Background
Breast cancer is one of the most prevalent malignancies among women worldwide [
1,
2]. According to a 2022 statistical report, approximately 2.9 million new cases of breast cancer are diagnosed globally, constituting 31% of all malignant neoplasms in women [
1]. Triple-negative breast cancer (TNBC), accounting for 10-20% of all cases of breast cancer [
3], has a higher recurrence rate, poorer prognosis, and greater rate of malignancy than other subtypes [
4]. Unfortunately, targeted and endocrine therapies have proven ineffective in TNBC patients [
5]. Therefore, it has become crucial to explore the mechanism underlying these diseases to identify promising therapeutic targets.
Long non-coding RNAs (LncRNAs) are transcripts of more than 200 nucleotides, that have no protein coding potential [
6]. Researchers have demonstrated the significant involvement of lncRNAs in the progression and metastasis of multiple malignant tumors [
7‐
10]. LncRNAs exhibit diverse molecular mechanisms, such as acting as miRNA sponges [
11], enhancing enhancer activity by physically binding to enhancer regions, binding with transcription factors to regulate gene expression [
12], binding to antisense mRNAs to influence their post-transcriptional regulation [
13], and interacting with one or more protein chaperones [
14]. Furthermore, lncRNAs can act as scaffold molecules for the organization of macromolecular complexes [
15]. These findings highlight the importance of lncRNAs in cancer research and underscore their potential as therapeutic targets.
RNA-binding proteins (RBPs) are involved in a wide variety of cellular processes, including gene expression regulation [
16‐
19]. Heterogeneous nuclear ribonucleoprotein K (HNRNPK) is a vital member of the HNRNP family that functions as an RNA and DNA binding protein and engages in complex interactions with molecular partners to participate in chromatin remodeling, transcription, mRNA splicing, export, and translation processes [
20‐
23]. Previous work has demonstrated that HNRNPK interacts with transcription factors or transcriptional repressor proteins involved in transcription, including p53, YBX1, and Zik1 [
20,
24,
25]. Despite some progress, the molecular partners and functional mechanisms of HNRNPK in TNBC have yet to be thoroughly explored.
The tricarboxylic acid (TCA) cycle is a central hub for cellular energy metabolism, macromolecular synthesis, and redox balance. Recent studies have provided profound insights into the pivotal role of the TCA cycle in cancer metabolism, suggesting that it is a promising focal point for cancer therapy [
26‐
28]. IDH2 is an essential rate-limiting enzyme in the TCA cycle that utilizes NADP+ mediators to convert isocitrate and α-ketoglutarate (α-KG) [
29]. IDH2 exerts a significant influence on the progression of multiple cancers, including breast cancer [
30‐
33]. However, the mechanism regulating the transcription of IDH2 has not been elucidated. It is imperative to explore the regulation of the TCA cycle and its rate-limiting enzymes.
In the present study, we revealed that the expression level of LINC00571 was markedly upregulated in TNBC samples and that LINC00571 facilitated the progression of TNBC. Specifically, LINC00571 acted as a scaffold, facilitating the interaction between HNRNPK and ILF2, thereby regulating the transcription of IDH2 and promoting the TCA cycle. Consequently, targeting this mechanism holds is a promising potential therapeutic strategy for TNBC.
Methods
Patients and specimens
Tumor and paracancerous tissues were obtained from TNBC patients who had not undergone chemotherapy or radiotherapy during surgery at Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, and demographic and clinicopathological information was collected concurrently. The Institutional Review Board of Tongji Medical College approved the human tissue study. All procedures were implemented in accordance with the 'Helsinki Declaration' rules. Written informed consent was obtained from all patients. The tumor tissues were confirmed as TNBC by pathological diagnosis, and the tissues were frozen in liquid nitrogen and stored at -80 °C.
Cell culture and reagents
The human normal mammary epithelial cell line (MCF10A) and TNBC cell lines MDA-MB-231 and BT-549 were purchased from the American Type Culture Collection (ATCC, USA). MCF10A cells were cultured in DMEM/F12 medium (Gibco, USA) supplemented with 5% horse serum (Gibco, USA), 20 ng/ml recombinant epidermal growth factor (PeproTech, USA), 0.5 μg/ml hydrocortisone (Sigma, USA), 10 μg/ml insulin (Sigma, USA), 0.1 μg/ml cholera toxin (Sigma, USA), and 1% antibiotics (Invitrogen, USA). MDA-MB-231 cells were cultured in DMEM medium (GIBCO, USA), and BT-549 cells were cultured in RPMI 1640 medium (GIBCO, USA) supplemented with 10% fetal bovine serum (GIBCO, USA). All cells used in this study were cultured in a 5% CO2 incubator at 37 °C.
