR site 1 F, 5′-CGGGGATTGGTAGGTAGCC-3′ and R, 5′-CTGGAGAGGTCGGGTAGC-3′; DANCR site 2 F, 5′-GGTGTCCCCACGAGCTTTG-3′ and R, 5′-AAATTGTTACGGTGCCCAGAC-3′; and DANCR site 3 F, 5′-CGCCCCGCTCAGGATCTTC-3′ and R, 5′-GCACTCACCGCGCAACTC-3′.

Dual-luciferase reporter assay

To produce reporter vectors with binding sites for miRNA, the complete 3′ untranslated regions (UTRs) of human DANCR and E2F1 were cloned. The full-length 3′UTR fragments from DANCR and E2F1 were amplified by PCR as aforementioned and inserted into the reporter luciferase expression vector pmiR-RB with Not1-Xho1 sites. 293T cells were cultured in DMEM containing 100 µg/ml penicillin/streptomycin and 10% FBS and miR-34c-5p mimic was used for the luciferase assay. 293T cells were transfected at 40–50% confluence using JetPRIME (Polyplus-transfection SA). For transfection, 20 mol/l hsa-miR-34c-5p mimic or NC, alongside 0.5 mg DANCR or E2F1 plasmid (Guangzhou RiboBio Co., Ltd.), was utilized. The results of the luciferase activity were determined after 48 h using a luminometer (GloMax™ 20/20; Promega Corporation) and a dual-luciferase reporter assay kit (Promega Corporation). The direct oligomer synthesis technique was employed to produce a nucleotide-substitution mutation in the 3′UTRs of DANCR and E2F1. Normalization of the firefly luciferase results was performed by comparison with Renilla luciferase activity.

Prediction of lncRNA localization and fluorescence in situ hybridization (FISH)

The lncRNA subcellular localization predictor database (lncLocator; http://www.csbio.sjtu.edu.cn/bioinf/lncLocator/) was used to analyze the subcellular localization of DANCR. FISH was performed using a RNA-FISH kit (Bes1002; Guangzhou BersinBio Biotechnology Co., Ltd.) as described in the manufacturer's instructions (19). The lncRNA probe for DANCR was obtained from Guangzhou Bersinbio Biotechnology Co., Ltd. MCF7 and MDA-MB-231 cells were harvested and fixed in 4% formaldehyde. After denaturation, probes were hybridized with cells for 20 h at 42°C. A DAPI stain was then applied to the nuclei and cells were observed using a fluorescence microscope (LSM800; Carl Zeiss AG).

Statistical analysis

All statistical tests were conducted utilizing R (version 3.5.3; RStudio, Inc.). The data were presented as the mean ± standard deviation from three independent replicates. The expression of DANCR in cancer tissues compared with normal tissues were analyzed using a paired t-test. The unpaired Student's t-test was used to compare variances between two groups. For evaluating statistical differences among multiple groups, a one-way ANOVA was performed, followed by a Tukey's Honestly Significant Difference post hoc test. P<0.05 was considered to indicate a statistically significant difference.

Results

Upregulation of DANCR correlates with poor prognosis in breast cancer

TCGA data were downloaded and analyzed to evaluate the expression levels of DANCR in human cancer. These results demonstrated that the expression levels of DANCR were upregulated in pan-cancer samples when compared with normal samples (Fig. 1A; Table SI). Additionally, it was demonstrated that the expression levels of DANCR were notably elevated in various types of malignancy, including bladder urothelial carcinoma (BLCA), cholangiocarcinoma, colon adenocarcinoma, liver hepatocellular carcinoma (LIHC), lung adenocarcinoma, lung squamous cell carcinoma, prostate adenocarcinoma (PRAD), rectum adenocarcinoma, uterine corpus endometrial carcinoma (UCEC) and breast invasive carcinoma (BRCA), when compared with their noncancerous tissue counterparts (Fig. 1B; Table SI). To verify this observation, RT-qPCR analysis of breast cancer tissues was conducted and yielded results consistent with the aforementioned findings (Fig. 1C; Table SII).

