H3K27 acetylation activated long noncoding RNA RP11-162G10.5 promotes breast cancer progression via the YBX1/GLO1 axis

Abstract

Purpose

Long noncoding RNAs (lncRNAs) orchestrate critical roles in human tumorigenesis. However, the regulatory mechanism of lncRNAs in tissue-specific expressions in breast cancer (BC) remains poorly understood. This study aims to investigate lncRNA role and mechanisms in BC.

Methods

RNA sequencing was used to explore differentially expressed lncRNAs in BC and adjacent tissues. H3K27 acetylation (H3K27ac) chromatin immune-precipitation sequencing (ChIP-seq) data of BC cells from the GEO dataset (GSE85158) was retrieved to identify the H3K27ac activated lncRNAs that were involved in tumorigenesis. RP11-162G10.5 was selected as the target lncRNA for further functional and mechanism study.

Results

In this study, we identified a novel lncRNA RP11-162G10.5, whose overexpression was specifically driven by H3K27ac in luminal breast cancer. And increased RP11-162G10.5 in BC is correlated with poor patient outcomes. RP11-162G10.5 promotes tumor cell proliferation in vitro and in vivo. Mechanistically, RP11-162G10.5 recruits transcriptional factor YBX1 to the GLO1 promoter, consequently activating GLO1 transcription to modulate the progression of BC.

Conclusions

Our findings suggest that the histone modification-activated lncRNA contributes to the oncogenesis of BC. Also, our data reveal a role for RP11-162G10.5 in BC tumorigenesis and may supply a strategy for targeting the RP11-162G10.5 as a potential biomarker and a therapeutic target for breast cancer patients.

Supplementary Information

The online version contains supplementary material available at 10.1007/s13402-022-00756-8.

Background

Breast cancer (BC) is currently the most common malignant cancer worldwide. The number of new cases has been increasing each year, posing a severe threat to women’s life and health [1, 2]. Although several treatments have been developed, the prognosis continues to be poor [3–5]. This may be because the molecular mechanisms underlying the occurrence and development of breast cancer are still not fully understood. Therefore, discovering new effective biomarkers and targets for breast cancer diagnosis and treatment is urgently needed [6].

Long noncoding RNAs (lncRNAs) are a class of RNAs with a length of more than 200 nucleotides, and without the ability to code for protein [7]. LncRNAs act as key roles in epigenetic regulation of gene transcription and translation, and control important biological processes, including human tumorigenesis [8, 9]. Moreover, lncRNAs exhibit tissue-specific expressions than protein coding genes [10]. Epigenetic alterations, such as histone acetylation have been established to play important roles in regulating tissue/cell -specific gene expression [11]. However, the key epigenetic alteration-regulated lncRNAs and their consequences in the progression of breast cancer is still unclear.

In our present study, we screened and identified a novel lncRNA RP11-162G10.5, whose overexpression was specifically driven by H3K27ac in luminal breast cancer. High levels of RP11-162G10.5 in BC tissues is associated with poor patient outcomes. RP11-162G10.5 overexpression promoted cell proliferation both in vitro and in vivo. Mechanistically, RP11-162G10.5 recruited transcriptional factor Y-box protein (YB1) to the GLO1 promoter, consequently inducing GLO1 transcription to modulate the progression of BC.

Methods

Patients and clinical samples

A total of 201 patients who underwent surgery at the Breast Tumor Center, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University were enrolled in this study. The study was approved by the Ethics Boards of the Sun Yat-Sen Memorial Hospital, and was performed in compliance with the Helsinki Declaration. All patients gave written informed consent for publication. The clinical pathological characteristics of BC patients are summarized in Table 1.

Table 1.

Correlation between RP11-162G10.5 expression and clinical characteristics of BC patients

	RP11-162G10.5
Variable	All cases	Low expression N = 98	High expression N = 103	P value
Age (years)				0.726
≤ 45	100	50 (51.0%)	50 (48.5%)
> 45	101	48 (49.0%)	53 (51.5%)
Tumor size (cm)				0.009**
≤ 2	98	57 (58.2%)	41 (39.8%)
> 2	103	41 (41.8%)	62 (60.2%)
Grade (Elston-Ellis)				0.333
I	36	14 (14.3%)	22 (21.4%)
II	82	44 (44.9%)	38 (36.9%)
III	83	40 (40.8%)	43 (41.7%)
TNM Stage (AJCC)a				0.033*
Tis	16	9(9.2%)	7 (6.8%)
I	70	40 (40.8%)	30 (29.1%)
II	105	48 (49.0%)	57 (55.3%)
III	10	1 (1.0%)	9 (8.7%)
Lymphatic metastasis				0.887
Negative	173	84 (85.7%)	89 (86.4%)
Positive	28	14 (14.3%)	14 (13.6%)

Abbreviations: TNM tumor node metastasis

* p < 0.05, ** p < 0.01

^aAmerican Joint Committee on Cancer (AJCC), patients were staged in accordance with the 8th Edition of the AJCC Cancer’s TNM Classification

Cell culture and transfection

Human breast cancer cell lines MCF-7, BT474, MDA-MB-231, and MDA-MB-468 were obtained from American Type Culture Collection (ATCC). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (HyClone, USA) in a humidified atmosphere containing 5%CO₂/95% air at 37 °C.

