Introduction
Luminal breast cancer (BC) is one of the most common subtypes of BC, accounting for approximately two-thirds of all BC cases [
1]. It can be divided into two types: luminal A and luminal B. Among them, luminal A is the most prevalent subtype, characterized by the positive expression of the estrogen receptor (ER) and progesterone receptor (PR). Luminal B, similar to luminal A, is characterized by ER
+ and PR
+, but is distinguished by its notably elevated expression of proliferation markers, specifically Ki67. Despite the availability of diverse therapeutic modalities, such as hormonal therapy and chemotherapy, luminal BC continues to present unique challenges [
2]. For instance, patients diagnosed with luminal BC exhibit higher rates of late disease recurrence and mortality than those diagnosed with other subtypes. Therefore, despite the better prognosis of patients with luminal BC, their survival rates do not surpass those of other subtypes in the long term (20 years), leading to a significant societal and medical burden [
3]. Hence, there is an urgent need to investigate the pivotal regulatory factors and signaling networks involved in luminal BC. Such research would contribute to the identification of novel biomarkers and development of targeted therapeutics, offering a solid scientific foundation.
Long noncoding RNA (lncRNAs) are a class of RNA molecules that have a transcription length of over 200 nucleotides and lack coding ability. LncRNAs play a significant role in various genome regulatory processes, including X chromosome inactivation, genomic imprinting, chromatin modification, transcription, splicing, translation, and degradation [
4]. Additionally, they serve as crucial regulators of cell proliferation, differentiation, apoptosis, adhesion, migration, and other biological processes [
5]. Some researchers have postulated that cell type-specific lncRNAs may significantly affect cancer progression. Thus, there is significant research interest in characterizing lncRNA involvement in the pathogenesis of luminal BC to identify novel targets and biomarkers for this subtype. Nevertheless, the precise biological function and underlying mechanisms of specific lncRNAs that are transcriptionally dysregulated in luminal BC remain unclear.
ER positivity is a molecular characteristic of luminal BC, with ERs, including ERα and ERβ. ERα plays a role in promoting the progression of luminal BC [
6]. To identify evidence of ER-associated oncogenic transcription factor (TFs) binding, researchers conducted transcription factor motif enrichment analyses were conducted on both normal and tumor-specific ER cistromes. The results demonstrated that the GRHL2 DNA-binding motif was one of the most enriched. Previous studies have identified GRHL2 as a potential oncogene associated with the progression of ER
+ luminal BCs. The absence of the GRHL2 motif in the normal ER cistrome indicates that GRHL2 may act as a transcription factor that cooperates with ER in driving ER
+ breast tumorigenesis [
7‐
9]. However, it remains unclear how ERα affects GRHL2 expression and its role in the regulatory networks of GRHL2.
In this study, we discovered a significant upregulation of lncRNA NCALD, specifically in luminal BC, which was closely correlated with poor prognosis. In addition, ERα and estrogen transcriptionally upregulate the expression of lncRNA NCALD by binding to the promoters of the SE-lncRNA NCALD. Subsequently, ERα and lncRNA NCALD form a complex that activate the transcription of GRHL2, which ultimately enhances luminal BC proliferation.
Materials and methods
Ethics approval and consent to participate
This study was approved by the ethics committee of Guangdong Provincial People’s Hospital. This animal study was approved by the Institutional Animal Care and Use Committee of Guangdong Provincial People’s Hospital.
Datasets and computational analysis
The Cistrome database browser (
http://cistrome.org/db/#/) [
10] was used to acquire ESR1 ChIP-seq data for luminal BC cell lines (MCF7:33491, T47D:81169, and ZR-75-1:68858), and SP1 ChIP-seq data for luminal BC cell lines (MCF7 Data ID:64351). The hTFtarget database was used to predict TFs for lncRNA NCALD [
11]. RNA-seq analysis was conducted using data downloaded from the GEO database, including datasets GSE70905, GSE58135, and GSE48216 [
12]. We utilized the RNA–Protein Interaction prediction (RPISeq) tool (
http://pridb.gdcb.iastate.edu/RPISeq/index.html) [
13] to predict the protein partners that interact with lncRNA NCALD.
