Abstract Although tamoxifen is commonly utilized as adjuvant therapy for Estrogen Receptor alpha (ERα)-positive breast cancer patients, approximately 30–50% of individuals treated with tamoxifen experience relapse. Therefore, it is essential to investigate additional factors besides ERα that influence the estrogen response. In this study, cross-analysis of databases were performed, and the results revealed a significant association between LINC00626 and ERα signaling as well as increased expression levels of this gene in tamoxifen-resistant cells. LINC00626 is a novel ERα-regulated long non-coding RNA (lncRNA) that has not yet been examined for its potential contribution to endocrine therapy resistance. This study revealed that the upregulation of LINC00626 in breast cancer was associated with poor overall survival in patients. Additionally, ERα signaling was found to transcriptionally regulate LINC00626 expression, thereby promoting cancer progression and enhancing resistance to tamoxifen in breast cancer cells via the regulation of UPF1 expression. Depletion of LINC00626 restored sensitivity to tamoxifen by activating the PERK-ATF4-CHOP signaling pathway via UPF1. These findings support the role of LINC00626 as a potential therapeutic target for combating tamoxifen resistance. Supplementary Information The online version contains supplementary material available at 10.1038/s41598-025-86287-2. Keywords: Tamoxifen resistance, Estrogen, Apoptosis, LINC00626, UPF1 Subject terms: Proteins, RNA Introduction Breast cancer is the most common type of cancer among women, and approximately 70–80% of breast cancer cases are pathologically classified as ERα-positive^[30]1,[31]2. To mitigate the role of ERα in ERα-positive breast cancer, different hormonal treatments have been utilized, including selective ERα modulators, selective ERα downregulators, and aromatase inhibitors^[32]3–[33]6. Despite the widespread use of tamoxifen as an adjuvant therapy for postmenopausal ERα-positive breast cancer patients, clinical challenges persist as approximately 30–50% of patients treated with tamoxifen experience relapse^[34]7–[35]9. The precise mechanisms underlying de novo and acquired resistance to tamoxifen treatment have yet to be fully elucidated. Since the human genome sequencing was completed, many long non-coding RNAs (lncRNAs) exceeding 200 base pairs in length have been identified as crucial components in cancer biology^[36]10,[37]11. In various cellular processes, lncRNAs regulate the stability of molecules by interacting with RNAs, proteins, or chromatin DNA^[38]12–[39]16. Despite recent advancements in understanding the regulation of lncRNAs by ERα and their roles in ERα-positive breast cancer cells^[40]17–[41]19, the mechanisms underlying tamoxifen resistance in ERα-positive breast cancer remain incompletely understood. Herein, ERα-regulated lncRNAs related to tamoxifen resistance were screened by cross-analyzing various databases, resulting in the discovery of LINC00626 as a new ERα-regulated lncRNA. UPF1 has been linked to the tumorigenesis and progression of various human cancers, such as endometrial cancer, glioblastoma, and colorectal cancer^[42]20–[43]22. Recent studies have highlighted the importance of the nonsense-mediated decay (NMD) pathway in regulating the unfolded protein response (UPR) pathway, with UPF1 serving as a pivotal NMD factor in humans^[44]23,[45]24. The ER stress-induced UPR pathway has been identified as a critical mechanism underlying resistance to endocrine therapy^[46]25. Pathway enrichment analysis in the present study revealed a correlation between LINC00626 and the ER stress response; no previous studies have examined the potential interaction between UPF1 and LINC00626 in ER stress. This study elucidated the transcriptional regulation of LINC00626 by ERα signaling and its roles in promoting cancer progression and enhancing resistance to tamoxifen in breast cancer cells. Mechanistically, LINC00626 facilitated cell proliferation and tamoxifen resistance through UPF1. Inhibition of LINC00626 restored sensitivity to tamoxifen by activating the PERK-ATF4-CHOP signaling pathway via UPF1. Overall, LINC00626 may serve as a candidate target for overcoming tamoxifen resistance. Methods Cell lines Human breast cancer cell lines (MCF-7, T47D, BT-474, BT-549, MDA-MB-231, MDA-MB-453 and MDA-MB-468) and a mammary epithelial cell line (MCF-10 A) were obtained from the American Type Culture Collection (ATCC). The HEK293T cell line used in this study was purchased from ATCC. The SUM149 and SUM159 cell lines were kind gifts from Professor Suling Liu’s laboratory (Fudan University, China). The MCF-7 and T47D TamR cell lines were obtained from Dr. Tao Zhu (USTC, China). Authentication of all the cell lines was conducted through STR genotyping, and routine mycoplasma testing was monitored via the Mycoplasma detection set from M&C Gene Technology, as detailed in the methods section. MCF-7, T47D and BT-474 cells were cultured in RPMI-1640 medium (Gibco) supplemented with 10% FBS (Gibco) at 37 °C with 5% CO[2]. BT-549 cells were cultured in RPMI-1640 medium supplemented with 10% FBS and 10 µg/ml insulin (Sigma) at 37 °C with 5% CO[2]. MDA-MB-231, MDA-MB-453, MCF-10 A and HEK293T cells were cultured in DMEM (Gibco) supplemented with 10% FBS at 37 °C with 5% CO[2]. MDA-MB-468 cells were cultured in Leibovitz L-15 medium supplemented with 10% FBS at 37 °C in the absence of CO[2]. SUM149 and SUM159 cells were cultured in Ham’s F12 medium (Gibco) supplemented with 5% FBS, 5 µg/ml insulin and 1 µg/ml hydrocortisone (Sigma) at 37 °C with 5% CO[2]. MCF-7 and T47D TamR cells were maintained in RPMI-1640 medium