According to the manufacturer’s protocol, specific siRNA, LNA (Locked Nucleic Acid, QIAGEN, Germany), and negative control were transfected with Lipofectamine 3000 reagent (Invitrogen, USA). After transfection, the cells were cultured for an additional 48 or 72 hours before further treatment. All sequences are listed in Table S1.

RNA isolation and RT-qPCR analysis

Total RNA was extracted from BC cells and sample tissues using TRIzol reagent (Invitrogen, USA). For qRT-PCR, RNA was reverse transcribed to cDNA using a Reverse Transcription Kit (Takara, Japan). Real-time PCR analyses were performed with SYBR Premix Ex TaqII (Takara, Japan). Results were normalized to the expression of β-actin. The primer sequences are listed in Table S2.

Plasmid constructs

The expression plasmids of RP11-162G10.5, antisense, and fragments of RP11-162G10.5 were synthesized and cloned into the expression vector pcDNA3.1 (IGE Biotechnology, China). The GLO1promoter-driven luciferase reporter plasmid was cloned into the expression vector pGL4.17.

Cytoplasm/nucleus fractionation

Separation of the nuclear and cytosolic fractions was performed using the NE-PER™ Nuclear and Cytoplasmic Extraction Reagent Kit (Thermo Scientific, USA), according to the manufacturer’s instructions.

Rapid amplification of cDNA ends (RACE)

The 5′ and 3’ RACE assays were performed following the instructions of a SMARTer^® RACE Kit (Takara Bio, USA) according to the manufacturer’s instructions. The RACE PCR products were separated on a 1% agarose gel and subjected to bidirectional sequencing. RP11-162G10.5-specific primers were designed and synthesized for the nested PCR.

Cell proliferation assay

Cell proliferation was measured with MTS and EdU assays, following the instructions. BC cells were transfected with LNA or overexpression plasmid, followed by seeding in 96-well plates at a density of 3 × 10³ cells per well and observed for 96 hours. The cells were then treated with MTS solution according to the manufacturer’s instructions from the MTS kit (APExBIO, USA). The number of viable cells was measured at OD490 with a microplate reader (BioTek, USA). For EdU assays, the solution was added to the cells according to the manufacturer’s instructions from the EdU kit (RIB&BIO, China). The images were obtained with an Olympus laser scanning microscope system.

Colony formation assay

Transfected cells were plated at a density of 1000 cells per well in 6-well plates and cultured with a complete medium for 2 weeks, and then stained with 1% crystal violet and counted.

RNA pull-down assays

The full-length sense and antisense of RP11-162G10.5 were prepared by in vitro transcription using MEGAscript^TM T7 Transcription kit (Invitrogen, USA) and were labeled with Bio-16-UTP (Invitrogen, USA). Then RNA pull-down assays were performed by a Pierce Magnetic RNA-Protein Pull-Down Kit (Thermo Scientific, USA) according to the manufacturer’s instruction.

RNA binding protein immunoprecipitation (RIP) assays

5 × 10⁶ – 1 × 10⁷ cells were lysed with lysis buffer containing 1 mL PierceTM IP lysis buffer (Thermo Scientific, USA), 10 μL Halt protease inhibitor (Thermo Scientific), and 10 μL EDTA (Thermo Scientific), and then incubated on ice for 30 min. The supernatant was retained after centrifugation at 16,000 ×g for 30 min. Then 10% of the supernatant was removed as “input”, and the remaining supernatant was divided into two parts incubated with target antibodies or negative control normal rabbit IgG overnight at 4 °C overnight with rotation. Next, RNA protein complexes were adsorbed by the Protein A Dynabeads^TM beads (Thermo Scientific) and detected by qRT-PCR. The antibody isotype rabbit IgG was used as negative control, and total RNAs was utilized as input control.

Chromatin isolation by RNA purification (ChIP) assays

ChIP assay was performed with an EZMagna ChIP Kit (Merck Millipore, USA), following the manufacturer’s instructions. A total of 1 × 10⁷ cells were collected and crosslinked in 1% formaldehyde solution for 5 min at room temperature, followed by the addition of 125 mM glycine for 10 min. The nuclear lysis was sheared by a sonication system-Bioruptor (Diagenode, Belgium) ranged from 200 to 400 bp. One microgram of normal IgG was used as the negative control. Acetyl-Histone H3 Lys27 (3,083,478, Merck Millipore, USA) and anti-YBX1 (ab76149, Abcam, USA) antibodies were used for each immunoprecipitation. The IPs were eluted and reverse cross-linked, after which the DNA fragments were purified. Immunoprecipitated and input DNAs were subjected to qRT–PCR analysis. The ChIP primer sequences are listed in Table S3.