Sample collection
Forty-eight pairs of frozen tumor and paracarcinoma tissues were collected from patients with BC immediately after surgery at the Department of Breast Surgery, Guangdong Provincial People’s Hospital, between December 1, 2020, and December 31, 2021. The patients provided informed consent, and the clinical processes were approved by the Ethics Committees of Guangdong Provincial People’s Hospital. The histological subgroups of the frozen tissues were determined using immunohistochemistry (IHC) to assess the expression of ERα and PR and fluorescence in situ hybridization (FISH) to evaluate the expression of HER2. All BC specimens were confirmed to have tumor cell percentages > 80%.
Cell lines and cell culture
MCF7 (RRID: CVCL 0031) and T47D (RRID: CVCL 0553) cell lines were obtained from Guangzhou Cellcook Biotech Co. Ltd. (Guangzhou, China). HCC1954 (RRID: CVCL 1259) was purchased from Cobioer (Nanjing, China). MDA-MB-231 (RRID: CVCL 0062), MDA-MB-468 (RRID: CVCL 0419), SKBR-3 (RRID: CVCL 0033), MCF10A (RRID: CVCL 0598), and ZR-75-1 (RRID: CVCL 0588) were acquired from Procell Life Science & Technology Co., Ltd. (Wuhan, China). All specimens were cultured in standardized media and conditions. All cell lines were subjected to STR authentication. All experiments were performed using mycoplasma-free cells.
Protein preparation and Western blot assay
Western blot assay was performed as previously described. Briefly, total protein was extracted from cells using RIPA buffer supplemented with protease and phosphatase inhibitors (Beyotime, Beijing). Protein aliquots were separated by SDS-PAGE and visualized using Immobilon Western HRP substrate (Millipore). The primary antibodies used are listed in Additional file
1: Table S1.
In situ hybridization (ISH) and fluorescence in situ hybridization (FISH)
ISH probes (1. 5′-CCTTTATGACCGAAGATGGAACTGAAATGCCATCCTGTTA-3,’ 2. 5'-ACCATCAGGTTGTAATTGTTCAGATCAGAAATTCCCAAGC-3,’ 3. 5′-AGCCTGGCACAGTTGGGCTTGAAACCATCTGTGTAAAGGG-3′) for the detection of digoxin-labeled lncRNA NCALD were designed and synthesized by BOSTER (Boster Biological Technology Ltd., Wuhan, China). A commercial BC tissue array was obtained from OUTDO (SHANGHAI OUTDO BIOTECH CO., Shanghai, China). Two independent pathologists evaluated the staining score of the lncRNA NCALD to determine its expression as either low or high. FISH was performed on the MCF7 and T47D cell lines. Cells were fixed in 4% formaldehyde for 15 min, washed with PBS, treated with pepsin, and dehydrated using ethanol. A FISH probe was added to the air-dried cells in hybridization buffer at a concentration of 40 nM. Following the hybridization process, the slides were thoroughly washed, dehydrated, and treated with Prolong Gold Antifade Reagent containing DAPI to facilitate the detection of nucleic acids. Fluorescence microscopy (DMI4000B, Leica) was used to observe immunofluorescence on the slides. The plugin colocalization finder of Image J was used for the quantitative analysis of colocalization. To evaluate the colocalization results quantitatively, Pearson's correlation coefficient and overlap coefficient, as proposed by Manders [
14,
15], were utilized [
16,
17].
RNAi and cell transfection
Knockdown of lncRNA NCALD in cell lines was accomplished using Ribo lncRNA Smart Silencers, which consist of three pairs of small interfering RNA (siRNA), three pairs of antisense oligonucleotides, and short hairpin RNA (shRNA). ESR1 knockdown cell lines were generated using siRNA and shRNA. Ribo-lncRNA Smart Silencers were developed by RiboBio Co. (Guangzhou, China). The siRNA and shRNA sequences used are listed in Supplementary Table S2. Lipofectamine 3000 transfection reagent (Thermo Fisher, L3000015) was used to transfect 50 nM siRNA or 2 μg shRNA in Opti-MEM (Gibco, #31985070) using reverse transfection techniques. RNA and proteins were harvested at 48 and 72 h, respectively, following siRNA transfection.