supplemented with 10% FBS and 1 µmol/L tamoxifen (Sigma) at 37 °C with 5% CO[2]. Tamoxifen-resistant cell lines ERα-positive breast cancer cell lines (MCF-7 and T47D) were subjected to a gradual increase in tamoxifen concentration from 0.1 µmol/L to 1 µmol/L over a period of at least 6 months to induce the development of tamoxifen-resistant cell lines. The cells were then cultured in medium containing 1 µmol/L tamoxifen. The establishment of tamoxifen-resistant cell lines was confirmed through MTT and colony formation assays. Estrogen deprivation and stimulation experiments To prepare hormone-deprived fetal bovine serum, dextran-coated charcoal was synthesized by combining 1.25 g charcoal and 12.5 mg dextran in 500 ml of a buffer solution containing 1.5 mmol/L MgCl[2], 10 mmol/L HEPES, and 0.25 mol/L sucrose (pH 7.4). The mixture was gently stirred overnight at 4 °C. All reagents were procured from Sigma. Subsequently, the dextran-coated charcoal was thoroughly mixed with the serum at 4 °C for at least 12 h. The resulting hormone-deprived fetal bovine serum was then filtered through a 0.22-µM filter (Millipore). For the estrogen deprivation assay, the cells were cultured in phenol red-free RPMI-1640 (Gibco) medium containing 5% estrogen-deprived serum for 6 days prior to the experiments. For the estrogen stimulation treatment, after the cells were cultured in medium containing 5% estrogen-deficient serum for 6 days, they were stimulated with estrogen (Sigma), tamoxifen (Sigma), or fulvestrant (Sigma), and the expression level of LINC00626 was subsequently detected via qRT-PCR. Proliferation assay The cell proliferation assays involved seeding 1,500 MCF-7 cells or 2,500 T47D cells in 96-well plates, followed by measuring cell viability (OD570 nm) via MTT assays for 5 days. To study tamoxifen resistance, 2,000 MCF-7 cells or 3,000 T47D cells were seeded in 96-well plates and exposed to different concentrations of tamoxifen (ranging from 0 to 10 µmol/L) in cell culture medium. For the colony formation experiments, 1,000 MCF-7 cells or 1,500 T47D cells were seeded in 6-well dishes, and images of the cell clusters were captured and counted after a period of 9 days. Cell transfection and lentiviral generation The RNA oligonucleotides were transfected via Lipofectamine 3000 from Invitrogen. To create the lentivirus system, 1 µg of plasmid expressing the target gene was transferred into HEK293T cells. The medium was replaced with DMEM supplemented with 10% FBS after transfection, and the lentivirus-containing medium was filtered through a 0.22-µM Millipore filter. The virus was subsequently collected once more two days later. The cell lines were treated with culture medium for two days, after which stably transfected cells were selected by 2 µg/mL puromycin. The full-length LINC00626 DNA fragment was cloned and inserted into the PSIN-GFP plasmid by Sangon Biotech (Shanghai, China). The shRNAs targeting LINC00626 were synthesized by Sangon Biotech and cloned and inserted into the pLKO.1 plasmid. All RNA oligonucleotides sequences are listed in Additional File 1. Western blot and real-time PCR The cells were lysed in RIPA buffer to extract total protein. The total protein concentration in the cells was determined via the Bio-Rad DC PROTEIN ASSAY kit. The images obtained from the Western blot experiment were collected and processed by GSG-2000II. Total RNA was isolated from cultured cell lines via Trizol (Invitrogen). In accordance with the guidelines provided by the manufacturer, 1 µg of RNA was used to synthesize cDNA via HiScript^® III RT SuperMix (Vazyme). ChamQ Universal SYBR qPCR Master Mix (Vazyme) was used for real-time PCR. The primers and antibodies used are listed in Additional Files 1 and 2. Chromatin immunoprecipitation assays Chromatin immunoprecipitation (ChIP) was conducted via a ChIP Assay Kit (Beyotime Biotechnology, China) following the manufacturer’s instructions. In summary, approximately 1 × 10^7 cells were crosslinked with 1% formaldehyde, and the reaction was quenched with glycine solution. The cells were subsequently lysed and subjected to sonication. Following centrifugation, the supernatant was incubated with 1 µg of ERα antibody or IgG at 4 °C overnight. The antibody-supernatant mixture was then incubated with protein A/G beads at 4 °C. Following a series of washes, the enriched DNA fragments were eluted. The enrichment of LINC00626 DNA was quantified via qPCR with specific primers (see Additional File 1). Luciferase reporter assays The cells were cotransfected with 0.2 µg of pGL3 Basic luciferase reporters and 0.02 µg of pRL-TK plasmid once the cell population reached approximately 60% confluence in 24-well plates. An internal transfection control was provided by the pRL-TK plasmid. After 2 days of transfection, the cells were collected, and the luciferase activities were measured via a Dual-Luciferase® reporter assay from Promega. Biotin pulldown assay Briefly, either sense or antisense biotin-labelled DNA oligonucleotides that match LINC00626 were incubated with cell lysates from breast cancer cells. Dynabeads coupled with streptavidin from Invitrogen were added to separate the RNA-protein complexes after two hours. Proteins were subsequently extracted through lysis buffer and analyzed via mass spectrometry or immunoblotting techniques. Biotin-labelled LINC00626 sense or antisense DNA probes were synthesized by Sangon Biotech, and the sequences of the probes are provided in Additional File 1. Mass spectrometry experiment An RNA pull-down assay was performed using biotin-labelled LINC00626 sense or antisense DNA probes. The resulting RNA-protein complexes were subsequently analyzed