Background
The microtubule-disrupting agent paclitaxel, a plant alkaloid developed from the bark of the Pacific yew tree,
Taxus brevifolia, is a first-line chemotherapeutic agent for solid tumors, such as lung, ovarian and breast cancer, and contributes to substantial improvement in patient survival [
1]. However, clinical responses to paclitaxel have shown variable sensitivity in cancer patients [
2]. Thus, elucidation of the underlying mechanisms of paclitaxel sensitivity and identification of reliable biomarkers that can predict the response to paclitaxel in human cancer are urgently required.
Autophagy is a homeostatic process for degrading cellular materials under cellular stress [
3]. Accumulating evidence has shown that autophagy is involved in modulating paclitaxel sensitivity. Tumors expressing diminished levels of autophagy-initiating genes are resistant to paclitaxel therapy, whereas induction of autophagy improves the antitumoral efficiency of the microtubule-disrupting agent paclitaxel [
4,
5]. Furthermore, several studies have proposed that autophagy-related genes/proteins could be investigated as possible prognostic or predictive markers of paclitaxel efficacy in the clinic [
6]. However, it is not clear which autophagy-initiating genes can be used as a predictive factor for paclitaxel response.
Long noncoding RNAs (lncRNAs) are a heterogeneous class of transcripts with a minimum length of 200 bases and limited protein-coding potential [
7]. LncRNAs exhibit a wide range of expression levels and distinct cellular localizations and are thus a large and diverse class of regulators [
8]. LncRNAs can function
in cis to regulate the expression of neighboring genes or
in trans to perform many roles in various modes [
9]. The landscape of lncRNAs dysregulated in autophagy and their molecular mechanisms in autophagy regulatory networks were summarized in our previous study in detail [
10]. Studies have demonstrated that lncRNAs such as
NBR2 and
APF can regulate autophagy processes with the aid of certain important proteins [
10]. Several lncRNAs have also recently been implicated in the modulation of drug resistance [
11]. However, the relationship between lncRNA expression and paclitaxel insensitivity caused by abnormal autophagy remains largely unexplored.
Eosinophil granule ontogeny transcript (
EGOT), as an antisense intronic long noncoding RNA (Ai-lncRNA), is expressed from
ITPR1, which is a ligand-gated ion channel that mediates calcium release from intracellular stores [
12,
13]. The biology function of Ai-lncRNAs including
EGOT is still largely unknown. In our previous study, we first reported that downregulation of
EGOT expression was correlated with advanced malignant status and worse prognosis in breast cancer [
14]. Here, we describe a previously unrecognized role of
EGOT in increasing sensitivity to paclitaxel via triggering autophagy. Mechanistically,
EGOT enhances autophagosome accumulation via the upregulation of ITPR1 expression
in cis and
in trans. On one hand,
EGOT upregulates ITPR1 levels via formation of a
pre-ITPR1/EGOT dsRNA that induces
pre-ITPR1 accumulation to increase ITPR1 protein expression
in cis. On the other hand,
EGOT recruits hnRNPH1 to enhance the alternative splicing of pre-ITPR1
in trans via two binding motifs in
EGOT segment 2 (324–645 nucleotides) in exon 1. We also uncover that hypoxia induces
EGOT transcriptional expression, while estrogen suppresses its expression directly. Finally,
EGOT is confirmed to be associated with a favorable prognosis and to enhance paclitaxel sensitivity in breast cancer patients and other human malignancies in our breast cancer cohort and public data cohorts. Given its significance in the autophagy signaling pathway,
EGOT may be act as a promising predictive biomarker for paclitaxel response, and proper regulation of
EGOT may be a novel synergistic strategy for enhancing paclitaxel sensitivity in human cancer.