Luciferase reporter assay

The indicated regions of the GLO1 promoter were directly inserted into the pGL4.10 luciferase reporter plasmid. All constructs were verified by DNA sequencing. The luciferase assays were performed using a Dual-luciferase reporter assay kit (Promega, USA) according to the manufacturer’s protocol. The relative luciferase activity was normalized with renilla luciferase activity.

Tumor xenografts

Female nude mice (4–6-weeks-old) were bred and maintained under SPF barrier facilities at the Animal Experiment Center of Sun Yat-sen University. All procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Sun Yat-sen University and conformed to the legal mandates and national guidelines for the care and maintenance of laboratory animals. MCF-7 cells stably transfected with sh-RP11-162G10.5 (sh-lnc1 and sh-lnc2) or sh-control (sh-NC) were re-suspended at 1 × 10⁷ cells/mL. To establish the subcutaneous xenograft tumor model, a total of 100 μL of suspended cells were injected subcutaneously into the right armpit of nude mice. Tumor growth was evaluated by monitoring tumor volume (TV = 1/2 length x width²) every 5 days for 1 month. When the xenografts became palpable (xenograft volume reached 200 mm³) the mice were killed, and their tumor xenografts were harvested for further evaluation.

Statistical analysis

All statistical analyses were performed using SPSS 25.0 software (IBM, SPSS, USA). The significance of differences between two groups was estimated by two-tailed Student’ s t-test. One-way ANOVA with Bonferroni’s test was applied for multiple comparisons. Overall survival rates were calculated by the Kaplan Meier method with the log-rank test. Survival data were evaluated using the univariate and multivariate Cox proportional hazards model. Variables with a value of P < 0.05 in univariate analysis were used in subsequent multivariate analyses based on Cox regression analyses. Two-sided P-values were calculated. A P value <0.05 was considered to be statistically significant.

Results

H3K27 acetylation activated RP11-162G10.5 is highly expressed in BC and is correlated with poor prognosis

To identify lncRNAs that potentially drive the tumorigenesis of BC, lncRNA sequencing was used to explore differentially expressed lncRNAs in breast cancer (BC) and adjacent tissues. A total of 329 lncRNAs were significantly expressed (fold change >2) (Fig. 1A). Given that tissue- and cell-specific expression patterns are often driven by epigenetic alterations [10], which can be marked with H3K27ac modifications to promoters, we retrieved H3K27ac ChIP-seq data of BC cells from the GEO dataset (GSE85158) to identify the H3K27ac activated lncRNAs that were involved in tumorigenesis. Focusing on the noncoding genome, we found that 6733 and 3569 lncRNA loci had H3K27ac modification in two samples of MCF-7 cells, respectively. We then intersected the lncRNA sequencing data with H3K27ac ChIP-seq data and noted that eight lncRNAs were overexpressed with higher H3K27ac peaks in MCF-7 cells (Fig. 1B and C). Among these lncRNAs, RP11-162G10.5 exhibited the highest expression and the highest H3K27ac signals (Fig. 1D). Subsequently, the eight lncRNAs were validated by qRT-PCR analysis. Among these candidate lncRNAs, RP11-162G10.5 showed the highest expression in MCF-7 cells (Fig. 1E). We then further measured the expression pattern of RP11-162G10.5 in 60 paired BC tissues and adjacent normal tissues by qRT-PCR. As expected, the expression of RP11-162G10.5 was elevated in 83.3% (50 of 60 paired) of BC tissues (Fig. 1F). In addition, RP11-162G10.5 were significantly up-regulated in MCF-7 and BT474 cells compared to other breast cancer cell lines (Fig. 1G). These results suggested that RP11-162G10.5 was specifically elevated in luminal subtype. Thus, RP11-162G10.5 was selected as the target lncRNA for further study.

Fig. 1.

RP11-162G10.5 is highly expressed in BC and is correlated with poor prognosis. (A) RNAseq analysis showed the differential expression of lncRNAs in breast cancer tissues and adjacent tissues. (B) The number of overlapped lncRNAs upregulated in tumor tissues and lncRNAs activated by H3K27ac in the GEO database. (C) Expression change of lncRNAs interested with H3K27ac ChIP-seq data; 8 lncRNAs were overexpressed with higher H3K27ac peaks. (D) RP11-162G10.5 exhibited the highest H3K27ac signals. (E) Validated lncRNAs by qRT-PCR analysis. (F) qRT-PCR analysis of RP11-162G10.5 expression in breast tumor tissues (n = 60) paired with adjacent tissues. (G) qRT-PCR analysis of RP11-162G10.5 expression in a normal human breast cell line (MCF10A) and breast cancer cell lines. (H-I) ISH analysis of RP11-162G10.5 expression in paraffin-embedded tumor sections of BC patients (n = 201). Representative images from clinical cases (H) and H-score (I) are shown. (J-K) H-score of TNM stage and tumor size. (L) Overall survival was analyzed and compared between patients with high (H score > 100, n = 103) and low (H-score ≤ 100, n = 98) levels of RP11-162G10.5 expression. (M) Multivariable Cox proportional-hazard analysis of prognostic variables was performed with BC patients (n = 201). Error bars represent standard deviations of three independent experiments. Ns, no significance, *p < 0.05, **p < 0.01, ***p < 0.001