via mass spectrometry (ProtTech, Suzhou, China). A subset of potential candidate proteins interacting with LINC00626 were identified through mass spectrometry screening, with the enriched peptide segments ranked according to their abundance. Then, RNA pull-down followed by immunoblot analysis was employed to confirm the proteins that interact with the LINC00626 DNA probes. RNA-IP A total of 1 × 10^7 cells were lysed in RIPA buffer supplemented with RNase inhibitor and proteinase inhibitor. The cell lysates were pretreated with protein A/G beads from Proteintech and then incubated with 1 µg of anti-UPF1 antibodies or anti-IgG. Protein A/G beads (Invitrogen) were introduced into the cellular lysates, and then RNA extraction was carried out via Trizol. The enriched RNAs were subsequently analyzed via qRT-PCR, and the antibodies used are listed in Additional File 2. Xenograft mouse model This work obtained approval for research ethics from the Association of Laboratory Animal Sciences at Anhui Medical University (LLSC20231697). The animals received care in accordance with the National Institutes of Health Guidelines. Four-week-old female BALB/c nude mice were purchased from SLAC Laboratory Animal Co. Ltd. (Shanghai, China). Female mice were implanted with a 60-day-release pellet containing 0.36 mg of 17β-estradiol in the orthotopic model. A total of 2 × 10^6 LINC00626-knockdown MCF-7 cells were combined with Matrigel (BD Biosciences) and then injected into the mammary fat pads of nude mice. A caliper was used to measure the diameter of the tumor, and the tumor volume was calculated via the following formula: volume = width^2×length/2, with measurements provided in cubic millimeter. Seven weeks after the orthotopic injection, the mice were euthanized, and the primary mammary tumors were collected. Following the completion of the experiments, all the mice utilized in the study were euthanized via a carbon dioxide chamber. Breast cancer specimens Breast cancer samples were harvested from the Second Affiliated Hospital of Anhui Medical University. The research was approved by the Biomedical Ethics Committee of the Second Affiliated Hospital of Anhui Medical University (No. YX2023-140). Written informed consent was obtained from all participants prior to their involvement in the study, and the study was carried out in accordance with the principles outlined in the Declaration of Helsinki. The clinical information can be found in Additional File 3. Data availability The expression data of lncRNA expression (FPKM) and survival information of The Cancer Genome Atlas (TCGA) breast cancer cohort were obtained from Sangerbox, a free online data analysis platform ([47]http://www.sangerbox.com/tool). The TCGA dataset comprises 1102 breast cancer tissue samples and 113 normal breast tissue samples (see Additional File 4). The sequence data used in this study were obtained from the Gene Expression Omnibus (GEO) under the accession number [48]GSE5840. The samples in the [49]GSE5840 dataset comprised MCF-7 cells, with data derived from four replicate experiments conducted using biologically independent samples^[50]26. Statistical analysis The R programming language was used to analyze data from the TCGA BRCA cohort and GEO dataset. Differentially expressed lncRNAs between samples from different groups were identified via the ‘limma’ package, and the cut-off criteria were as follows: log[2]-fold change (log[2]FC)|>2.0 and FDR < 0.05. Gene Ontology (GO) functional enrichment analysis was conducted via the ‘clusterProfiler’ R package, with a significance threshold established at P < 0.05. Survival curves were generated via the Kaplan-Meier plotter platform, and univariate analysis was conducted via the log-rank test. Analysis of the data was performed via GraphPad Prism 7 software to evaluate distinctions among different cohorts. Student’s t test or ANOVA was used to assess between-group differences, with significant differences indicated by a P-value less than 0.05 (unless otherwise stated). SPSS was used to conduct the chi-square test. Each experiment was conducted at least three times. Results Identification of LINC00626 as a potential biomarker in breast cancer To identify the key lncRNAs related to tamoxifen resistance, the R software was used to analyze data from The Cancer Genome Atlas (TCGA) databases. A total of 228 differentially expressed lncRNAs were identified by comparing lncRNA expression levels in cancer versus normal samples (Fig. [51]1A). Additionally, by examining lncRNA levels in MCF-7 cells treated with 17β-estradiol versus DMSO in the [52]GSE5840 database and comparing the expression levels of lncRNAs in tamoxifen-resistant versus tamoxifen-sensitive cells, 117 differentially expressed lncRNAs were further identified (Fig. [53]1B and C). The Venn diagram illustrates six candidate lncRNAs that are differentially expressed in breast cancer tissue, are regulated by estrogen, and are differentially expressed in tamoxifen-resistant cells (Fig. [54]1D). Kaplan-Meier survival analysis revealed that patients with elevated expression levels of HAGLROS, LINC01213, LINC00518, and LINC00626 presented a shorter overall survival rate (Fig. [55]S1A-F). Additionally, qRT-PCR revealed that only LINC00626 expression was significantly upregulated in MCF-7 TamR cells (Fig. [56]S1G). Therefore, this study ultimately identified LINC00626 for further investigation. Fig. 1. [57]Fig. 1 [58]Open in a new tab Identification of ERα-regulated lncRNAs in tamoxifen-resistant samples. (A) Heat map visualization of the 228 most differentially expressed lncRNAs (cancer vs. normal) in the TCGA cohorts (n = 1215). (B) Heat map representation