Materials and methods
Breast specimens and clinical assessments
Eligible patients with a histological diagnosis of breast cancer who had received neither chemotherapy nor radiotherapy before surgical resection were recruited to the present study. In total, 258 breast cancer tissues and 258 normal tissues, along with 15 adjuvant chemotherapy regimens, were obtained from Harbin Medical University Cancer Hospital/Center in Harbin, China. All samples were frozen in liquid nitrogen immediately after surgical resection, and only tumors with > 80% tumor cells were selected for RNA extraction. Two independent senior pathologists confirmed the pathological diagnosis and molecular subtype of each cancer tissue. This study conformed to the clinical research guidelines and was approved by the research ethics committee of Harbin Medical University Cancer Hospital. We obtained written informed consent from all patients. For 184 patients, telephone follow-up was performed to verify survival status after primary treatment. Among them, 159 patients received paclitaxel treatment.
Cell culture and treatments
The human cervical cancer cell line HeLa and 10 breast cancer cell lines (MCF7, T47D, UACC-812, SK-BR-3, MDA-MB-453, MDA-MB-231, Hs578T, HCC70, BT549, and MDA-MB-468) were obtained from the Chinese Academy of Sciences Cell Bank and Cellbio (China), respectively. All cell lines were periodically authenticated (Cellbio). Unless specifically indicated, cells were cultured in DMEM (R10–017-CV, Corning, USA), RPMI-1640 (R10–040-CM, Corning) or Leibovitz’s L15 (PYG0038, Boster, China) medium supplemented with 10% fetal bovine serum (0500, ScienCell, USA) at 37 °C with 5% CO
2 or air and 95% humidity. In estrogen-related experiments, MCF7 and T47D cells were washed three times with phenol red-free DMEM and subjected to hormone deprivation for up to 3 days with 10% activated charcoal-absorbed fetal calf serum (FCS; P30–2302, PAN) before proceeding to the next steps. The reagents used were 17 β-oestradiol (E8875, Sigma, USA), the estrogen receptor antagonist tamoxifen (T5648, Sigma) and ICI 182780 (Asc-131, Ascent Scientific, USA). Autophagosome and LC3 experiments were performed as previously described [
15] and were performed in one of two ways. Cells (10–20 × 10
4 cells/ml) were plated in medium containing 10% serum. After 24 h, the medium was changed to that containing 0.1% serum, and the cells were collected 48 h later with or without 100 mM CQ (ab142116, Abcam, USA) treatment for 1 h before collection. Alternatively, the cells were cultured with EBSS (E2888, Sigma) for the indicated durations before collection. For the ubiquitination assay, HeLa cells were treated with MG132 (10 μM/l) (ab141003, Abcam) for 40 min. Protein levels were detected by immunoblotting and quantified by densitometry. For the CHX chase assay, HeLa cells were incubated with 50 μg/ml CHX (Catalogue Number HY-12320, MCE, USA) for the indicated durations (0,15, 30, 60, 120, and 180 min) as previously described [
16]. For transcriptional inhibition experiments, actinomycin D (6 μg/ml) (A1410, Sigma) was added to the cells, and samples were harvested at the indicated time points (0, 0.5, 1, 2, 4, and 8 h) as previously described [
17].
Total RNA and miRNA were extracted from cells using the E.Z.N.A.® Total RNA Kit I (Catalogue Number R6834–01, Omega Bio-Tek, USA). First-strand cDNA was prepared with the Transcriptor First Strand cDNA Synthesis Kit (Catalogue Number 04897030001, Roche, USA). Real-time PCR was performed using FastStart Universal SYBR Green Master (ROX) (Catalogue Number 04913914001, Roche) on a 7500 Fast Real-Time PCR system (ABI, USA). For quantification of gene expression, we used the 2
-ΔΔCt method.
GAPDH expression was used for normalization. The primer sequences were synthesized by Shanghai Generary Biotech Co., Ltd. and are included in Additional file
1: Table S1.