RNA extraction and quantitative real-time PCR analysis
Total RNA was extracted from cell lines or fresh tissues using TRIzol reagent (Invitrogen, USA). cDNA was synthesized using a PrimeScript RT kit (TaKaRa, Japan). qRT‒PCR was performed using the StepOnePlus Real-Time PCR System (Applied Biosystems, USA). A StepOnePlus Real-Time PCR System (Applied Biosystems, USA) was used for qRT‒PCR. The mRNA internal controls used were GAPDH and U6. Relative RNA abundance was calculated using the standard 2-ΔΔCt method. Relative RNA abundance was calculated using the standard 2-ΔΔCt method with the following formula: ∆Ct = Ct (gene of interest) – Ct (gene of internal controls), ∆∆Ct = ∆Ct (sample) – ∆Ct (control average), and fold gene expression = 2^-(∆∆Ct). On the resulting bar chart, the y-axis shows the fold change in mRNA expression relative to the control. The primers used in the study were synthesized by TSINGKE Biotech (Beijing, China) and are shown in Additional file 13.
Subcellular fractionation
Nuclear and cytoplasmic separation were performed using a PARIS Kit (Life Technologies, USA) according to the manufacturer’s instructions.
RNA fluorescence in situ hybridization
RNA fluorescence in situ hybridization (FISH) was performed on TNBC cell lines using a Fluorescence In Situ Hybridization Kit (RiboBio, China). Antisense and sense probes containing the LINC00571 linker sequence were synthesized. The cells were seeded on confocal dishes. Cells were fixed in 4% paraformaldehyde (Thermo Scientific, Rockford, IL, USA) for 15 minutes, washed three times for 5 minutes per wash with PBS (Sigma, Aldrich, USA), incubated with 0.5% Triton X-100/PBS osmotic solution at room temperature for 5-10 minutes, and then washed three times with PBS for 5 minutes per wash. The cells were incubated with the specific probe at 37 °C overnight. Cell nuclei were stained with DAPI. Fluorescence images were captured using a Nikon A1R-si laser scanning confocal microscope (Nikon, Japan). The probe sequence used for FISH is shown in Additional file
13.
Plasmid construction and stable transfection
Short hairpin RNAs targeting LINC00571, HNRNPK, ILF2, and IDH2 (Additional file
13) were synthesized by TSINGKE and cloned into pLKO.1-puro (Sigma‒Aldrich). Human cDNAs for LINC00571, HNRNPK, ILF2, and IDH2 were synthesized by TSINGKE (Wuhan, China) and cloned into pLVX-puro (Takara Bio, Japan) and p3XFLAG-CMV-10 (Sigma-Aldrich) vectors to construct overexpression plasmids. Truncated HNRNPK was amplified using specific primers (Additional file
13) and subcloned into the p3XFLAG CMV-10 vector. The plasmids were transfected into breast cancer cells by using Lipofectamine 3000 (Invitrogen, USA) according to the manufacturer’s instructions. Stable cell lines were screened using neomycin or puromycin (Invitrogen, USA). Interfering shRNA (shNC) and empty vector were utilized as controls (Additional file
13).
CCK8 assay
A Cell Counting Kit-8 (CCK8) assay was performed according to the manufacturer’s protocol to evaluate the proliferation efficiency of the TNBC cells. At a density of 5000 cells/well, TNBC cells were seeded into 96-well plates. Then, 10% CCK-8 solution (Dojindo, Japan) was added to the 96-well plates, which were incubated in a 37 °C incubator for 1 hour. The spectral absorbance of each well at 450 nm was measured on a microplate reader (Thermo Fisher, USA).
After relevant transfection with the relevant plasmids, TNBC cells (1000 cells/well) were seeded into 6-well plates and incubated at 37 °C. After approximately two weeks, the cell colonies were washed with PBS, fixed with 4% paraformaldehyde (Thermo Scientific, USA) for 10 minutes, stained with 0.2% crystal violet (Solarbio, China) for 15 minutes, imaged and counted.