of the 117 most differentially expressed lncRNAs in response to 17β-estradiol versus DMSO treatment (n = 8). (C) Heat map representation of the 117 most differentially expressed lncRNAs in tamoxifen-resistant versus tamoxifen-sensitive samples (n = 8). (D) Schematic diagram of six potential lncRNAs in breast cancer. The blue circle represents the 228 top differentially expressed lncRNAs in cancer versus normal samples, while the red and green circles indicate the 117 lncRNAs regulated by estrogen and differentially expressed in tamoxifen-resistant cells. (E) LINC00626 expression was significantly higher in cancer samples (n = 681) compared to normal samples (n = 53). (F) The expression of LINC00626 in 681 cases of breast cancer samples relative to the average expression in normal samples. (G,H) The subcellular localization and expression levels of LINC00626 in breast cancer samples (n = 37) and normal-adjacent samples (n = 30) were detected by RNA FISH (red). DAPI (blue) for nuclear DNA (Scale bar: 20 μm and 100 μm). ***P < 0.001 (Student’s t test in (E) and (H); ANOVA test in (F)). LINC00626 is located on human chromosome 1q24.2 and possesses three exons and two introns. To investigate the involvement of LINC00626 in breast cancer progression, we analyzed the expression of LINC00626 in 53 normal tissues and 681 breast cancer samples obtained from TCGA datasets. The results revealed a significant increase in LINC00626 levels in breast cancer samples compared to normal tissues (Fig. [59]1E). Furthermore, the expression of LINC00626 was greater in 82.4% (561/681) of the breast cancer tissues than in normal tissues (Fig. [60]1F). Subsequently, qRT-PCR was conducted on 19 normal-adjacent samples and 55 breast cancer samples, thus validating the elevated levels of LINC00626 in breast cancer (Fig. [61]S1H). Fluorescence in situ hybridization (FISH) was performed and yielded additional evidence supporting the high expression of LINC00626 in breast cancer samples relative to normal-adjacent samples, with predominant localization in the cytoplasm (Fig. [62]1G and H). Additionally, the validation of LINC00626 was confirmed in a mammary epithelial cell line (MCF-10 A) and nine breast cancer cell lines (T47D, MCF-7, BT474, BT549, MDA-MB-231, MDA-MB-453, MDA-MB-468, SUM149 and SUM159). The expression of LINC00626 was significantly greater in breast cancer cell lines than in a mammary epithelial cell line (Fig. [63]S1I). Therefore, these findings suggest that LINC00626 is upregulated in breast cancer. LINC00626 is an estrogen-responsive gene To investigate whether LINC00626 is estrogen-responsive, the expression of LINC00626 was examined in MCF-7 cells under conditions of hormone deprivation, and the results revealed a progressive reduction in LINC00626 expression (Fig. [64]2A). Under estrogen-replete conditions, stimulation with estrogen led to a significant increase in LINC00626 levels in a time-dependent manner (Fig. [65]2B). Furthermore, the expression level of LINC00626 was significantly elevated following estrogen stimulation in estrogen-deprived MCF-7 cells. This increase was strongly attenuated by the antagonism of ERα with either tamoxifen or fulvestrant (Fig. [66]2C). Fig. 2. [67]Fig. 2 [68]Open in a new tab LINC00626 is transcriptionally regulated by ERα. (A) LINC00626 expression was analyzed by qRT-PCR at various time points after estrogen deprivation in MCF-7 cells. (B) The qRT-PCR expression of LINC00626 at specific time points following stimulation with 10 nmol/L E2 in MCF-7 cells. (C) qRT-PCR analysis of LINC00626 following treatment with 10 nmol/L E2, 1 µmol/L Tam and/or 1 µmol/L Fulv for 24 h in estrogen-deprived MCF-7 cells. (D) Schematic diagram of the ESR1 binding motif and schematic illustration of two predicted conserved ERE sites on the upstream regions of the LINC00626 gene. The red box denoted the anticipated ERα binding sites, while the white lines indicated the promoter regions harboring the D1 and D2 domains cloned into luciferase reporter plasmids. (E) ChIP products from ERα antibody or IgG in cells treated with 1 µmol/L Tam or vehicle for 24 h were amplified by qPCR. (F) Schematic representation of the pGL3-based wild-type (D1 and D2) and mutant (D1 MUT and D2 MUT) reporter constructs. (G) The regulation of LINC00626 promoter activity by ERα was investigated through luciferase reporter assay. Reporter activity was measured following transfection of HEK293T cells with plasmids containing ESR1 or vector. TamR: Tamoxifen-resistant cells; Tam: Tamoxifen; E2: estrogen; Fulv: fulvestrant. Results are shown as mean ± SD obtained from three independent experiments. ***P < 0.001; ns, not significant (ANOVA test). To confirm the direct connection between ERα and the promoter area of LINC00626, two anticipated conserved estrogen response element (ERE) locations situated 3kbps upstream of the LINC00626 gene (hereafter termed the D1 and D2 sites) were discovered through the JASPAR database ([69]https://jaspar.elixir.no/) and rVista 2.0 ([70]https://rvista.dcode.org/, Fig. [71]2D)^[72]27,[73]28. ChIP assays validated the direct interaction of ERα with the chromatin region containing the D1 and D2 sites (Fig. [74]2E). Tamoxifen treatment inhibited the binding of ERα to the D1 and D2 sites in MCF-7 cells (Fig. [75]2E). In contrast, there was a significant increase in enrichment at these locations in the MCF-7 TamR cells (Fig. [76]2E). To clarify whether ERα regulates LINC00626 transcription, DNA fragments with the original or mutant sequence were cloned and inserted into luciferase reporter plasmids (Fig. [77]2F). The overexpression of ERα significantly increased the luciferase activity of the