Lentiviral packaging and infection
Lentivirus packaging was performed by Shanghai Genechem (China). To knock down lncRNA NCALD and ESR1, we cloned two validated hairpins (lncRNA NCALD target sequences: ACCGAAGATGGAACTGAAA, ACAGTTGGGCTTGAAACCAT; ESR1 target sequence: TGATCAAACGCTCTAAGAA, TCCGAGTATGATCCTACCA) specifically targeting the human lncRNA NCALD and ESR1 transcript into the hU6-MCS-CMV-puromycin vector. To achieve overexpression of GRHL2 and ESR1, the complete transcripts of these genes were inserted into the CMV-MCS-3FLAG-SV40-puromycin vector. Similarly, the lncRNA NCALD was inserted into the CMV-MCS-SV40-puromycin vector. The infected MCF7 and T47D cells were cultured in selection medium containing 1.0 μg/mL puromycin for 72 h post-infection. The cells were collected for downstream analyses.
RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA was extracted from both BC cell lines and tissues using an RNAeasy Fast Tissue/Cell Kit (TIANGEN, Beijing). Complementary DNA (cDNA) was reverse-transcribed using the RiboSCRIPT mRNA/lncRNA qRT-PCR Starter Kit (Ribobio, Guangzhou, China). SYBR Green PCR Master Mix was used to amplify the cDNA aliquots. GAPDH served as an endogenous control. The sequences of the sense and antisense primers used are listed in Additional file
1: Table S3.
Cytoplasmic/nuclear RNA fractionation
Cytoplasmic and nuclear RNA fractionation was performed using a cytoplasmic and nuclear RNA purification kit (Norgen, 21,000). RNA was reverse-transcribed and quantified by qRT-PCR. The results for the lncRNA NCALD are illustrated in Additional file
1: Fig. S1.
Flow cytometry analysis
MCF-7 and T-47D cells stably transfected with shRNA or overexpression vector were collected. We conducted a cell cycle arrest assay using propidium iodide staining followed by flow cytometry as previously described [
18]. Three biological replicates were established, and the data collected accurately represented all the experiments.
Cell proliferation and colony formation assays
A total of 2000 cells were seeded in 96-well plates with 10% FBS-containing culture medium and incubated for 5 days for the CCK-8 assay. Cell viability was assessed daily using a CCK-8 kit. Approximately 1000 cells were seeded into six-well plates and cultured for 2 weeks to conduct the colony formation assay. Colonies were fixed with 4% paraformaldehyde and stained with crystal violet. The ImageJ software was used to count the colonies. Replicates were established for all the assays.
Annexin V apoptosis assay
After a 15-min staining period at room temperature in the dark using FITC and PE (BD Biosciences), the cells were analyzed by flow cytometry within 1 h (BD Biosciences). Cell apoptosis analysis was performed using FlowJo software (BD Biosciences).
Xenograft in a nude mouse model
Considering the limited capacity of human cell lines to develop tumors in mice, 5-week-old NOD-SCID mice with severe immune deficiencies were selected. Under the guidance of animal protection organizations and principles of animal welfare, the reduction, refinement, replacement, and responsibility (4R) principle is currently promoted for animal experiments. Based on a previous study [
18], five mice that satisfied these criteria were carefully selected for biological replication and 4R. Four different types of MCF7 cells, each with distinct treatment, were injected into female NOD-SCID mice. Tumor volume was measured and evaluated approximately two–three times per week using the formula: length × width
2 × 0.5. Animals were used in research conducted at the Guangdong Provincial People's Hospital after obtaining approval from the Institutional Animal Care and Use Committee.
Dual-luciferase reporter assay
The promoter of the lncRNA NCALD was defined as the region located 2 kb upstream of the transcription start site. To investigate the promoter activity of lncRNA NCALD, we generated promoter regions of different lengths: − 1460 to + 100, 1/2, 1/4, and 1/8 of the full-length promoter region. Since the − 1460 to + 100 region contains an ERα-binding site, we constructed a mutant − 1460– + 100 region to investigate the regulatory role of ERα in lncRNA NCALD. The truncated wild-type and mutated sequences of the promoter were cloned into a pGL3-basic vector (Promega) to generate recombinant plasmids. The plasmids were constructed by Synbio Technologies (Suzhou, China). MCF7 and T47D cells were seeded into 24-well plates. After 24 h, 0.2 ng phRL-CMV, 0.5 μg of recombinant plasmids, and 50 nM ESR1 siRNA were transfected using Lipofectamine 3000 (Thermo Fisher Scientific). The cells were harvested 48 h post-transfection and luciferase activity was measured using a dual-luciferase reporter assay system (Promega). Three technical replicates were used for each assay.