Furthermore, to validate the clinic pathological factors of RP11-162G10.5 expression in BC patients, we detected RP11-162G10.5 levels by in-situ hybridization (ISH) and found higher RP11-162G10.5 levels in luminal BC tumor tissue than in normal adjacent tissues (Fig. 1H and I). In addition, high RP11-162G10.5 expression was closely correlated with larger tumor size and higher pathological tumor stage (Fig. 1J, K and Table 1). Importantly, Kaplan-Meier analysis demonstrated that BC patients with elevated RP11-162G10.5 expression had shorter overall survival (OS) (Fig. 1L), indicating that increased RP11-162G10.5 in BC was associated with poor patient outcomes. Furthermore, the univariate and multivariate Cox regression model revealed that RP11-162G10.5 expression and tumor size were independent factors associated with poor prognosis in BC (Fig. 1M and Table 2). In short, RP11-162G10.5 served as an important oncogene, and its high expression was associated with poor prognosis.

Table 2.

Univariate and multivariate analysis of Overall Survival (OS) in BC patients (n = 201)

Clinical Variables	Case number	P value	HR	95% CI
Univariate analysis
RP11-162G10.5 expression (high versus low)	103/98	0.006**	2.684	1.336–5.390
Age (> 45 versus ≤45)	101/100	0.068	2.030	0.948–4.347
Tumor size (> 2 cm versus ≤2 cm)	103/98	0.006**	2.887	1.360–6.126
TNM stage (II + III versus I)	115/70	0.043*	2.360	1.029–5.413
Grade (III versus I + II)	83/118	0.381	1.336	0.699–2.552
LNM (Positive versus Negative)	28/173	0.781	1.144	0.443–2.951
Multivariate analysis
RP11-162G10.5 expression (high versus low)	103/98	0.046*	2.090	1.013–4.313
Tumor size (> 2 cm versus ≤2 cm)	103/98	0.022*	2.671	1.151–6.197
TNM stage (II + III versus I)	115/70	0.083	2.099	0.909–4.847

Abbreviations: LNM lymph node metastasis, HR hazard ratio; 95% CI 95% confidence interval, TNM tumor node metastasis. * p < 0.05,  ** p < 0.01

We further performed cytoplasm/nucleus RNA fractionation from a subcellular distribution assay. RP11-162G10.5 was mainly located in the nucleus in the luminal type of BC cells (Fig. 2A and B). This finding was further validated by fluorescence in situ hybridization (FISH) analysis (Fig. S1A). Next, rapid amplification of cDNA ends (RACE) was performed to identify the full sequence of RP11-162G10.5. The full-length sequence is 925 nt, 99 nt longer than the sequence in the UCSC database (826 bp, with poly (A) tail, Fig. 2C). Then we explored the mechanism of RP11-162G10.5 overexpression in BC, particularly in Luminal subtype. We firstly discovered high enrichment of H3K27Ac at the promoter of RP11-162G10.5 by using UCSC Genome Bioinformatics Site (http://genome.ucsc.edu/). Then, we observed a gain of H3K27Ac at the promoter of RP11-162G10.5 gene in MCF-7 and BT474 cells by ChIP assays (Fig. 2D). More importantly, the expression of RP11-162G10.5 was increased when MCF-7 and BT474 cells were treated with a total histone deacetylase inhibitor, trichostatin A (TSA), and a subtype of histone deacetylase inhibitor MS-275 (Fig. 2E), confirming that abnormal histone acetylation modification at the promoter of RP11-162G10.5 regulates its expression. MS-275 has been used as the histone deacetylase-1 (HDAC1) and histone deacetylase-3 (HDAC3) inhibitors. Therefore, we detected the expression of RP11-162G10.5 by silencing HDAC1 and HDAC3 in BC cells. qPCR analysis showed that the knockdown of HDAC1 and HDAC3 activated RP11-162G10.5 expression in MCF-7 and BT474 cells (Fig. 2F), suggesting that HDAC1 and HDAC3 mainly regulate the expression of RP11-162G10.5. Collectively, these data identified that the level of RP11-162G10.5 was frequently elevated in BC. Abnormal gain of histone sites H3K27ac of the promoter region of RP11-162G10.5 gene might partially account for the significant activation of RP11-162G10.5.

Fig. 2.