Figure 1. Expression of DANCR is upregulated and correlated with a poor prognosis. (A) Expression level of DANCR was significantly higher in pan-cancer tissues when compared with normal tissues. (B) The Cancer Genome Atlas results demonstrated that the expression level of DANCR was significantly higher in UCEC, SKCM, READ, PRAD, LUSC, LUAD, LIHC, ESCA, COAD, CHOL, BRCA and BLCA and markedly higher in THYM, STAD, PAAD and CESC compared with normal tissues. (C) Expression level of DANCR was higher in breast cancer tissues compared with normal tissue (n=5/group). Higher DANCR expression level had a significant positive association with (D) poor overall survival, a marked positive association with poor disease-specific survival and a significant positive association with (E) advanced stage in pan-cancer. (F) Higher expression level of DANCR was significantly related to poorer overall survival in BRCA. (G) DANCR expression was upregulated in triple-negative and HER-2 enriched subtypes. *P<0.05; **P<0.01; *****P<0.0001. DANCR, differentiation antagonizing non-protein coding RNA; UCEC, uterine corpus endometrial carcinoma; THYM, thymoma; STAD, stomach adenocarcinoma; SKCM, skin cutaneous melanoma; READ, rectum adenocarcinoma; PRAD, prostate adenocarcinoma; PAAD, pancreatic adenocarcinoma; LUSC, lung squamous cell carcinoma; LUAD, lung adenocarcinoma; LIHC, liver hepatocellular carcinoma; ESCA, esophageal carcinoma; COAD, colon adenocarcinoma; CHOL, cholangiocarcinoma; CESC, cervical squamous cell carcinoma and endocervical adenocarcinoma; BRCA, breast invasive carcinoma; BLCA, bladder urothelial carcinoma; TNB, triple-negative breast cancer; Her-2, human epidermal growth factor receptor 2; LumA, luminal A; LumB, luminal B; HR, hazard ratio; PAM 50, PAM 50 molecular subtype; num(T), number of tumor tissues; num(N), number of normal tissues.

The relationship between DANCR expression levels and patient survival status was further investigated using a log-rank test and Kaplan-Meier analysis based on pan-cancer samples. The results demonstrated that, compared with patients with low DANCR expression, patients with high expression had markedly lower disease-specific survival and overall survival (OS) (Fig. 1D). Furthermore, DANCR expression was significantly higher in advanced TNM stages than in stage I (Fig. 1E). Patients with high DANCR expression had significantly lower OS compared with patients with low DANCR expression, especially in patients with breast cancer (Fig. 1F). Furthermore, compared with other subtypes of breast cancer, the expression of DANCR was significantly upregulated in the triple negative subtype (Fig. 1G). In addition, various cancer types present in the TCGA data cohort were examined. The analysis demonstrated a notable correlation between elevated levels of DANCR in BRCA, kidney renal clear cell carcinoma, LIHC, sarcoma and skin cutaneous melanoma and a decline in OS rates (Figs. 1F and S1). However, the expression of DANCR in Head and Neck squamous cell carcinoma (HNSC), Kidney Chromophobe, Pheochromocytoma and Paraganglioma, Stomach adenocarcinoma, Thyroid carcinoma (THCA) and UCEC was not linked to OS (Fig. S1).

DANCR promoted tumor growth

To elucidate the role of DANCR in the phenotype of breast cancer cells, loss-of-function experiments were conducted on MCF7 and MDA-MB-231 breast cancer cells. After transfection with siRNA, the expression of DANCR in these cells was significantly decreased compared with control cells (Fig. 2A). In addition, after stable silencing of DANCR, the EdU assay showed a decrease in proliferation compared with control cells, whereas the opposite results were determined after overexpression of DANCR (Fig. 2B). These findings suggested that DANCR knockdown can inhibit breast cancer cell proliferation, implying a potential oncogenic role in tumorigenesis.

Figure 2. DANCR promoted tumor growth. (A) Transfection efficacy of siDANCR in MCF7 and MDA-MB-231 cells. (B) EdU assay results demonstrated that silencing DANCR inhibited tumor cell proliferation, while overexpression of DANCR promoted tumor cell proliferation. Magnification, ×100. (C) Transwell invasion assay results showed that knockdown of DANCR inhibited the invasion ability of tumor cells, while overexpression of DANCR promoted tumor cell invasion ability. Magnification, ×200. (D) Wound healing assay results demonstrated that silencing DANCR suppressed tumor cell migration, while overexpression of DANCR promoted tumor cell migration. Magnification, ×100. Data were presented as the mean ± standard deviation (n=3). **P<0.01; ***P<0.001; *****P<0.0001. DANCR, differentiation antagonizing non-protein coding RNA; si, small interfering RNA; sh, short hairpin RNA; NC, negative control; CON, blank control group; EdU, 5-ethynyl-2′-deoxyuridine.