Lentivirus production and infection
Recombinant lentiviruses expressing the lncRNA
EGOT, shEGOT, shITPR1, shESR,
EGOT-MS2, Flag-MCP2 and controls were constructed by Sangon Biotech Company (China). Concentrated viruses were used to infect 5 × 10
5 cells in a 6-well plate with 4–6 μg/ml polybrene (107,689, Sigma). The infected cells were then subjected to selection with 1 μg/ml puromycin (Catalogue Number 540411, Calbiochem, USA) for two weeks. Stable overexpression cell lines or knockdown cell lines were identified using qRT-PCR or western blotting. The shRNA sequences are provided in Additional file
1: Table S1.
Co-immunoprecipitation, western blot assay and antibodies
Co-immunoprecipitation assays were carried out by using the Pierce™ Crosslink Magnetic IP/Co-IP Kit (Catalogue Number 88805, Thermo Fisher, USA) according to the manufacturer’s protocol. Western blotting was performed according to the previously described procedures [
18]. Anti-LC3B (ab48394, 1:1000), anti-IP3R (ab5804, 1:1000) antibody, anti-SQSTM1/P62 (ab91526, 1:1000) antibody, anti-hnRNPH1 (ab10374, 1:1000) antibody, anti-HMGB1 (ab79823, 1:1000) antibody, anti-RIP140 (ab42126,1:500) antibody, goat anti-rabbit IgG H&L (HRP) (ab6721, 1:10,000), and goat anti-mouse IgG H&L (HRP) (ab6789, 1:10,000) were obtained from Abcam. Anti-tubulin (sc-73,242, 1:1000) antibody was purchased from Santa Cruz Biotechnology. Anti-estrogen receptor (WL00940, 1:1000) antibody and anti-GAPDH (WL01114, 1:1000) antibody were obtained from Wanleibio (China). Anti-Bax (D2E11, 1:1000) (5023) antibody, anti-Bcl2 (2872, 1:1000) antibody and c-Jun (60A8) Rabbit mAb (#9165, 1:500) antibody were obtained from Cell Signaling Technologies (CST). Total cell lysates were prepared using a 1 × sodium dodecyl sulfate buffer. Identical quantities of proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto nitrocellulose filter membranes. After incubation with specific antibodies, the blots were incubated with goat anti-rabbit IgG H&L (HRP) and goat anti-mouse IgG H&L (HRP) for 1 h at room temperature. The proteins were detected using a FluorChem HD2 (Protein Simple, USA).
Tandem mRFP-GFP fluorescence microscopy
Tandem monomeric RFP-GFP-tagged LC3 (tfLC3) (HB-AP2100001, Hanbio, China) was used to monitor autophagic flux as previously reported. LC3-II relocalized to the autophagosomal membranes during autophagy. Thus, the accumulation of mRFP-GFP-LC3 puncta is an effective way to detect autophagosomes. When tfLC3 is located in autolysosomes, this form of LC3 displays only red fluorescence since the GFP signal is sensitive to the acidic condition in the lysosome lumen, whereas the RFP signal is more stable. To evaluate tandem fluorescent LC3 puncta, 48 h after tfLC3 transfection, cells were washed once with 1 × PBS, incubated with EBSS (E2888, Sigma) for the indicated durations and then directly sent out for confocal microscopy analysis. Images of samples were captured using a Zeiss LSM 710 confocal microscope system (Carl Zeiss, Germany) and processed with ZEN LE software (Carl Zeiss).
Subcellular fractionation
Nuclear/cytoplasmic isolation was carried out by using the NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Catalogue Number 78835, Thermo Fisher) according to the manufacturer’s protocol. Subcellular fractions were prepared as follows. Cytoplasmic and nuclear fractions were divided for RNA extraction. GAPDH and U1 were used as qRT-PCR markers of cytoplasmic and nuclear RNAs, respectively.
Transmission electron microscopy
Conventional electron microscopy was performed as previously described [
19]. In brief, cells were fixed with 2.5% glutaraldehyde and then postfixed with 1% osmium tetroxide, dehydrated in a graded ethanol series and embedded in Embed 812 resin. Ultrathin sections were mounted on copper grids and then double-stained with uranyl acetate and lead citrate. The samples were examined and photographed with an FEI Tecnai spirit transmission electron microscope.