RP11-162G10.5 cellular location, distribution, full-length, and histone acetylation regulation. (A-B) The expression level of RP11-162G10.5 in the subcellular fractions of MCF-7 (A) and BT474 cells (B) detected by qRT-PCR. MALAT1 and β-actin were used as nuclear and cytoplasmic markers, respectively. (C) The full sequence of RP11-162G10.5 was confirmed by Race. (D) The UCSC Genome Bioinformatics Site (http://genome.ucsc.edu/) showed high enrichment of H3K27Ac at the promoter of RP11-162G10.5. ChIP assays detected the level of H3K27Ac at the promoter of RP11-162G10.5 in BC cells. (E-F) The expression level of RP11-162G10.5 treated with deacetylase inhibitors was detected by qRT-PCR. **p < 0.01, ***p < 0.001

RP11-162G10.5 regulates BC cell proliferation in vitro and in vivo

To explore the role of RP11-162G10.5 in BC, the LNA-mediated knockdown and plasmid-mediated overexpression were used to exogenously manipulate the expression of RP11-162G10.5 in BC cell lines (Fig. 3A and Fig. S2A). MTS assays showed that knockdown of RP11-162G10.5 significantly inhibited cell proliferation (Fig. 3B and C). In contrast, overexpressed RP11-162G10.5 promoted cell proliferation (Fig. S2B and S2C). This was further confirmed by colony formation assay (Fig. 3D and E; Fig. S2D and S2E) and EdU assays (Fig. 3F). In addition, the cell apoptosis analysis displayed that silencing RP11-162G10.5 increased cell apoptosis (Fig. 3G), which was consistent with the result of Edu proliferation assays. These results suggest that RP11-162G10.5 might promote BC progression by regulating cancer cell proliferation and apoptosis.

Fig. 3.

RP11-162G10.5 regulates BC cell proliferation in vitro. (A) The efficiency of RP11-162G10.5 knockdown in MCF-7 and BT474 cells was verified by qRT-PCR assays. (B-C) Cell viability after RP11-162G10.5 knockdown in MCF-7 (B) and BT474 (C) cells was assessed by MTS assays. (D-E) The effect of RP11-162G10.5 knockdown on colony formation was counted in MCF-7 and BT474 cells (D). The histogram analysis (E) showed the mean ± SD of colonies from three independent experiments. (F) Representative images and histogram analysis of EdU assays after RP11-162G10.5 knockdown in MCF-7 and BT474 cells. (G) The apoptosis of MCF-7 and BT474 cells analyzed by flow cytometry. ***p < 0.001

To further validate the oncogenic roles of RP11-162G10.5 in BC progression in vivo, a subcutaneous xenograft tumor model was established. MCF-7 cells with stable expression of sh-RP11-162G10.5 (sh-lnc1 and sh-lnc2) or sh-control (sh-NC) were injected into the right armpits of nude mice (n = 5 per group) for the formation of BC xenografts. Within a long evaluation period of 30 days post the injection, knockdown of RP11-162G10.5 markedly suppressed tumor growth and tumor weight (Fig. 4A-C). In support of the oncogenic role of RP11-162G10.5, Ki67 staining showed that downregulation of RP11-162G10.5 inhibited tumor cell proliferation in vivo (Fig. 4D and E). These findings indicate that RP11-162G10.5 maybe an oncogenic lncRNA in breast cancer and regulate BC cell proliferation.

Fig. 4.

RP11-162G10.5 regulates BC growth in vivo. (A) Gross appearance of xenograft tumors in sh-NC, sh-lnc1, and sh-lnc2 groups (n = 5). (B-C) Tumor volumes (B) and weights (C) were measured in the indicated groups (n = 5). (D-E) Representative images (D) of IHC for Ki-67. Histogram analysis (E) revealed that RP11-162G10.5 was associated with Ki-67 expression (n = 5)

RP11-162G10.5 directly interacts with YBX1 protein to promote BC cell proliferation

It has been reported that lncRNAs orchestrated biological functions by interacting with target proteins [10]. Therefore, to identify RP11-162G10.5-binding proteins, RNA pull-down assays were performed by using biotin-labeled RP11-162G10.5 and antisense control in MCF-7 cells (Fig. 5A). Subsequent silver staining showed a distinct band weighted at ~50 kDa. The specific band was cut off and subjected to mass spectrometry (MS). As a result, Y-box binding protein 1 (YBX1) was identified as a specific RP11-162G10.5-interacting protein from the candidate proteins. Western blot analysis indicated that RP11-162G10.5 was associated with YBX1 (Fig. 5B). Whether this candidate protein could directly interact with RP11-162G10.5 in vivo was further evaluated by RIP assays. The results displayed a significant interaction of RP11-162G10.5 with YBX1 in MCF-7 cells (Fig. 5C). These data suggested that RP11-162G10.5 may promote BC proliferation by interacting with transcriptional factor YBX1. In addition, catRAPID algorithm predicted a specific binding motif (200 nt–400 nt) of RP11-162G10.5 that interacts with YBX1 (Fig. 5D). To further identify the YBX1 binding motif of RP11-162G10.5, a series of truncations were constructed based on the secondary structure of RP11-162G10.5 (Fig. S3A and S3B). The RNA pull-down assay displayed that RP11-162G10.5 truncation containing nt 350–400 retained the similar capability to bind to YBX1 as efficiently as the full-length lncRNA (Fig. 5E and F).