EdU proliferation assay
The assays were performed with an EdU incorporation assay kit (RiboBio, China) according to the manufacturer’s instructions. Transfected cells (2 × 104 cells/well) were seeded into 96-well plates. After 24 hours, 100 μl of medium containing 50 mM EdU was added to each well, and the cells were then incubated for 2 hours at 37 °C. The cells were then fixed with 4% paraformaldehyde and stained with Hoechst and Apollo reaction cocktail.
Flow cytometry analysis
An Annexin V-FITC/PI Apoptosis Detection Kit (Vazyme, China) was used to evaluate cell apoptosis. The cells were harvested and washed twice with 4 °C precooled PBS. The cell precipitates were resuspended in 200 µL of 1× binding buffer, stained with 5 µL of Annexin V-FITC/PI, and protected from light for 15 minutes at room temperature. PI/RNase Staining Buffer (BD Biosciences, USA) was used to assess the cell cycle distribution. The cells were collected and washed with precooled PBS at 4 °C. The cell precipitates were fixed overnight at -20 °C in 75% ethanol, washed twice with pre-cooled PBS at 4 °C, stained with 200 µl of PI/RNase Staining Buffer, and protected from light for 15 minutes at room temperature. Apoptosis and cell cycle stage were valuated via flow cytometry and analyzed via FlowJo software.
Western blot analysis
The proteins were extracted and separated via 10% SDS‒PAGE gel and transferred to 0.22 µm PVDF membranes (Millipore, USA). The membrane was blocked with 5% skim milk powder and incubated with specific antibodies at 4 °C overnight. The membrane was then incubated with the appropriate secondary antibody, and protein bands were detected using an enhanced chemiluminescence (ECL) detection system (Bio-Rad, USA), and images were acquired using a Bio Spectrum 600 imaging system (UVP). GAPDH was used as a control. The antibodies used included primary antibodies against HNRNPK (11426-1-AP, Proteintech, China), ILF2 (ab113205, Abcam, UK), ILF2 (67685-1-Ig, Proteintech, China), GAPDH (60004-1-Ig, Proteintech, China), IDH2 (15932-1-AP, Proteintech, China), Flag (AE005, ABclonal, China), ubiquitin (10201-2-AP, Proteintech, China). HRP-conjugated secondary goat anti-mouse (SA00001-1, Proteintech, China), and goat anti-rabbit (SA00001-2, Proteintech, China).
Tumor xenograft model
All animal experiments were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals, and approved by the Animal Care Committee of Tongji Medical College. For subcutaneous xenograft tumor models, MDA-MB-231 cells (5×106) were injected into the upper back of 4-week-old female BALB/c nude mice (n=5 per group). For orthotopic breast tumors models, 3 × 106 MDA-MB-231 cells were injected into the mammary fat pad of 8-week-old female BALB/c nude mice (n=5 per group). Tumor sizes were measured using a caliper, and tumor volume was evaluated = length × (width^2)/2.
Immunohistochemistry
Immunohistochemical staining and quantitative evaluation were carried out with antibodies specific for Ki-67 (GB111499, Servicebio, China) and PCNA (GB11010; Servicebio, China). According to the percentage of positive cancer cells, the degree of positivity was measured.
TUNEL assay
TUNEL staining was performed on xenograft tumor tissue according to the guidelines of the TUNEL Apoptosis Detection Kit (Vazyme, China). Images were captured using an Olympus FSX100 microscope (Hungary).
Measurement of oxygen consumption rate
The oxygen consumption rate (OCR) was measured using an XFe96 Extracellular Flux Analyzer (Seahorse Bioscience, Agilent). A total of 2 ×104 cells were seeded in XFe96 cell culture microplates precoated with 20 mg/mL poly-Dlysine, to reach 90% confluence before measurements. The medium was replaced with XF base medium (Seahorse Bioscience), and the cells were incubated at 37 ℃ without CO2 for 45 minutes prior to the assay. The OCR was measured under basal respiration, upon sequential treatment with 1 μM oligomycin, 0.5 μM carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP), and 1 μM rotenone plus antimycin A. The OCR was normalized to the cell index as measured by crystal violet staining at the end of the experiment.
Lactate level detection
A total of 5 × 106 cells were harvested and the lactate content was determined according to the instructions of the Lactate Content Assay Kit (Solarbio, BC2230, China). To construct a standard curve, the concentration of each standard solution was used as the x-axis, and the corresponding absorbance values (ΔA standard) served as the y-axis. The standard curve was then used to obtain the standard equation, y=kx+b. We subsequently calculated the lactate content by entering the ΔA measurement values into the following equation: lactate content (μmol/106 cells) = 1.1875 × [(ΔA measurement – b)/k]/ cell count.