RNA-FISH assay
To detect
EGOT and
pre-ITPR1 mRNAs, we used the QuantiGene® ViewRNA ISH Cell Assay Kit (Catalogue Number QVC0001, Thermo Fisher) to perform the QuantiGene ViewRNA FISH assay according to the manufacturer’s protocol.
EGOT,
ITPR1 and
ACTB (as control) hybridization was carried out using cy3, cy5, and 488-nm DNA-oligonucleotide probes in a moist chamber, respectively. After digestion with a working protease solution, slides were incubated with RNase III (AM2290, Life Technologies, USA) or RNase A (AM2272, Life Technologies) for 2 h if RNase enzymatic activity was to be determined. Standard immunofluorescence and imaging were performed by confocal microscopy. The details of the probe sets and corresponding gene sequences are provided in Additional file
1: Table S2.
TUNEL assay
To detect apoptosis in sections of tumor tissues, TUNEL assay was performed according to the manufacturer’s instructions (Catalogue Number 11684795910, Roche) as previously described [
20]. The sections were analyzed by fluorescence microscopy (Olympus, Japan).
RNA pull-down assay
The Flag-MS2bp-MS2bs-based RNA pull-down assay was carried out by using the Anti-Flag M2 Affinity Gel (Catalogue Number A2220, Sigma) as previously described [
21]. In short, lentivirus Flag-MS2bp and lentivirus EGOT MS2bs were cotransfected into breast cancer cells, and the cells were harvested after 48 h. Approximately 1 × 10
7 cells were lysed in soft lysis buffer (20 mM Tris-Cl, pH 8.0, 10 mM NaCl, 1 mM EDTA, and 0.5% NP-40) containing RNasin (80 units/ml). Fifty microliters of Anti-Flag M2 Magnetic beads was added to each binding reaction tube and incubated at 4 °C overnight. The beads were washed three times with lysis buffer and boiled in 1x loading buffer for 10 min. Finally, the retrieved proteins were analyzed by a NanoLC-ESI-MS/MS system (ProtTech, China).
Chromatin immunoprecipitation (ChIP) and ChIP-seq
Analysis of genome-wide NRIP1 and AP-1 occupancy was carried out using specific and internally validated antibodies. We purchased the EZ-ChIP™ Chromatin Immunoprecipitation Kit (Catalogue Number #17–371, Millipore, USA) to perform ChIP and ChIP-seq using MCF7 cells as previously described [
22]. Briefly, MCF7 cells were subjected to hormone deprivation for up to 3 days and then treated with 1 nM E2 or ethanol (control) for 6 h. Approximately 2 × 10
7 cells were used for each ChIP or ChIP-Seq assay. Chromatin DNA precipitated by polyclonal antibodies against AP-1 or NRIP1 was purified with the Universal DNA purification kit (DP214, Tiangen, China) according to the manufacturer’s protocol. Rabbit anti-RIP140 (ab42126, Abcam) and rabbit anti-c-JUN (60A8) (Catalogue Number #9165, CST) antibodies and rabbit IgG (sc-2027, Santa Cruz, USA) were used. ChIP-PCR enrichment of target loci was normalized to input DNA and reported as % input ± s.e.m. ChIP libraries were prepared using ChIP DNA according to the BGISEQ-500ChIP-Seq library preparation protocol. In-depth whole-genome DNA sequencing was performed by BGI (Shenzhen, China). ChIP-seq data were deposited in the NCBI SRA: SRP149488 (
https://www.ncbi.nlm.nih.gov/sra/SRP149488).
Transient transfection of cells
For downregulation of NRIP1 and hnRNPH1 expression, siRNAs targeting NRIP1 and hnRNPH1 were purchased from Sigma. Cancer cells were transfected with 100 pM siRNA using Lipofectamine 2000 (11668–019, Invitrogen, USA). The siRNA sequences are provided in Additional file
1: Table S1. After HeLa and T47D cells (5 × 10
5 cells/well) were cultured in 6-well plates for 24 h, siRNA or the corresponding controls were transfected into the cells by Lipofectamine 2000 reagent in accordance with the manufacturer’s protocol. Total RNA was extracted 48 h after transfection.