Fig. 5.

RP11-162G10.5 directly interacts with YBX1 protein to promote BC cell proliferation. (A) RP11-162G10.5 sense and antisense RNAs were used in pull-down assays in MCF-7 cells, followed by electrophoresis and silver staining. The band in the red frame specifically precipitated by RP11-162G10.5 but not by antisense RNA was submitted for mass spectrometric detection. (B) Western blotting analysis of proteins from RP11-162G10.5 pull-down assays revealed that RP11-162G10.5 specifically interacts with YBX1. (C) RIP assays showed that RP11-162G10.5 binds to YBX1. (D) The potential binding sequence of RP11-162G10.5 for YBX1 is predicted by catRAPID algorithm, demonstrating a specific binding area of RP11-162G10.5. (E-F) Nucleotide 350–400 fragment contains the binding motif of RP11-162G10.5 with YBX1. (G-H) The qPCR and Western blot assays detected the expression of YBX1 after the knockdown of RP11-162G10.5. (I) MTS assays showed that knockdown of YBX1 could suppress cell proliferation and reverse RP11-162G10.5-mediated growth promotion. Ns, no significance, *p < 0.05, **p < 0.01

YBX1 is a member of the DNA and RNA binding protein family, with an ancient and conservative cold shock domain [12, 13]. YBX1 has been reported to act as a transcription factor, having an established role in tumorigenesis [14, 15]. To explore how YBX1 was regulated by RP11-162G10.5, we firstly examined the levels of YBX1in MCF-7 cells and found that knockdown of RP11-162G10.5 did not affect the mRNA or protein levels of YBX1 (Fig. 5G and H). Additionally, we explored whether the interaction between RP11-162G10.5 and YBX1 contributed to BC progression and discovered that depletion of YBX1 expression could partly rescue si-lncRNA RP11-162G10.5-mediated inhibition of cell proliferation (Fig. 5I), indicating that YBX1 has a key role in RP11-162G10.5 mediated BC cell proliferation. Collectively, RP11-162G10.5 interacted with YBX1 and may promote BC progression.

RP11-162G10.5 promotes GLO1 transcription by recruiting YBX1 to GLO1 promoter

To ascertain the downstream signal of RP11-162G10.5/YBX1-associated pathway which contributes to breast cancer progression, RNA transcriptome sequencing in MCF-7 cells transfected with control LNA or RP11-162G10.5 LNA was performed. A total of 752 genes with highly significant expression changes post-LNA-mediated knockdown of RP11-162G10.5 treatment was observed (fold change ≥2) (Fig. 6A). Among them, 549 differentially up-regulated genes and 203 down-regulated genes were identified, which were further analyzed by KEGG enrichment (Table S4). A pyruvate metabolism pathway was one of the most significantly regulated pathways (p < 0.05), with three target genes of the pyruvate metabolism pathway were identified (Fig. 6B, Table S5). Among these three target genes, the downregulated fold-change of GLO1 expressions between the control group and si-lncRNA group has the most significant differences (Table S5, Fig. 6C). Thus, we choose GLO1/pyruvate metabolism signal for further research. Aberrant activation of GLO1 in tumor cells gives rise to dysregulation of a series of genes mainly involved in the regulation of proliferation and apoptosis [16–19]. We further explored the function of GLO1 in breast cancer, and found that down-regulation of GLO1 could inhibit the proliferation of MCF-7 cells (Fig. S4A). Kaplan-Meier analysis revealed that BC patients with high levels of GLO1 expression displayed a decline in OS compared to those with low levels of GLO1 from the GEO database (Fig. S4B). Further qRT-PCR analysis revealed that knockdown of RP11-162G10.5 significantly inhibited GLO1 expression in MCF-7 cells (Fig. 6D), whereas the overexpression of RP11-162G10.5 upregulated GLO1 levels (Fig. 6E). Additionally, the knockdown of GLO1 expression could partly rescue overexpressed RP11-162G10.5-mediated upregulation of GLO1 levels in MCF-7 cells (Fig. 6E). More importantly, the knockdown of GLO1 could partly reverse the promotion effects of overexpressed RP11-162G10.5 levels in breast cancer cell proliferation (Fig. 6F). Collectively, these results confirmed that the increased expression of RP11-162G10.5 in breast cancer enhanced the malignant behaviors of tumor cells by regulating of GLO1 expression.

Fig. 6.