plasmids containing the D1 or D2 site (Fig. [78]2G). Additionally, mutation of the D1 or D2 site completely abrogated estrogen responsiveness (Fig. [79]2G). Taken together, these data collectively indicate that LINC00626 is subject to transcriptional regulation by ERα signaling. LINC00626 enhances the proliferation of breast cancer cells To characterize LINC00626 from a functional perspective, shRNA was used to deplete this gene in ERα-positive MCF-7 cells (Fig. [80]3A). MTT and colony formation assays were performed to determine the potential role of LINC00626 in the proliferation of cancer cells (Fig. [81]3B and C). The depletion of LINC00626 significantly inhibited cell viability and colony formation capacity. Silencing of LINC00626 also led to a greater proportion of cells undergoing early apoptosis, as shown by flow cytometry analysis (Fig. [82]3D). In contrast, the overexpression of LINC00626 led to significant increases in the proliferation and colony formation of MCF-7 cells (Fig. [83]3E-G). Additionally, a xenograft model was used to assess the oncogenic role of LINC00626 in vivo. Consistent with these findings in vitro, LINC00626 depletion led to a significant reduction in tumor volume compared with that in the control group (Fig. [84]3H). Fig. 3. [85]Fig. 3 [86]Open in a new tab LINC00626 enhances cell proliferation both in vitro and in vivo. (A) The level of LINC00626 knockdown using two independent shRNAs or vector was examined in MCF-7 cells. (B,C) Cell viability of MCF-7 cells transfected with plasmids containing two independent shRNAs or vector was determined by MTT (B) and colony formation (C) assays. (D) Early apoptotic (Annexin V-FITC+/PI-) population in MCF-7 cells with LINC00626 depletion was determined by flow cytometry. (E) The upregulation of LINC00626 expression using lentiviruses in MCF-7 cells. (F,G) Cell viability of MCF-7 cells transfected with lentiviruses expressing LINC00626 or vector was assessed by MTT (F) and colony formation (G) assays. (H) 2 × 10^6 MCF-7 cells transfected with LINC00626 shRNA or vector were orthotopically injected into nude mice (n = 4 for each group). The photographs of xenograft tumors were taken 7 weeks after injection. Results are shown as mean ± SD obtained from three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001 (Student’s t test in (E) and (G); ANOVA test in (A–D,F,H)). In this study, we further investigated the impact of LINC00626 on ERα-positive T47D cells and found that reducing LINC00626 resulted in a significant reduction in cell proliferation and colony formation (Fig. [87]4A-C). The silencing of LINC00626 resulted in an increased proportion of T47D cells undergoing early apoptosis (Fig. [88]4D). Conversely, the overexpression of LINC00626 in T47D cells was observed to increase cell proliferation and colony formation capacity (Fig. [89]4E-G). Collectively, these data suggest that LINC00626 has a significant effect on the growth of breast cancer cells. Fig. 4. [90]Fig. 4 [91]Open in a new tab LINC00626 promotes the proliferation of T47D cells. (A) The level of LINC00626 following knockdown of lncRNA using two independent shRNAs in T47D cells. (B,C) Cell viability of T47D cells transfected with plasmids containing two independent shRNAs or vector was determined by MTT (B) and colony formation (C) assays. (D) Early apoptotic population in T47D cells following LINC00626 depletion was determined by flow cytometry. (E) The level of LINC00626 following overexpression of lncRNA using lentiviruses in T47D cells. (F,G) Cell viability of LINC00626 overexpressing T47D cells was determined by MTT (F) and colony formation (G) assays. Results are shown as mean ± SD obtained from three independent experiments. **P < 0.01; ***P < 0.001 (Student’s t test in (E) and (G); ANOVA test in (A–D,F)). LINC00626 promotes tamoxifen resistance in breast cancer cells Since ERα regulates LINC00626 transcription, we further investigated its impact on tamoxifen resistance^[92]29. The overexpression of LINC00626 was shown to decrease the responsiveness of breast cancer cells to tamoxifen (Fig. [93]5A and B). The resistance of MCF-7 TamR and T47D TamR cells to tamoxifen treatment was confirmed through MTT and colony formation assays (Fig. [94]S2A-D). Notably, compared with that in the corresponding parental cells, LINC00626 was found to be upregulated in TamR cells (Fig. [95]5C). In contrast, the absence of LINC00626 in TamR cells led to significant recovery of tamoxifen sensitivity (Fig. [96]5D and E), indicating that the upregulation of LINC00626 in TamR cells may play a role in maintaining tamoxifen resistance. Fig. 5. [97]Fig. 5 [98]Open in a new tab LINC00626 increases the resistance of breast cancer cells to tamoxifen. (A) MCF-7 cells transfected with lentiviruses expressing LINC00626 or vector were treated with varying concentrations of tamoxifen. (B) Cell viability was determined by MTT assay in LINC00626 overexpressing T47D cells flowing tamoxifen treatment. (C) The expression of LINC00626 was examined in both parental and TamR cells. (D) MCF-7 parental and TamR cells were transfected with plasmids containing shRNAs or vector, followed by treatment with 1 µmol/L tamoxifen. (E) T47D parental and TamR cells were treated with 1 µmol/L tamoxifen or vehicle after transfected with plasmids containing two shRNAs or vector. Results are shown as mean ± SD obtained from three independent experiments. ***P < 0.001 (Student’s t test in (C), ANOVA test in (A,B,D,E)). LINC00626 upregulates UPF1 through direct interaction A selection of potential candidate proteins that interact with LINC00626 was analyzed through mass spectrometry screening (Fig. [99]6A). Subsequent RNA