Subsequently, an evaluation was conducted to determine the impact of DANCR on breast cancer cell migration and invasion through the implementation of wound healing and Transwell assays. The findings demonstrated that the introduction of shDANCR into MCF7 and MDA-MB-231 cells via stable transfection significantly inhibited both invasive and migratory cells compared with control cells (Fig. 2C and D). By contrast, the overexpression of DANCR significantly enhanced the invasion (Fig. 2C) and migration (Fig. 2D) of breast cancer cells in vitro compared with controls.

DANCR modulated breast cancer cell progression by regulating miR-34c-5p

The typical regulatory role of lncRNAs is to host miRNAs and serve as a miRNA ‘sponge’. In the present study, LncLocator and FISH were used to predict and verify the subcellular localization of DANCR. The findings demonstrated that the cytoplasm of both the MCF7 and MDA-MB-231 cell lines contained DANCR (Fig. 3A and B). Moreover, bioinformatics analysis demonstrated that miR-34c-5p contained a sequence matching DANCR at the 3′UTR (Fig. 3C). According to the luciferase assay results, miR-34c-5p mimics caused a significant decrease in luciferase activity compared with negative controls, which indicated a strong affinity between DANCR 3′UTR and miR-34c (Fig. 3C). Compared with MCF10A cells, the expression of miR-34c-5p was significantly diminished in breast cancer cell lines (Fig. 3D). Additionally, the upregulation of DANCR resulted in the downregulation of miR-34c-5p (Fig. S3A). The present study indicated that miR-34c-5p was negatively correlated with DANCR and targeted the 3′UTR of DANCR.

Figure 3. DANCR modulated breast cancer cells progression via regulating miR-34c-5p. (A) Lnclocator predicted that DANCR was mainly located in the MCF7 cytoplasm. (B) Distribution of DANCR (green) in MCF7 and MDA-MB-231 cells as detected by fluorescence in situ hybridization assay (nuclei stained blue with DAPI). Magnification, ×400. (C) A luciferase reporter assay was used to assess the interactions between miR-34c-5p and its binding sites or mutated binding sites in the 3′ untranslated regions of DANCR in 293T cells. (D) Expression levels of miR-34c-5p were higher in breast cancer cell lines compared with the MCF10A cell line. (E) Overexpression of DANCR upregulated the expression level of miR-34c-5p. (F) Knockdown of DANCR downregulated the expression level of miR-34c-5p. (G) The proliferation capacity of miR-34c-5p mimic, E2F1 siRNA, DANCR shRNA and DANCR shRNA + miR-34c inhibitor transfected MCF7 and MDA-MB-231 cells were assessed by EdU assay. Magnification, ×100. (H) The invasion capacity of miR-34c-5p mimic, E2F1 siRNA, DANCR shRNA and DANCR shRNA plus miR-34c inhibitor transfected MCF7 and MDA-MB-231 cells were assessed by Transwell assay. Magnification, ×200. (I) The migratory capacity of miR-34c-5p mimic, E2F1 siRNA, DANCR shRNA and DANCR shRNA + miR-34c inhibitor transfected MCF7 and MDA-MB-231 cells were assessed by wound healing assay. Magnification, ×100. Data were presented as the mean ± standard deviation (n=3). *P<0.05; **P<0.01; ***P<0.001; *****P<0.0001. DANCR, differentiation antagonizing non-protein coding RNA; E2F1, E2F transcription factor 1; WT, wild type; mut, mutant; miR, microRNA; si, small interfering RNA; sh, short hairpin RNA; NC, negative control; CON, blank control group.

Given that DANCR has previously been identified as an oncogenic lncRNA in breast cancer (20), how it interacts with miR-34c-5p to control pathological course was studied. Loss-of-function and rescue tests were conducted in MCF7 and MDA-MB-231 cells. According to these experiments, siDANCR transfection of MCF7 and MDA-MB-231 cells significantly enhanced the expression of miR-34c-5p compared with negative controls (Fig. 3E and F). In addition, EdU analysis demonstrated that, compared with in the shDANCR transfection group, shDANCR co-transfection with the miR-34c-5p inhibitor significantly reversed the reduced proliferation of MCF7 and MDA-MB-231 breast cancer cells (Fig. 3G). Furthermore, DANCR knockdown significantly reduced cell migration and invasion rates compared with controls and this effect was significantly reversed by the miR-34c-5p inhibitor (Fig. 3H and I). Overall, these findings suggested a potential antagonistic relationship between DANCR and miR-34c-5p in breast cancer, whereby miR-34c-5p inhibition may help to lessen the inhibitory effect of DANCR knockdown on cell proliferation, migration and invasion.