Animal experiment
Female athymic BALB/c nude mice (5 weeks old) were obtained from Beijing Vital River Laboratory Animal Technology Co, Ltd. (Beijing, China). All of the animal experiments were performed according to approved protocols and in accordance with the guidelines of the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council). The protocol was approved by the Institutional Animal Care and Use Committee of Center of Harbin Medical University. Approximately 8 × 106 UACC-812 cells resuspended in 0.2 ml of 25% phenol red-free Matrigel (Catalogue Number 356234, Corning) with 0.9% NaCl were injected in the axilla of five-week-old BALB/c mice. After the tumors grew to approximately 300–500 mm3 in size, 15 mg/kg paclitaxel was administered once every 4 days for a total of three courses. Tumor volume was measured once every 2 days by using calipers at the indicated time points. The tumor volume was estimated by the following formula: length × width × width/2. The whole body weight of mice was measured once every 2 days as indicated. All mice were euthanized by intraperitoneal injection of 200 mg/kg pentobarbital at the end of the experiment.
Immunohistochemistry
Immunohistochemical (IHC) detection of ITPR1 and LC3B was performed on each slide. Each section was incubated with anti-LC3B (1:200) antibody or anti-IP3R (1:200) antibody solution. The proportion and intensity of ITPR1 and LC3 staining were evaluated in a series of 10 randomly selected high-power fields (magnification, 400×), which were considered to represent the average expression. The IHC staining intensity was graded as 0 (no staining), 1 (weak staining = light yellow), 2 (moderate staining = yellow brown) or 3 (strong staining = brown). The proportion of positively stained tumor cells in a field was scored as 0 (no positive tumor cells), 1 (fewer than 10% positive tumor cells), 2 (10–50% positive tumor cells) or 3 (more than 50% positive tumor cells). The staining index (SI) for each sample was obtained by multiplying the intensity and proportion values, with a score of less than 4 being classified as low expression.
Library preparation for lncRNA sequencing
A total of 3 μg of RNA per sample was used for downstream RNA sample preparation. Ribosomal RNA was removed using the Ribo-Zero™ Gold kit (Epicentre, Wisconsin, USA). Subsequently, sequencing libraries were generated according to the manufacturer’s recommendations, and the libraries were sequenced on an Illumina HiSeq 2500 platform to generate 100-bp paired-end reads. Raw sequencing and processed RNASeq data from this study have been deposited into the NCBI GEO database under accession number GSE71651 (
http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=obcxosaur xoppwx & acc = GSE71651).
Access and analysis of public data
GDS723, GDS4121, GDS1453 and GSE11324 were downloaded from GEO datasets (
http://www.ncbi.nlm.nih.gov/geo/) and processed according to our previous study [
23]. Genome-wide
EGOT and
ITPR1 expression profiles and clinical pathology information for human cancers were downloaded from The Cancer Genome Atlas (TCGA) (
https://tcga-data.nci.nih.gov/), International Cancer Genome Consortium (ICGC) (
http://icgc.org/) and Cancer Cell Line Encyclopedia (CCLE) (
http://www.broadinstitute.org/ccle). All transcripts were normalized by log
2 transformation. The expression of
EGOT or
ITPR1 was dichotomized using a study-specific median expression as the cut-off to define high values (at or above the median) versus low values (below the median). Guilt-by-association analysis was performed according to our previous study [
23]. Correlations between genes were assessed by Pearson correlation coefficients. Unpaired Student’s t-tests were used to detect significant differences among tumors or between tumor and normal samples. The overall survival (OS) and relapse-free survival (RFS) were calculated as the time from surgery until the occurrence of death and relapse, respectively. The log-rank test was used to examine the survival difference between different patient groups. All statistical tests were two-sided, and
P < 0.05 indicated statistical significance.