pull-down assays followed by immunoblot analysis confirmed that only UPF1 was precipitated with biotin-labelled LINC00626 antisense DNA probes (Fig. [100]6B). The absence of a strong connection between LINC00626 and β-ACTIN indicates that LINC00626 does not typically bind to proteins indiscriminately (Fig. [101]6B). Additionally, the UPF1 antibody enriched LINC00626 in the RIP assay (Fig. [102]6C). Furthermore, the physical association between LINC00626 and UPF1 was evident in MCF-7 TamR cells (Fig. [103]6D). Fig. 6. [104]Fig. 6 [105]Open in a new tab LINC00626 upregulates UPF1 through direct interaction. (A) The protein interaction of LINC00626 was analyzed through mass spectrometry screening. (B) MCF-7 cell lysates were subjected to biotin pull-down assay using in vitro synthesized biotin-labeled sense or antisense DNA probes against LINC00626. (C) MCF-7 cell lysates were immunoprecipitated with UPF1 antibody or IgG. (D) Biotin pull-down assay in MCF-7 TamR cell lysates analyzed the physical association between LINC00626 and UPF1. (E) UPF1 expression was higher in tumors samples (n = 1102) compared to normal samples (n = 113). (F) The protein level of UPF1 in LINC00626-depleted MCF-7 cells was assessed by western blotting, β-ACTIN was used as control. (G) Immunoblot assessment of the UPF1 levels in MCF-7 cells with forced expression of LINC00626. Results are shown as mean ± SD obtained from three independent experiments. ***P < 0.001 (Student’s t test in (B,C,E)). UPF1 is an important participant in the NMD pathway and is essential for maintaining the quality of mRNA transcripts in eukaryotic cells^[106]30,[107]31. To examine the role of UPF1 in the progression of breast cancer, the expression of UPF1 was assessed in 113 normal tissues and 1102 breast cancer samples obtained from TCGA datasets. The findings demonstrated a significant elevation in UPF1 expression levels in breast cancer samples compared with normal tissues (Fig. [108]6E). Notably, the expression of UPF1 was significantly reduced in LINC00626-depleted MCF-7 cells, whereas the expression of UPF1 increased with the overexpression of LINC00626 (Fig. [109]6F and G). The depletion of LINC00626 activates the PERK-ATF4-CHOP signaling pathway UPF1 is a key participant in the NMD pathway, and previous studies have shown a strong connection between UPF1 and the development and progression of various human cancers^[110]32. NMD degrades mRNAs encoding elements of the unfolded protein response to inhibit UPR activation during innocuous ER stress^[111]33–[112]35. Consequently, there is speculation regarding the potential regulatory role of LINC00626 in the ER stress response through UPF1. An analysis of the TCGA breast cancer RNA-seq cohort identified the candidate coding genes that were differentially correlated with LINC00626 levels (Fig. [113]7A and B). Pathway enrichment analysis revealed that the response to ER stress and UPR pathways were significantly enriched among differentially expressed genes associated with LINC00626 (Fig. [114]7C). Consistent with these findings, the depletion of LINC00626 led to the upregulation of ATF4, CHOP, and PERK phosphorylation, whereas XBP1-s and AFT6 expression remained unaffected (Fig. [115]7D and E). Taken together, these data revealed that depletion of LINC00626 activated the PERK-ATF4-CHOP signaling pathway. Fig. 7. [116]Fig. 7 [117]Open in a new tab The depletion of LINC00626 activates the PERK-ATF4-CHOP signaling pathway. (A) Volcano plot shows the differences of gene expression relative to the LINC00626 levels (log[2]FC > 0.5 and P < 0.05). (B) Heat map representation of 1500 differentially expressed genes correlated with LINC00626 levels. (C) Gene ontology pathway analysis of differentially expressed genes relevant to LINC00626 from TCGA database. (D) p-PERK, ATF4, and CHOP levels were determined in LINC00626-depleted MCF-7 cells by western blotting, β-ACTIN was used as input control. (E) XBP1-s and ATF6 levels were determined in MCF-7 cells transfected with plasmids containing two independent shRNAs or vector. Results are shown as mean ± SD obtained from three independent experiments. UPF1 regulates LINC00626–induced tamoxifen resistance in breast cancer This investigation sought to elucidate the impact of UPF1 in MCF-7 cells through MTT and colony formation tests, and revealed that reducing UPF1 significantly inhibited cell viability and colony formation capacity (Fig. S3A-C). To explore the potential role of UPF1 in mediating the cellular functions of LINC00626, we overexpressed LINC00626 while simultaneously depleting UPF1. The data revealed that the depletion of UPF1 led to the restoration of increased cell viability induced by the overexpression of LINC00626 (Fig. [118]8A and B). The ability of LINC00626 to sustain resistance to tamoxifen via UPF1 was assessed via a viability assay. The overexpression of LINC00626 was shown to decrease sensitivity to tamoxifen, a phenomenon that was reversed by UPF1 depletion (Fig. [119]8C). Moreover, depletion of either LINC00626 or UPF1 in TamR cells led to a significant restoration of sensitivity to tamoxifen (Fig. [120]8D). Interestingly, a positive correlation between LINC00626 and UPF1 was similarly observed in MCF-7 TamR cells (Fig. [121]8E and F). Moreover, the levels of ATF4, CHOP, and PERK phosphorylation were increased in MCF-7 TamR cells following the inhibition of LINC00626 (Fig. [122]8G). Conversely, the overexpression of UPF1 reversed the elevated expression levels of these proteins induced by LINC00626 depletion (Fig. [123]8G). Collectively, these data suggest that the estrogen-inducible LINC00626 promotes the proliferation of breast cancer cells