E2F1 acted as the target of miR-34c-5p

DANCR expression was shown to be significantly positively correlated with E2F1 gene expression by bioinformatics analysis (Fig. 4A) and downstream gene pathways, such as E2F1, were found to be significantly related to cell cycle and cell division (Fig. 4B). Additionally, it was expected that the 3′UTR of E2F1 contained binding sites complementary to miR-34c-5p (Fig. 4C). The binding of miR-34c-5p to the 3′UTR of E2F1 was confirmed using a luciferase reporter assay (Fig. 4C). These results showed that transfection with the miR-34c inhibitor significantly increased E2F1 expression in MCF7 and MDA-MB-231 cells compared with negative controls (Fig. 4D). In addition, when knocking down DANCR, both E2F1 mRNA and protein expression levels were significantly decreased compared with controls (Fig. 4E and F). However, this effect was reversed when cells were co-transfected with shDANCR and miR-34c-5p inhibitor (Fig. 4E and F).

Figure 4. E2F1 acted as the target of miR-34c-5p. (A) The Cancer Genome Atlas data showed that E2F1 positively correlated with DANCR in breast cancer. (B) GO and KEGG enrichment analysis of key genes of DANCR downstream pathway. (C) A luciferase reporter assay was used to assess the interactions between miR-34c-5p and its binding sites or mutated binding sites in the 3′ untranslated regions of E2F1 in 293T cells. (D) Downregulated miR-34c-5p promoted E2F1 expression. (E) Knockdown of DANCR upregulated the expression levels of E2F1. (F) E2F1 expression changes cells transfected with in miR-34c mimic, DANCR shRNA and DANCR shRNA + miR-34c inhibitor as detected by Western blotting. Data were presented as the mean ± standard deviation (n=3). **P<0.01; ***P<0.001; *****P<0.0001. DANCR, differentiation antagonizing non-protein coding RNA; E2F1, E2F transcription factor 1; WT, wild type; MUT, mutant; miR, microRNA; si, small interfering RNA; sh, short hairpin RNA; KEGG, Kyoto Encyclopedia of Genes and Genomes; GO, Gene Ontology; NC, negative control; CON, blank control group.

These findings showed a negative association between the expression of miR-34c-5p and the expression of E2F1 and DANCR. There was also a strong positive association between E2F1 expression and DANCR expression. Consequently, this study potentially elucidated a regulatory pathways of DANCR/miR-34c-5p/E2F1 in breast cancer.

E2F1 regulated the DANCR promoter region and activated its expression

The present study proposed that TF binding to the promoter region of the lncRNA DANCR enhanced its expression. To validate this, the online database JASPAR was used to predict TF binding to the DANCR promoter region (Fig. 5A). In addition, GO and KEGG analyses were performed to identify the basic functions and pathways of DANCR promoter region-binding TFs via DAVID Functional Annotation Bioinformatics Microarray Analysis. These results suggested that the transcription factor E2F1, which may serve an important role in breast cancer, could bind to the functional region of the DANCR promoter (Fig. 5B). E2F1 may bind to the promoter region of DANCR in MCF7 and MDA-MB-231 breast cancer cell lines. This theory was supported by ChIP-seq data for E2F1 from ENCODE (Fig. 5C). Additionally, the ChIP data demonstrated that the DANCR promoter had a far higher affinity for E2F1 than for IgG (Fig. 5D).