ATP measurement
The culture medium was first aspirated, and subsequently, 5 x 106 cells were collected. The ATP concentration within the cells was detected using an ATP Content Assay Kit (Solarbio, BC0305, China). The ATP content (μmol/106 cells) was calculated with the following formula: ATP content (μmol/106 cells) = 0.125 × ΔA (sample)/ ΔA (standard). In this equation, ΔA (sample) represents the change in absorbance measured for the sample, while ΔA (standard) denotes the change in absorbance measured for the standard.
Citrate and α-KG level detection
A total of 5 x 106 cells were harvested and the concentration. of citrate was determined using a Citric Acid Content Assay Kit (Solarbio, BC2155, China). The citrate content (μmol/104 cells) was calculated as follows: citrate content (μmol/104 cells) = 2 × ΔA (sample - blank)/ ΔA (standard - blank)/ cell count. Similarly, the α-ketoglutarate (α-KG) content was measured using a α-Ketoglutaric Acid (α-KG) Content Assay Kit (Solarbio, BC5425, China) according to the following formula: α-KG content (nmol/106 cells) = 475 × ΔA (sample - blank)/ ΔA (standard - blank)/ cell count × sample dilution factor.
RNA pull-down and mass spectrometry
Full-length LINC00571 was synthesized by in vitro transcription using the T7 High Yield RNA Transcription Kit (Vazyme, China) and labelled with biotin-14-dCTP according to the manufacturer's protocol (Invitrogen, Grand Island, NY, USA). TNBC cells (2 x 107) were harvested and lysed to extract protein. The lysates, biotin-labelled RNA and streptavidin C1 magnetic beads (Invitrogen, USA) were incubated for 12 hours at 4 °C, after which the beads were washed four times with lysis buffer. Purified nuclear proteins were analysed by boiling the RNA‒protein binding mixture in SDS buffer followed by mass spectrometry.
Silver staining and mass spectrometry analysis
Silver staining was performed using a PAGE Gel Silver Staining Kit (Solarbio, China) according to the manufacturer’s protocol, after which a mass spectrometry analysis was performed to analyze the purified nuclear proteins. Liquid chromatography‒mass spectrometry (LC‒MS) (Novogene, China) was used for mass spectrometry analysis.
RNA immunoprecipitation assay
RNA immunoprecipitation (RIP) assays were performed according to the instructions of the Thermo Scientific RIP Kit (Thermo, Waltham, MA, USA) using specific antibodies against HNRNPK (11426-1-AP, Proteintech, China), ILF2 (ab113205, Abcam, UK), Flag (ab45766, Abcam, UK), or IgG (30000-0-AP, Proteintech, China) and Protein A/G magnetic beads (Life Technologies, USA) for RNA immunoprecipitation. Input and coprecipitated RNA were detected via qRT‒PCR analysis.
Immunofluorescence analysis
Cells were seeded on confocal dishes. The cells were fixed in 4% paraformaldehyde (Thermo Scientific, Rockford, IL, USA) for 15 minutes, washed three times with PBS (Sigma‒Aldrich, USA), incubated with 0.5% Triton X-100/PBS at room temperature for 5-10 minutes, and then washed three times with PBS. The cells were blocked with 1% BSA for 30 minutes at 37 °C and then incubated with HNRNPK antibody at 4 °C overnight. On the second day, the cells were washed with PBS and incubated with an anti-ILF2 antibody overnight at 4 °C. On the third day, the cells were washed with PBS and incubated with the appropriate secondary antibody at 37 °C for 30 minutes. Cell nuclei were stained with DAPI. Fluorescence images were captured using a Nikon A1R-si laser scanning confocal microscope (Nikon, Japan).
Coimmunoprecipitation assay
A total of 107 cells were subjected to cell lysis by NP40 supplemented with protease inhibitors for 30 minutes on ice. The cell lysates were incubated with protein A/G agarose (Thermo Fisher Scientific, #20421, USA) and antibodies or IgG at 4 °C overnight. The cell lysates were then washed three times with PBST buffer and eluted at 95 °C for 10 minutes. The eluted proteins were detected by Western blotting. The following antibodies were used for coimmunoprecipitation (Co-IP) analysis: HNRNPK (11426-1-AP, Proteintech, China), ILF2 (ab113205, Abcam, UK), and IgG (30000-0-AP, Proteintech, China).