and maintains resistance to tamoxifen by regulating UPF1. The depletion of LINC00626 restored sensitivity to tamoxifen by triggering the PERK-ATF4-CHOP signaling pathway via UPF1 (Fig. [124]8H). Fig. 8. [125]Fig. 8 [126]Open in a new tab UPF1 plays a critical role in regulating LINC00626–mediated tamoxifen resistance in breast cancer. (A,B) Cell viability was determined by MTT (A) and colony formation (B) assays in MCF-7 cells overexpressing LINC00626 co-transfected with UPF1 shRNA or vector. (C) Cell viability was determined using the MTT assay in MCF-7 cells overexpressing LINC00626 co-transfected with UPF1 shRNA or vector treatment with varying concentrations of tamoxifen. (D) Cell viability was assessed in MCF-7 TamR cells transfected with LINC00626 shRNA and/or UPF1 shRNA flowing tamoxifen treatment. (E) The protein levels of UPF1 were evaluated in LINC00626-depleted MCF-7 TamR cells using western blotting. (F) Immunoblot assessment of the UPF1 levels in MCF-7 TamR cells with forced expression of LINC00626. (G) The expression levels of p-PERK, ATF4, and CHOP were assessed in MCF-7 TamR cells co-transfected with plasmids containing shLINC00626 and/or UPF1 using western blot analysis. (H) Proposed schematic diagram illustrating the role of estrogen-inducible LINC00626 in tamoxifen-resistant breast cancer cells. The depletion of LINC00626 resulted in restored tamoxifen sensitivity by activating the PERK-ATF4-CHOP signaling pathway through UPF1. Results are shown as mean ± SD obtained from three independent experiments. ***P < 0.001 (ANOVA test). Discussion Approximately 70% of breast cancer cases are categorized as ERα-positive breast cancer, thus making endocrine therapy a crucial treatment option for this disease^[127]36,[128]37. Despite the widespread use of tamoxifen for postmenopausal ERα-positive breast cancer patients, its efficacy is limited, as 30–50% of patients treated with this agent experience relapse^[129]38,[130]39. Therefore, it is essential to identify additional factors besides ERα that play a significant role in the estrogen response. In this study, multiple databases were analyzed to identify ERα-regulated lncRNAs associated with tamoxifen resistance. The results revealed that LINC00626 is a novel ERα-regulated lncRNA. Despite recent advancements in elucidating the regulation of lncRNAs by ERα, there is limited knowledge regarding LINC00626 and its role in contributing to tamoxifen resistance in ERα-positive breast cancer^[131]40–[132]42. This study elucidated the transcriptional regulation of LINC00626 by ERα signaling and its roles in promoting cancer progression and enhancing resistance to tamoxifen in breast cancer cells. In clinical research, investigations of lncRNA tumor markers have yielded significant findings^[133]43. A retrospective multicentre study was conducted on samples from patients with nasopharyngeal carcinoma, leading to the identification of nine lncRNA markers associated with immune heterogeneity in this type of cancer. These markers have the potential to predict the long-term recurrence risk of nasopharyngeal carcinoma^[134]44. Furthermore, research conducted on animal models and human tumor cells have demonstrated that the suppression of MALAT1 via antisense oligonucleotides (ASOs) can effectively inhibit tumor growth and metastasis. Consequently, MALAT1 has emerged as a promising target for future cancer drug development^[135]45. Our results indicated that targeting LINC00626 with shRNA effectively restored tamoxifen sensitivity in tamoxifen-resistant cells via UPF1. Thus, targeting LINC00626 may present a novel therapeutic strategy to enhance current treatment options for tamoxifen resistance. In subsequent experiments, the feasibility of targeting LINC00626 for the treatment of tamoxifen-resistant cases will be assessed via ASO interventions. This will be evaluated through studies involving animal models and clinical tissue samples. Dysregulation of UPF1 has been shown to impact carcinogenesis in multiple tumors, leading to increased proliferation and metastasis. Specifically, compared with normal tissues increased UPF1 expression has been detected in various cancer types, particularly in breast cancer tissues derived from patients^[136]46. Additionally, MACC1-AS1 has been shown to increase the self-renewal capability of non-small cell lung cancer cells by upregulating UPF1^[137]47. The PVT1-UPF1 axis controls the aggressive traits of endometrial cancer stem cells^[138]20. Furthermore, UPF1 contributes to the development of chemoresistance in colorectal cancer by controlling TOP2A function and preserving stem cell characteristics^[139]21. Conversely, decreased levels of UPF1 have been reported in certain tumors such as gastric cancer^[140]48, thus indicating the dual role of UPF1 in both promoting and inhibiting tumorigenesis. Analysis of the TCGA database revealed elevated levels of UPF1 expression in breast cancer tissues. This study illustrated that UPF1 deficiency diminished the impact on cell viability and colony formation capacity. Furthermore, LINC00626 was found to increase the proliferation of breast cancer cells and sustain resistance to tamoxifen through the regulation of UPF1. The NMD pathway plays a crucial role in preserving the quality of mRNA transcripts in eukaryotic cells, with UPF1 identified as a key factor in this pathway^[141]49. Recent evidence indicates that NMD plays a critical role in modulating the ER stress response by suppressing the activation of the UPR^[142]50,[143]51. During ER stress, the phosphorylation of the initial factor eIF2α can suppress NMD, whereas certain proteins involved in maintaining oxidative stress