Downstream genes of RP11-162G10.5. (A) Volcano plot analysis for all genes with altered expression after knockdown of RP11-162G10.5. (B) KEGG pathway analysis of significant expression after knockdown of RP11-162G10.5. (C) The upregulation and down-regulation of the top 30 genes were analyzed in heatmap after knockdown of RP11-162G10.5. (D) Altered mRNA levels of GLO1 were detected by qRT-PCR in knockdown RP11-162G10.5. (E) Relative expression was determined in RP11-162G10.5-overexpressing cells simultaneously transfected with GLO1 siRNA by qRT-PCR. (F) MTS assays showed that knockdown of GLO1 could suppress cell proliferation and reverse RP11-162G10.5-mediated growth promotion

To further investigate the regulation of YBX1 on GLO1 expression, the level of GLO1 in MCF7 cells was detected when YBX1 was manipulated through transfection with siYBX1. Western blot analysis showed that knockdown of YBX1 inhibited GLO1 expression (Fig. 7A). Furthermore, the upregulation of GLO1 levels by overexpressing RP11-162G10.5 in MCF-7 cells was reversed by the treatment of siYBX1 (Fig. 7B). YBX1 has been reported to regulate gene transcription via binding to the promoter regions of target genes [20, 21]. Next, we performed the ChIP assay using YBX1-specific antibodies. Compared to the sample bounded with IgG, we found YBX1 bound complex showed a remarkable enrichment in the GLO1 promoter (Fig. 7C). Then, we examined whether RP11-162G10.5 knockdown affected YBX1 enrichments in the promoter regions of GLO1gene. ChIP assays followed by qPCR analysis demonstrated that silencing of RP11-162G10.5 in MCF7 cells inhibited the enrichment of YBX1 in the promoter of GLO1 gene (Fig. 7D). On the contrary, the elevated expression of RP11-162G10.5 promoted YBX1 binding to the GLO1 promoter (Fig. 7E). In addition, the silence of YBX1 expression reversed the binding of YBX1 to the GLO1 promoter via overexpression of RP11-162G10.5 in MCF7 cells (Fig. 7E). Further luciferase assay revealed that GLO1 transcriptional activity could be downregulated by siRP11-162G10.5 (Fig. 7F), and increased by overexpression of RP11-162G10.5 (Fig. 7G). Additionally, the inhibition of YBX1 expression affected the transcriptional activity of GLO1 via overexpression of RP11-162G10.5 in MCF7 cells (Fig. 7G). Collectively, these findings demonstrated that the elevated expression of RP11-162G10.5 in breast cancer promoted tumor cell proliferation by transcriptional activation of GLO1 through interaction with YBX1.

Fig. 7.

RP11-162G10.5 interacts with YBX1 to promote GLO1 transcription. (A) The effects of si-YBX1 on GLO1 expression detected by Western blot. (B) The promotion of GLO1 by RP11-162G10.5 was reversed by knockdown of YBX1, based on qRT-PCR. (C) ChIP assays with anti-YBX1 antibody or IgG were performed to verify the binding between YBX1 and the GLO1 promoter. (D) ChIP assays with anti-YBX1 antibody or IgG were performed after the knockdown of RP11-162G10.5. (E) ChIP assays with anti-YBX1 antibody or IgG were performed after overexpression of RP11-162G10.5 and si-YBX1. (F) Luciferase activity assays were performed after the knockdown of RP11-162G10.5. After 48 hours of transfection, firefly luciferase activity was detected and normalized by Renilla luciferase activity. (G) Luciferase activity assays were performed after overexpression of RP11-162G10.5 and si-YBX1. After 48 hours of transfection, firefly luciferase activity was detected and normalized by Renilla luciferase activity. (H) A proposed model medicated by RP11-162G10.5 in proliferation progression of BC

Discussion

LncRNAs exert crucial biological processes in the inflammatory response, cell proliferation, differentiation and survival [22], which has been observed in diverse types of cancers, including breast cancers [23, 24]. Our research group has previously identified several breast cancer-associated lncRNAs (e.g., Uc003xsl.1, NKILA, AFAP1) involved in regulating tumor cell proliferation, migration, invasion and therapeutic resistance, etc. [25–27]. However, the regulatory mechanism of lncRNAs in tissue-specific expressions in breast cancers remains poorly understood. Epigenetic alterations, such as histone acetylation, have been reported to play critical roles in regulating tissue- and cell-specific gene expression [28–30]. Whether epigenetic modification could also regulate lncRNA expression in breast cancers, which tend to display higher tissue-specific expressions than protein coding genes [31, 32], remains unknown. Hence, in this study, through integrative analysis of RNA-seq and H3k27ac ChIP-Seq data, we identified a lncRNA RP11-162G10.5, whose overexpression was specifically driven by H3K27ac in luminal breast cancer. RP11-162G10.5 overexpression promoted tumor malignancy progression, and high levels of RP11-162G10.5 in breast cancer tissues correlated with poor outcomes in luminal breast cancer patients. Mechanistically, RP11-162G10.5 recruits transcriptional factor YBX1 to the GLO1 promoter, consequently activating GLO1 transcription to modulate the progression of breast cancer.