Figure 5. E2F1 regulated the DANCR promoter region and activates its expression. (A) JASPAR predicted that E2F1 could bind with the promoter of DANCR. (B) GO/KEGG enrichment analysis of transcription factors predicted to bind to DANCR. (C) ENCODE validated the binding region of E2F1 to the DANCR promoter. (D) Chromatin immunoprecipitation assay showed the binding of E2F1 and DANCR promoter region in MCF7 and MDA-MB-231 cells. Data were presented as the mean ± standard deviation (n=3). (E) Hypomethylation occurred at the DANCR promoter locus in breast and pan-cancer tissues. (F) The methylation level at the DANCR promoter site was inversely proportional to DANCR expression in breast and pan-cancer samples. (G) H3K27ac and H3K4me3 were significantly enriched at the DANCR locus in breast cancer cell lines. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. DANCR, differentiation antagonizing non-protein coding RNA; E2F1, E2F transcription factor 1; KEGG, Kyoto Encyclopedia of Genes and Genomes; GO, Gene Ontology; PRAD, prostate adenocarcinoma; HNSC, head and neck squamous cell carcinoma; BRCA, breast invasive carcinoma; BLCA, bladder urothelial carcinoma; bp, base pairs; IP, immunoprecipitation group; DM500, DNA marker.

The pathway by which DANCR is upregulated in human cancer was also investigated and it was predicted that cancer samples would have less DNA methylation enrichment at the DANCR promoter locus compared with normal samples. In contrast to non-cancer tissues, these findings demonstrated that the DANCR promoter region was significantly hypomethylated in pan-cancer, BRCA, BLCA, PRAD and HNSC samples (Figs. 5E and S2; Table SIII). Moreover, in pan-cancer samples and most types of malignancies tested, the degree of DANCR promoter region methylation was significantly inversely correlated with DANCR expression (Figs. 5F and S2; Tables SIII and SIV).

Additionally, the role of H3K27ac and H3K4me3 modifications in upregulating DANCR expression in human cancer was investigated. These results demonstrated that both modifications were significantly enriched at the DANCR locus in breast cancer cell lines (Fig. 5G), which indicated their potential contribution to the upregulation of DANCR in breast cancer. Taken together, these findings suggested that TF binding, hypomethylation and H3K27ac and H3K4me3 modifications may collectively contribute to the upregulation of DANCR in breast cancer.

Discussion

In human esophageal squamous cell carcinoma and osteosarcoma, the lncRNA DANCR serves a crucial role, according to previously published studies (21,22). DANCR upregulation has been identified as a prognostic biomarker in both pancreatic and colorectal cancer (23,24). In our previous research, we reported that DANCR serves a tumor-promoting role both in vivo and in vitro in breast cancer (20). Mechanistically, DANCR targets miR-216a-5p, thereby regulating the expression of proteins such as Nanog, SOX2 and OCT4 to promote breast cancer progression. In the present study, the results demonstrated that partial inhibition of DANCR significantly reduced the proliferation, migration and invasion of MDA-MB-231 and MCF7 cells, which may have an impact on the tumor cell cycle.

Previous research has indicated that lncRNAs are involved in the epigenetic modification of multiple diseases, including via both transcriptional and post-transcriptional regulation (25,26). Notably, it has been reported that lncRNAs serve a crucial role in the etiology, proliferation, metastasis and recurrence of tumors (27–30). Currently, the most well-known mechanism by which lncRNAs participate in disease etiology is by serving as ceRNAs for miRNAs (31,32). In the present study, through computational approaches, it was determined that miR-34c-5p bound to the 3′UTR of DANCR. Furthermore, functional experiments showed the ability of miR-34c-5p to inhibit the oncogenic effects of DANCR on breast cancer cells, which suggests that the regulatory effect of DANCR on these cells may be mediated by its sequestration of miR-34c-5p. The regulation of gene expression by miRNAs occurs through binding to particular locations in the 3′UTR of target mRNAs and lncRNA-miRNA crosstalk is essential for the indirect control of gene expression (33–36). When lncRNAs are in the cytoplasm, they participate in modulating mRNA stability, regulating mRNA translation, serving as ceRNAs and functioning as precursors of miRNAs (31). In the present study, the subcellular grading confirmed that DANCR is mainly located in the cytoplasm of breast cancer cell lines. Furthermore, the inhibitory effect of shDANCR on E2F1 could be reversed by miR-34c-5p inhibitor, which suggests that DANCR could potentially enhance the expression of E2F1 by absorbing miR-34c-5p as ceRNA in cancer. Additionally, the present study demonstrated the involvement of the DANCR/miR-34c-5p/E2F1 feedback loop in the occurrence and development of breast cancer (Fig. 6). By modulating the expression of the oncogene E2F1, DANCR may serve as a potential therapeutic target for breast cancer. The results of the present study suggested that manipulating the expression of DANCR, as a ceRNA, may competitively regulate the expression of E2F1, thereby regulating the biological function of breast cancer cells. However, in the future, additional in vivo experiments and clinical trials are necessary to clarify the potential of DANCR as a therapeutic target for breast cancer.