homeostasis, such as ATF4 and CHOP, are significantly upregulated as a result of NMD inhibition^[144]52. This research revealed that LINC00626 upregulated UPF1 through direct interaction, and the depletion of LINC00626 resulted in the upregulation of ATF4, CHOP, and PERK phosphorylation, with no effect on XBP1-s and AFT6 expression. LINC00626 may interact with UPF1 to suppress PERK-ATF4-CHOP transcription, and the specific mechanism by which the LINC00626-UPF1 complex regulates the UPR stress pathway will be further studied. ER stress plays a role in many cellular processes including protein processing, lipid synthesis, and calcium storage and release^[145]53. When the demand for proper protein folding is unmet, cells initiate ER stress, resulting in the activation of the UPR. The UPR is primarily mediated by three ER stress sensors: PERK, ATF6, and IRE1α^[146]54. ER stress response activation in cancer cells serves as a prosurvival adaptive mechanism that facilitates tumor development, progression, and resistance to drugs^[147]55,[148]56. Targeting ER stress addiction could be a potential therapeutic strategy in cancer treatment by either suppressing the ER stress response to disrupt the prosurvival mechanism or inducing additional ER stress to activate proapoptotic signaling pathways^[149]57. Recent evidence indicates that the levels of activated IRE1α, PERK, and eIF2α are significantly lower in TamR breast cancer cells than in the corresponding parental cells. The activation of the UPR stress pathway imparts sensitivity to tamoxifen in TamR breast cancer cells by inducing apoptosis through the activation of the p38/JNK pathways^[150]58–[151]60. This study revealed that the inhibition of LINC00626 led to apoptotic cell death followed by the suppression of UPF1 and the activation of the PERK-ATF4-CHOP ER stress signaling pathways. The current study found that LINC00626 expression was elevated in ERα-positive breast cancer and even more elevated in tamoxifen-resistant cells. The transcriptional regulation of LINC00626 by ERα signaling was found to promote the proliferation of breast cancer cells and sustain resistance to tamoxifen through UPF1. Our results indicated that targeting LINC00626 with shRNA effectively restored tamoxifen sensitivity in tamoxifen-resistant cells by activating the PERK-ATF4-CHOP signaling pathway via UPF1. Thus, targeting LINC00626 may present a novel therapeutic strategy to enhance current treatment options for tamoxifen resistance. Conclusions Our study revealed that the estrogen-inducible gene LINC00626 is a key factor in promoting cancer progression and resistance to tamoxifen through interaction with UPF1. Inhibition of LINC00626 results in the restoration of tamoxifen sensitivity by activating the PERK-ATF4-CHOP signaling pathway via UPF1. Electronic supplementary material Below is the link to the electronic supplementary material. [152]Supplementary Material 1^ (1.6MB, pdf) [153]Supplementary Material 2^ (1.4MB, pdf) Abbreviations ERα Estrogen Receptor alpha LINC00626 Long intergenic non-protein coding RNA 626 UPF1 UPF1 RNA helicase and ATPase PERK Eukaryotic translation initiation factor 2 alpha kinase 3 ATF4 Activating transcription factor 4 ATF6 Activating transcription factor 6 IRE1α Inositol requiring enzymes 1α CHOP DNA damage inducible transcript 3 SERMs Selective ERα modulators SERDs Selective ERα downregulators AI Aromatase inhibitors lncRNAs Long non-coding RNAs RBPs RNA binding proteins NMD Nonsense-mediated decay pathway ER Endoplasmic Reticulum UPR Unfolded Protein Response pathway TCGA The Cancer Genome Atlas databases ERE Estrogen response element ChIP Chromatin immunoprecipitation assay TamR Tamoxifen-resistant cells RIP RNA immunoprecipitation ISH In situ hybridization GO Gene ontology pathway E2 Estrogen TAM Tamoxifen Fulv Fulvestrant Author contributions H.Y., W.H., and M.Y. developed the study concept and design; H.Y. and M.Y. developed the methodology and wrote, reviewed, and revised the paper; H.Y. provided the acquisition, analysis, and interpretation of data and statistical analysis; H.Y. and L.Z. provided technical and material support. All authors reviewed the manuscript. Funding This work was supported by National Natural Science Foundation of China (82303329), Project funded by China Postdoctoral Science Foundation (2022M720198), Key Scientific Research Foundation of the Education Department of Anhui Province (2023AH053180), and Anhui Medical University Foundation (2022xkj023). Data availability The sequence data in this study are obtained from the Gene Expression Omnibus (GEO) under the accession numbers [154]GSE5840. All data generated or analysed during this study are included in this published article. Declarations Competing interests The authors declare no competing interests. Ethics declarations All experiments conducted in this study were approved by the Ethics Committee of the Second Affiliated Hospital of Anhui Medical University (Ethical number: YX2023-140). Written informed consent was obtained from all participants prior to their involvement in the study, which was carried out in adherence to the principles outlined in the Declaration of Helsinki. This work has obtained approval for research ethics from the Association of Laboratory Animal Sciences at Anhui Medical University (LLSC20231697), ensuring that all procedures were performed in accordance with ethical guidelines. The study is reported in accordance with ARRIVE guidelines. Footnotes Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Contributor Information Hui Yuan, Email: yuanhui@ahmu.edu.cn. Wei Hu, Email: huwei@ahmu.edu.cn. Min Yang, Email: yangmin@ahmu.edu.cn. References