LncRNAs are known to regulate signal transduction or transcription by directly interacting with their counterpart proteins in these processes [33]. In this study, YBX1 was identified to be a key protein that directly binds lncRNA RP11-162G10.5. YBX1 has a nucleic acid-binding common domain in the gene promoter, CCAAT-box, which is a high consensus sequence in eukaryotes. YBX1 plays an important role in modulating cell signaling transduction, transcription, and tumorigenesis [34–36]. Furthermore, YBX1 serves as a transcriptional activator, binding to the promoter region of genes, and mediates the transcription of target genes [10]. Studies have shown that YBX1 directly activates cap-independent translation of mRNAs encoding Snail1 and other transcriptional factors involved in the down-regulation of cell proliferation-related genes and activation of mesenchymal genes [20]. Moreover, YBX1-induced activation of PIK3CA transcription upregulates PIK3CA expression and activates PI3K/AKT signal pathway, thus promoting the growth of tumor cells [13, 37]. In this study, we found that epigenetic interaction of RP11-162G10.5 with YBX1 is essential to promote breast cancer progression. Three lines of evidence support this conclusion: (i) knockdown of RP11-162G10.5 in MCF7 cells did not affect the mRNA or protein levels of YBX1; (ii) silence of YBX1 inhibited the proliferation of MCF7 cells; (iii) knockdown of YBX1 expression could partly rescue si-lncRNA RP11-162G10.5-mediated inhibition of tumor cell proliferation.

YBX1 drives the expression of a diverse set of genes [34–36]. RNA-seq analysis identified glyoxalase 1 (GLO1) was the downstream effector of RP11-162G10.5/YBX1 axis. GLO1 is a member of the glyoxalase system located in the cytoplasm of eukaryotes, which catalyzes the glutathione-dependent removal of the endogenous reactive dicarbonyl metabolite, methylglyoxal (MG) [38]. GLO1 is the critical enzyme in the anti-glycation defense. MG is formed mainly as a side product of anaerobic glycolysis, and is an oncometabolite involved in metabolic reprogramming. In the non-malignant state, GLO1 acts as a tumor suppressor protein. Increased GLO1 expression facilitates the growth of tumors with high glycolytic activity, and is considered as a biomarker of tumor growth. GLO1-mediated suppression of MG glycation has an important role in tumor growth and multidrug resistance [39]. Over-activation of GLO1 was found in many different types of cancers [40–42]. This study also found high expression of GLO1 in breast cancer, and the knockdown of GLO1 inhibited tumor cell proliferation. RP11-162G10.5 induced GLO1 transcription by recruiting YBX1 to the GLO1 promoter region, and then triggered the transcriptional activation of GLO1, thus facilitating breast cancer cell proliferation.

In conclusion, abnormal histone modification-mediated activation of a novel lncRNA RP11-162G10.5 promotes breast cancer cell proliferation by transcriptional activation of GLO1 through interaction with YBX1. These data revealed a role of RP11-162G10.5 in breast cancer tumorigenesis, and provide a strategy for targeting RP11-162G10.5 as a potential biomarker and a therapeutic target for patients with breast cancer (Fig. 7H).

Authors’ contributions

Conception and design: Y. Xu, H.R. Yao. Development of methodology: Y. Xu, N. Xie, R.H. Zhang. Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): Y. Xu, N. Xie, R.H. Zhang, Z.F. Bi. Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): Y. Xu, N. Xie, R.H. Zhang, W. Ren, K.Y. You. Writing, review, and/or revision of the manuscript: N. Xie, Y. Xu, H.R. Yao. Administrative, technical, or material support (i.e., reporting or organizing data, constructing database): H.R. Yao, H. Hu. Study supervision: H.R. Yao, Y. Xu.

Funding

This study was supported by the National Natural Science Foundation of China (Grant No. 81972471, 82203787, 82173232), Natural Science Foundation of Guangdong Province, China (No. 2020A1515010115). Guangzhou Municipal Science and Technology Project (Grant No. 202201010959, 202206010078, 202201020574), Sun Yat-Sen University Clinical Research 5010 Program (Grant No. 2018007), Sun Yat-Sen Clinical Research Cultivating Program (Grant No. SYS-C-201801), Guangdong Medical Science and Technology Program (Grant No. A2020558).

Data availability

The datasets analyzed here are available from the Gene Expression Omnibus data base (GSE85158) and all other data generated and/or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethical approval and consent to participate

All human studies were conducted under the approval of the Ethics Committees of the Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University. The written informed consent was obtained from all participants. All animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) of Sun Yat-sen University and conformed to the legal mandates and national guidelines for the care and maintenance of laboratory animals.

Competing interests

The authors declare no potential conflicts of interest.