Electrophoretic mobility shift assays (EMSAs) and supershift assays
Biotin labeling was performed on double-stranded DNA oligonucleotides containing the ERα-binding site discovered in the lncRNA NCALD promoter (sense:5′-GACCGGGCCCAGTGACCCGCGGGC-3′), as well as ERα-mutated oligonucleotides (sense:5′-GACCATATACAATTTAAAGCGGGC-3′). Biotin labeling was performed by Viagene Biotech Inc. (Changzhou, China). The assay was performed as previously described. To perform the supershift assay, 1 µg of anti-ERα antibodies and normal goat IgG were preincubated with 4 µg of nuclear extracts in binding buffer for 20 min at ambient temperature before adding the oligonucleotide probes.
ChIP-qPCR assay
DNA–protein complexes were immunoprecipitated from wild-type MCF7 cells, MCF7 cells with stable knockdown of ERα and lncRNA NCALD, and MCF7 cells treated with an RNA Pol II inhibitor (α-amanitin) using a ChIP assay kit (Millipore, Bedford, MA, USA). The complexes were immunoprecipitated using polyclonal antibodies against ERα (Santa Cruz, sc-8002X) or normal IgG (non-specific DNA-binding control). Immunoprecipitated DNA was purified and subjected to real-time PCR analysis. The primer sequences used for the real-time PCR amplification of GRHL2 were forward (F):5′-TTTCCCAGTCCAAAGGTCAC-3′ and reverse (R):5′-GCTCAGCCTCTTCCTGTCTC-3.’ The primer sequences used for real-time PCR amplification of the lncRNA NCALD were as follows: F:5′-CCAGCTACCCATGAAGCTACTTAG-3′ and R:5′-CATCTCTTGCCTGCCTTCAGA-3.’ The real-time PCR results were analyzed using the 2−ΔΔCT method.
RNA pulldown assay
Biotin-labeled RNA was transcribed and purified using the Biotin RNA Labeling Mix and T7 RNA polymerase. Biotin-labeled RNA was heated in RNA immunoprecipitation (RIP) buffer for one hour and combined with the cytoplasmic extract. The mixture was subsequently incubated with streptavidin–agarose beads. TRIzol reagent was used to extract the beads for qPCR analysis.
RNA immunoprecipitation (RIP)
Cells transfected with the specified plasmids were harvested and lysed using lysis buffer. RIP buffer supplemented with magnetic beads was then added to the cell lysates. They were conjugated to the beads either by conjugation with antibodies or anti-IgG as a control. Isolated RNA was digested with DNase I and proteinase K, followed by immunoprecipitation. Subsequently, enrichment of purified RNAs was evaluated using qRT-PCR.
Statistical analysis
Statistical analysis was conducted using SPSS version 20.0 (SPSS, Inc., Chicago, IL, USA) and Prism version 8.0 (GraphPad Software, Inc., La Jolla, CA, USA) statistical software packages. Student's t-test was used to analyze the experimental data, and all data are presented as the mean ± standard deviation unless otherwise specified. Statistical significance was set at a threshold of P < 0.05.
Discussion
In this study, we discovered that lncRNA NCALD was promoted by ERα. Specific enrichment promotes the proliferation of luminal BCs. Moreover, overexpression of lncRNA NCALD was identified as a standalone prognostic indicator in luminal BC. Moreover, it has been demonstrated that the lncRNA NCALD promotes proliferation in luminal BC by interacting with ERα, which stimulates the transcription of GRHL2.