Statistical analysis
Data are presented as the mean ± s.d. of at least three independent experiments for each cellular experimental group and at least five independent experiments for each animal group. Student’s t-tests were used to determine statistically significant differences between groups. Correlations between EGOT expression and a pathological response were determined by the chi-square test. All statistical tests were two-sided, and P < 0.05 indicated statistical significance. Statistical analysis was performed using R.3.4 graphics software and GraphPad Prism software (GraphPad Software, USA).
Discussion
Regarding the function of Ai-lncRNA
EGOT, we first reported that low levels of
EGOT expression were significantly correlated with increased tumor size, lymph node metastasis, and Ki-67 expression in human breast cancer [
14]. In this study, we identified an uncharacterized lncRNA,
EGOT, and its roles in enhancing paclitaxel sensitivity by triggering autophagosome accumulation. Mechanistically,
EGOT regulates ITPR1 expression
in cis and
in trans, leading to the activation of autophagy signaling. We also found that
EGOT is transcriptionally regulated by various stress conditions that are associated with paclitaxel resistance. Overall, we provide compelling evidence that in response to stress,
EGOT activates autophagy via ITPR1, which sensitizes paclitaxel cytotoxicity in human cancer (Additional file
2: Figure S8).
Several studies have investigated the role of macrophages in decreasing the cytotoxic effects of chemotherapy and demonstrated a therapeutic window for autophagy inhibition in cancer therapy and prevention [
36,
37]. Paradoxically, a previous study showed that autophagy enhances paclitaxel-induced cell death and that reduced autophagy may contribute to clinical chemotherapeutic resistance in primary breast tumors [
6]. Of note, paclitaxel blocks the activation of PI3K and Vps34 and inhibits the movement and maturation of autophagosomes to induce cell death [
6]. In vitro, autophagosome accumulation sensitizes cells to paclitaxel toxicity [
6]. Using in vivo and in vitro experiments in this study, we found that
EGOT overexpression activated autophagy signaling, which sensitizes cells to paclitaxel, whereas
EGOT knockdown has the opposite effect, in accordance with this previous study. Mechanistically,
EGOT upregulates the expression of ITPR1, which promotes autophagosome accumulation and sensitizes cells to paclitaxel toxicity, thereby highlighting a novel function for these molecules.
Loss or low expression of autophagy-related genes, indicating a decrease of autophagic flux, was associated with poor prognosis [
38,
39]. High expression of
EGOT is significantly associated with favorable OS and RFS in patients in the HMUCC cohort and TCGA and ICGC public datasets. Furthermore, recent study showed that autophagy-related genes/proteins could be possible predictive markers for paclitaxel efficacy in the clinic [
6]. High expression of
EGOT is significantly associated with enhanced paclitaxel sensitivity in patients in the HMUCC cohort and TCGA and ICGC public datasets, while the lack of
EGOT expression indicates resistance to paclitaxel therapy. In addition, high expression of
EGOT indicated an increased complete pathological response ratio following treatment with paclitaxel-containing adjuvant chemotherapy regimens, in contrast to patients with low
EGOT expression in the HMUCC cohort. However, paclitaxel-containing adjuvant chemotherapy sensitivity has only been evaluated in a small study cohort; thus, a multicenter clinical trial with a larger patient number will be performed in a future study to validate these results. Hence, our study provides a proof of concept showing that
EGOT may be a predictive marker of the clinical efficacy of paclitaxel treatment.