Figure 6. Role of DANCR in breast cancer progression in the present study. The transcription factor E2F1 could bind to the promoter region of DANCR, activating its transcription. DANCR, in turn, promoted the progression of breast cancer by competitively binding with miR-34c-5p, which led to the upregulation of E2F1 expression. DANCR, differentiation antagonizing non-protein coding RNA; E2F1, E2F transcription factor 1; miR, microRNA.

The results of the present study showed that knocking down E2F1 reduced cell migration and invasion. Therefore, E2F1, as a protein regulated by DANCR, may be involved in the G1/S transition of the mitochondrial cell cycle, regulation of the cell cycle, regulation of the mitochondrial cell cycle and G2/M transition of the mitochondrial cell cycle. E2F1, as a TF that binds to the DANCR promoter region, may be involved in the regulation of transcription of G1/S phase of the mitotic cell cycle, interphase of the mitotic cell cycle, cell cycle regulation, G1/S transition of the mitotic cell cycle and the G1 phase of the mitotic cell cycle.

Based on the analysis of the binding region between the TF E2F1 and DANCR, further analysis was performed on biological behaviors that E2F1 may be involved in by activating DANCR transcription. These results demonstrated that the regulation of DANCR by the TF E2F1 may be involved in various types of cancer, such as small cell lung cancer, prostate cancer and thyroid cancer, as well as signaling pathways, such as the MAPK signaling pathway, the Toll-like receptor pathway and the Wnt signaling pathway. Furthermore, E2F1 may be involved in regulating cell proliferation, cell death and the cell cycle. Therefore, it could potentially be hypothesized that the TF E2F1 binds to the DANCR promoter functional region and serves an important role in cancer.

Our previous study reported that miR-34c-5p can mediate liver and lung metastasis of breast cancer by regulating G protein-coupled receptor kinase interacting protein 1 (GIT1) (37). In addition, GIT1 can mediate the development of estrogen receptor-negative breast cancer by regulating the Notch pathway (38). Another study reported that UBTF promotes melanoma cell proliferation and cell cycle progression by promoting GIT1 transcription, thereby activating MEK1/2-ERK1/2 signaling pathways (39). Therefore, E2F1 and miR-34c-5p may regulate the progression of breast cancer by affecting the cell cycle.

Further validation of the present findings are required to address a number of limitations of the present study. Firstly, it is imperative to note that the assessment of DANCR expression levels in a limited sample size of breast cancer specimens requires further investigation with a more extensive sample size to establish a definitive correlation between the expression of DANCR/miR-34c/E2F1 and clinical parameters. Furthermore, it is necessary to confirm the protein concentration of E2F1 and the expression level of DANCR across a wider range of cell models and in vivo studies. Finally, the genes of interest identified in the present study via bioinformatics analysis, which may have the potential to become significant contributors to the development of breast cancer, warrant further investigation to validate the results.

Nevertheless, the present study demonstrated the potential role of DANCR in breast cancer progression. The formation of the DANCR/miR-34c/E2F1 feedback loop, facilitated by the binding of E2F1 to the DANCR promoter region, may provide a promising avenue for the precise treatment of breast cancer in the future.

Supplementary Material

Supporting Data

Supporting Data

Acknowledgements

Not applicable.

Funding

This work was supported by the First Affiliated Hospital of Harbin Medical University Fund for Distinguished Young Medical Scholars (grant no. 2021J17), the Beijing Medical Award Foundation (grant no. YXJL-2021-0302-0287), and the Postgraduate Research and Practice Innovation Program of Harbin Medical University (grant no. YJSCX2023-63HYD).

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors' contributions

SY and WT confirm the authenticity of all the raw data. SY and WT designed and directed experimental studies. SY, LT, JD, LJ, PX, WZ and WT performed sequencing data analysis. SY, LT, JD, LJ, PX and WZ performed experimental studies. SY, LJ and WT acquired patient samples. WT provided financial support. SY and WT provided project guidance. SY, LT, JD, LJ, PX, WZ and WT wrote the manuscript, which all authors reviewed. All authors read and approved the final version of the manuscript.

Ethics approval and consent to participate

The First Hospital of Harbin Medical University granted ethical approval for the present study and all patients provided their written informed consent (approval no.: 202438; Harbin, China).

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References