Cell-type-specific lncRNAs play pivotal roles in cancer progression [
23]. For instance, six lncRNAs are specifically expressed in hepatocellular carcinoma (HCC) cells but not in the liver or other normal tissues. These lncRNAs have been found to influence HCC [
24]. Furthermore, researchers have investigated the subtype-specific and cell-type-specific expression of lncRNAs in BC through bioinformatics analysis [
25]. However, their potential clinical value remains unexplored. In this study, we used tissues and cell lines representing diverse molecular subtypes of BC to identify potential lncRNAs. Our findings revealed that lncRNA NCALD was specifically overexpressed in luminal BC. Furthermore, enrichment of lncRNA NCALD was associated with high-grade BC. It also functions as an independent prognostic marker for luminal BC, indicating its potential as a useful marker for this subtype of BC. The potential reasons for this expression pattern may be related to two factors. On the one hand, previous studies suggested that super enhancers play prominent roles in driving the expression of cell-type-specific genes. It was observed that a super enhancer cluster in luminal and her2 breast cancers but not in normal and basal-like breast cancers was discovered within lncRNA NCALD’s promoter and gene body. The ChIA-PET data of MCF7 cells suggested that the super enhancer interacted with the promoter of lncRNA NCALD (data not shown). Furthermore, in cancer, lncRNA expression is often regulated by the occupancy of subtype-specific TFs at their promoter regions. For instance, the regulation of DSCAM-AS1 by ERα renders it specific to cell lines expressing ERα and exerts an impact on cellular functions [
26]. Furthermore, FOXA1 and STAT3 have been shown to elevate the expression of lncRNAs in luminal BC [
27,
28]. To identify the master transcription factor of lncRNA NCALD, the promoter region of lncRNA NCALD was analyzed using ChIPBase [
19] and hTFtarget [
11]. The prediction score for ERα was high, and its expression was positively correlated with lncRNA NCALD. ERα, a molecular characteristic of luminal BC, primarily affects cellular function via two pathways. It can activate MAPK, PI3K, and other signaling pathways to enhance cell proliferation and suppress apoptosis [
29]. Furthermore, it serves as a critical transcription factor capable of activating oncogene transcription by binding to the ER element [
30]. In our study, we identified a novel finding that the lncRNA NCALD is responsive to estrogen stimulation, and its transcription is activated by ERα. These results provide an explanation for the reduced expression of the lncRNA NCALD in HER2 or Basal-like cells, which is potentially caused by the absence of ERα regulation.
As lncRNA NCALD is associated with the clinical characteristics and prognosis of luminal BC, we propose that it may exert an impact on the cellular functions of luminal BC. The results indicated that the lncRNA NCALD enhanced cell proliferation in vivo and in vitro. This finding was consistent with the GEO data, which demonstrated that the expression of lncRNA NCALD was elevated in BC tissues compared to that in non-malignant or normal breast tissues.
Our ultimate objective was to identify the specific target gene of lncRNA NCALD and elucidate its regulatory mechanisms. We analyzed the correlation between the lncRNA NCALD and other genes. We observed a positive correlation between GRHL2 and NCALD lncRNAs. While the correlation between GRHL2 and lncRNA NCALD is not particularly strong, both genes are specifically expressed in luminal BC and exhibit a close relationship with ERα [
31]. Furthermore, our findings implied that the overexpression of GRHL2 stimulated cell proliferation in luminal BC, which was in line with previous studies demonstrating the subtype-specific role of GRHL2 in BC [
10].
Furthermore, our findings revealed that inhibiting the expression of the lncRNA NCALD reversed the cellular effects caused by the overexpression of GRHL2. GRHL2 is a tumorigenic ER-cooperating transcription factor [
22]. Several studies have indicated that downregulation of ERα can suppress the expression of GRHL2. However, the rationale behind the lack of upregulation of GRHL2 expression upon ERα overexpression has not been investigated [
22,
31]. Our study demonstrated that knockdown of either lncRNA or ERα downregulated GRHL2, whereas only the concurrent overexpression of lncRNA NCALD and ERα upregulated GRHL2 expression. The lncRNA NCALD serves as a scaffold for recruiting ERα to the GRHL2 promoter. Moreover, we discovered interactions between the lncRNA NCALD and ERα regions, which suggested that in the future, we can develop inhibitors targeting lncRNA NCALD fragment, which destroy its scaffold effect and prevent ERα from binding to GRHL2 promoter region, and thus inhibit the expression of GRHL2 to provide a new therapy for luminal breast cancer.
Some limitations of the study include the relatively small sample size and further validation in larger cohorts and in vivo models would be necessary in our future research. Besides, the expression of lncRNA NCALD in serum of luminal breast cancer patients was also unclear and its implications for liquid biopsy applications should be explored in the following study.
In summary, we discovered a new lncRNA, NCALD, that exhibited specific upregulation in luminal BC and displayed a positive correlation with poor prognosis. The unique expression pattern of the lncRNA NCALD can be attributed primarily to ERα stimulation. lncRNA NCALD facilitates cell proliferation by regulating GRHL2. It can also directly interact with ERα and bind to the GRHL2 promoter, consequently affecting its expression.
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