LncRNAs can function
in cis to regulate the expression of neighboring genes or
in trans to carry out many roles by various modes [
9]. Compared to other category of long non-coding RNAs, the function of Ai-lncRNAs including
EGOT is still largely unknown. In this study, we clearly demonstrated that
EGOT regulates ITPR1 expression both
in cis and
in trans. On one hand,
EGOT regulates ITPR1 levels via a unique regulatory mechanism involving the formation of a
pre-ITPR1/
EGOT dsRNA that induces
pre-ITPR1 accumulation to increase ITPR1 protein expression
in cis. However, we should mention that although it was initially proposed that lncRNA mainly functions to regulate neighboring gene transcription, other studies have shown that many lncRNAs do not exert such function [
40,
41]. Thus, whether
EGOT regulates any other neighboring gene transcription
in cis awaits further investigation. Furthermore,
EGOT recruits hnRNPH1 to promote ITPR1 expression
in trans. hnRNPH1, a splicing factor, has been shown to regulate alternative splicing and polyadenylation [
42,
43].
EGOT fragment 2 (324–645 nucleotides) in exon 1 was found to bind
pre-ITPR1 mRNA and the hnRNPH1 protein, thereby mediating alternative splicing of
pre-ITPR1 mRNA. hnRNPH1 has been shown to bind G-rich sequences interspersed with adenosines around exons [
28,
42].
ITPR1 contains 59 exons and multiple GGGA/C/G motifs distributed in these exons. Thus, we propose that hnRNPH1 mediates
pre-ITPR1 splicing in human cancer by binding these GGGA/C/G motifs. Taken together, these findings broaden the understanding of the autophagy-associated lncRNA landscape and provide novel insights into the exploration of the mechanism of autophagy regulation in human cancer.
Our data also show that
EGOT is positively and negatively transcriptionally regulated by hypoxia and estrogen-related stress, respectively, in human cancer. Studies have shown that hypoxia can promote tumor invasion, metastasis, autophagy, and angiogenesis, can regulate tumor metabolism and is closely related to the poor prognosis of cancers [
44,
45]. Hypoxia is associated with the expression of numerous noncoding RNAs, including miRNAs and lncRNAs [
46]. In this study,
EGOT was induced in vitro by subjecting different cancer cell lines to hypoxia. Apart from its positive regulation by hypoxia,
EGOT can be transcriptionally repressed by estrogen. Estrogen, a paracrine mediator throughout life, is an important factor in tumorigenesis in humans. Estrogen binds to estrogen receptors to control vast gene networks that are involved in glycolysis, glutaminolysis, oxidative phosphorylation, nutrient sensing and biosynthesis pathways in cancer [
47]. Several lncRNAs have been shown to be regulated by estrogen in breast cancer [
22,
48]. In this study,
EGOT was found to be a direct target of AP-1/NRIP1-mediated transcriptional repression complex in the presence of estrogen. Previous studies have shown that NRIP1 is an obligate co-factor of the estrogen receptor, and germline single-nucleotide polymorphisms (SNPs) near NRIP1 have been associated with ER-positive breast cancer [
49]. Estrogen mediates NRIP1 induction, which subsequently interacts with estrogen receptor AP-1 complexes to directly repress adjacent target genes, including
BCAS4,
IRX4,
GUSB and
MUC1 [
35]. In this study,
EGOT was inhibited by estrogen at physiological concentrations (0–10
− 9 M), which led to a gradual decrease in
EGOT expression; meanwhile NRIP1 expression was gradually increased with time. Knockdown of NRIP1 reversed the estrogen-mediated transcriptional repression of
EGOT expression. Interestingly, a previous study reported that the expression of ITPR1 protein decreased in an estrogen receptor-dependent manner and that the growth of MCF7 cells induced by estrogen was sensitive to pharmacological inhibitors of ITPR1 [
16]. Our study provides a proof of concept indicating that
EGOT is transcriptionally repressed by the AP-1/NRIP1 complex in the presence of estrogen and that this axis may send regulatory feedback signals to repress the excessive activation of autophagy.
In conclusion, these findings broaden comprehensive understanding of the biology function of Ai-lncRNAs. Moreover, our findings demonstrate that EGOT my act as a clinical biomarker of paclitaxel response and that proper regulation of EGOT may be a novel synergistic strategy for enhancing paclitaxel sensitivity, thereby enhancing its clinical benefits for cancer patients.