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LINC00504 Promotes the Malignant Biological Behavior of Breast Cancer Cells by Upregulating HMGB3 via Decoying MicroRNA-876-3p

Authors Yu H, Dong L, Wang H, Zhang Y, Wang Z, Wang C, Xia H, Bao H

Received 9 August 2020

Accepted for publication 26 January 2021

Published 22 February 2021 Volume 2021:13 Pages 1803—1815

DOI https://doi.org/10.2147/CMAR.S276290

Checked for plagiarism Yes

Review by Single anonymous peer review

Peer reviewer comments 3

Editor who approved publication: Professor Bilikere Dwarakanath


Hao Yu,1 Liqian Dong,2 Hongyu Wang,3 Yang Zhang,3 Zhuo Wang,3 Can Wang,3 Hong Xia,1 Huizheng Bao1

1Department of Hematology, Jilin Cancer Hospital, Changchun, Jilin, 130012, People’s Republic of China; 2Department of Nephrology, Jilin Province FAW General Hospital, Changchun, Jilin, 130013, People’s Republic of China; 3Department of Medical Oncology, Jilin Cancer Hospital, Changchun, Jilin, 130012, People’s Republic of China

Correspondence: Huizheng Bao
Department of Hematology, Jilin Cancer Hospital, 1018 Huguang Road, Changchun, Jilin, 130012, People’s Republic of China
Email hemat_baohuizheng@163.com

Purpose: Long intergenic non-protein coding RNA 504 (LINC00504) is a long non-coding RNA that has an important regulatory role in a variety of human cancers. In this study, LINC00504 expression in breast cancer tissues and cell lines was detected. Studies were also conducted to determine the impact of LINC00504 on the tumor behavior of breast cancer cells. The potential mechanisms underlying the oncogenic role of LINC00504 in breast cancer cells were elucidated in detail.
Methods: Expression of LINC00504 in breast cancer was analyzed by quantitative real-time polymerase chain reaction. The effects of LINC00504 on proliferation, apoptosis, in vitro migration and invasion, and in vivo tumor growth were elucidated using Cell Counting Kit-8 assay, flow cytometry, Transwell assays, and tumor xenograft models, respectively. Bioinformatics analyses in conjunction with RNA immunoprecipitation, luciferase reporter assays, and rescue experiments were conducted to investigate the underlying molecular mechanisms.
Results: LINC00504 was upregulated in breast cancer tissues and cell lines. Knocking down LINC00504 suppressed breast cancer cell proliferation, migration, and invasion and facilitated apoptosis in vitro. In addition, tumor growth in vivo was significantly inhibited by LINC00504 depletion. Regarding the underlying mechanism, LIN00504 could function as a competing endogenous RNA in breast cancer by sponging microRNA-876-3p (miR-876-3p), resulting in the upregulation of high mobility group box 3 (HMGB3). Rescue experiments further revealed that miR-876-3p downregulation or HMGB3 upregulation effectively reversed the inhibitory effects of LIN00504 deficiency on breast cancer cells.
Conclusion: The LIN00504-miR-876-3p-HMGB3 axis shows carcinogenic effects in modulating the biological behavior of breast cancer cells. This pathway may represent an effective target for CRC diagnosis and anticancer therapy.

Keywords: high mobility group box 3, long intergenic non-protein coding RNA 504, therapeutic target, ceRNA

Introduction

Breast cancer accounts for approximately 29% of all malignant tumors in women and is the second leading cause of cancer-related mortalities among women worldwide.1,2 Annually, there are approximately1.7 million new breast cancer diagnoses and 500,000 deaths.3 Currently, surgical excision remains the primary therapeutic intervention for breast cancer.4 Treatment and patient prognosis for breast cancer patients has significantly improved over the past decade with the use of radiochemotherapy, endocrine therapy, and other anticancer treatments. However, improving long-term survival is still a challenge, particularly in patients that are diagnosed at an advanced stage.5 The 5-year survival rate for advanced-stage breast cancer patients is only 6%.6 Metastasis and tumor recurrence, which significantly impair treatment, are the major causes of poor outcome.7 Therefore, identifying novel and effective treatment strategies for preventing and treating breast cancer is an active area of research.