Luciferase reporter
PGL3-basic luciferase reporter plasmids were transfected into breast cancer cells by using Lipofectamine 3000 (Invitrogen, USA) according to the manufacturer’s instructions. After 24-36 hours of transfection, the cells were lysed, and firefly luciferase and Renilla luciferase activities were measured using a Luciferase Reporter Gene Assay Kit (Promega, USA) according to the manufacturer's recommendations.
Statistical analysis
SPSS v.24.0 software (IBM, Armonk, NY, USA) was used for statistical analysis. The mean ± standard deviation was used to present the experimental results. Student’s t or one-way ANOVA was used to detect differences between groups, and the chi-square test was used to analyze the expression and clinical characteristics of LINC00571. p value < 0.05 was considered statistically significant.
Discussion
With the advances in deep sequencing technology, numerous lncRNAs have been identified in mammalian cells and tissues [
36,
37]. However, the biological mechanisms underlying lncRNAs in TNBC have not been comprehensively elucidated. This study revealed that LINC00571 interacted with the proteins HNRNPK and ILF2. LINC00571 acts as a molecular scaffold that regulates ILF2 stability through HNRNPK. This interaction leads to enhanced IDH2 transcription, promoting the progression of TNBC. These findings unveil a novel regulatory role for LINC00571 in TNBC development, highlighting its significance in breast cancer progression.
Emerging research has consistently indicated the aberrant expression of lncRNAs in cancer [
38‐
40], highlighting their potential as valuable targets for cancer diagnosis and treatment. The functionality of lncRNAs is closely tied to their cellular localization, relative abundance, and interactions with other molecules [
41‐
43]. In this study, we elucidated the predominant nuclear localization of LINC00571. Gain- and loss-of-function studies demonstrated that LINC00571 promotes TNBC cell viability. LINC00571, a novel long non-coding RNA, functions as a scaffold molecule that forms a complex with HNRNPK and ILF2, thereby providing a novel model for understanding the regulatory mechanisms of TNBC. We also observed a minor fraction of LINC00571 in the cytoplasm, the function of which requires further exploration.
HNRNPK, which features the KH 1, KH 2, and KH 3 structural domains, plays a critical role in nucleic acid recognition, promoting its binding to both RNA and DNA [
44]. Recent research revealed that the long non-coding RNA SINEUP facilitates the assembly of translation initiation complexes via interactions with PTBP1 and HNRNPK [
45]. Lnc-FAM84B-4 interacts with HNRNPK, promoting cancer progression by suppressing the expression of the MAPK phosphatase DUSP1 [
46]. In this study, LINC00571 was identified as a new binding partner of HNRNPK that interacts with the KH3 structural domain of HNRNPK. In addition, our results showed that the interaction between HNRNPK and ILF2 leads to a decrease in the ubiquitination of ILF2, thereby increasing its stability. Although previous research has shown that CRBN modulates the ubiquitination of ILF2 [
47], our study significantly extends the current understanding of the regulatory mechanisms underlying ILF2 protein stability.
Although cancer cells have previously believed to mainly use glycolysis, the role of the TCA cycle in cancer metabolism and tumorigenesis has not been emphasized until recently [
30]. IDH2 is a key enzyme in the TCA cycle that catalyzes the conversion of isocitrate to α-KG, a critical step in cellular metabolism and energy production [
48]. Previous studies have demonstrated that Nrf2 and EZH2 regulate IDH2 transcription [
49,
50]. In this study, we found that ILF2 functions as a transcriptional regulator to promote IDH2 transcription and the TCA cycle. However, the specific transcriptional binding sites of ILF2 require further investigation. Moreover, α-KG functions in regulating prosurvival signaling [
51]. Our results suggest that upregulation of IDH2 may stimulate oxidative TCA cycling, increase α-KG production and promote TNBC progression. Thus, this study highlights that the TCA cycle has an important role in the progression of TNBC.
In the present study, we verified the protumor effects of the LINC00571/HNRNPK/ILF2 complex in both vitro and in vivo. However, the study has certain limitations. First, immune responses within the model are neglected as immunodeficient nude mice are used. Since we did not identify a potential mouse homologue of human LINC00571 (data not shown), we could not directly test the significance of the LINC00571 complex in the immune microenvironment of BALB/c mice. Second, we did not validate whether our models can be generalized or extended to other types of tumor cells.
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