Long non-coding RNAs (lncRNAs) represent RNA transcripts with a length exceeding 200 nucleotides that are usually devoid of protein-coding capacity.8 LncRNAs were previously regarded as “noise” resulting from transcription, but in recent years, they have been attributed to important functions associated with RNA processing and transcriptional regulation of gene expression.9 In addition, many studies have identified regulatory roles for lncRNAs in tumorigenesis and tumor development.10–12 In breast cancer, an increasing number of lncRNAs have been identified that are differentially expressed and closely associated with oncogenesis.13–15 These dysregulated lncRNAs can exert pro- or anti-oncogenic effects on breast cancer cells and influence numerous biological behaviors.16

MicroRNAs (miRNAs) are a group of highly conserved, small non-coding RNA transcripts consisting of approximately 17–24 nucleotides.17 They post-transcriptionally regulate gene expression by base pairing with 3ʹ-untranslated regions (3ʹ-UTRs) of complementary target mRNAs. This results in transcription suppression and/or mRNA degradation.18 Recently, the competing endogenous RNA (ceRNA) theory was proposed in which lncRNAs function as ‘molecular sponges’ for specific miRNAs and thereby increasing the expression of miRNA target genes.19 Therefore, investigating lncRNAs and miRNAs may lead to the identification of promising biological markers for breast cancer diagnosis, prevention, and therapy.

Long intergenic non-protein coding RNA 504 (LINC00504) is a lncRNA reported to exert important regulatory roles in non-small cell lung,20 ovarian,21 and colon22 cancers. Currently, the expression status and function of LINC00504 in breast cancer has not been investigated. In this study, we examined the expression of LINC00504 in breast cancer cell lines and tissues. Next, we determined the effect of attenuating LINC00504 expression on several facets of malignant behavior in breast cancer cells including proliferation, apoptosis, in vitro migration, invasion, and in vivo tumor growth.

Materials and Methods

Collection of Tissue Specimens

Fifty-seven breast cancer patients admitted to Jilin Cancer Hospital were selected. Breast cancer tissues and corresponding adjacent normal tissues were collected and stored in liquid nitrogen. Enrollment criteria included patients that were diagnosed with primary breast cancer who had not been treated with chemotherapy, radiotherapy, or other anticancer treatments prior to surgery. This study was approved by the Ethics Committee of Jilin Cancer Hospital (2017.0615) and was conducted in accordance with the Declaration of Helsinki. All patients that agreed to participate in this study signed an informed consent form.

Cell Culture

The human immortalized breast epithelial cell line (MCF-10A) and five breast cancer cell lines (BT-474, MCF-7, MDA-MB-231, SKBR3 and T-47D) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). MEGMTM Mammary Epithelial Cell Growth Medium BulletKitTM (Lonza/Clonetics Corporation, Walkersville, MD, USA), supplemented with 100 ng/mL cholera toxin, was used for MCF-10A cell culture. BT-474 cells were grown in Hybri-Care Medium (ATCC) containing 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and 1% penicillin/streptomycin (Gibco; Thermo Fisher Scientific, Inc.). MCF-7, MDA-MB-231, SKBR3 and T-47D cell lines were, respectively, maintained in Eagle’s Minimum Essential Medium (ATCC), Leibovitz’s L-15 Medium (Gibco; Thermo Fisher Scientific, Inc.), McCoy’s 5a Medium (Gibco; Thermo Fisher Scientific, Inc.), and RPMI-1640 Medium (Gibco; Thermo Fisher Scientific, Inc.). The other culture conditions were the same as that of BT-474 cells. All cells were kept at 37°C in an incubator in a saturated humidified atmosphere supplied with 5% CO2.

Transient Cell Transfections

Small interfering RNAs (siRNAs) specifically designed for LINC00504 (si-LINC00504) and negative control (NC) siRNA (si-NC) were obtained from Genepharma Co., Ltd. (Shanghai, China). The miR-876-3p mimic, miRNA mimic negative control (miR-NC), miR-876-3p inhibitor and NC inhibitor were also purchased from Genepharma Co., Ltd. The pcDNA3.1 plasmid was engineered to overexpress HMGB3 (pcDNA3.1-HMGB3) (Sangon Biotech Co., Ltd.; Shanghai, China). The empty pcDNA3.1 plasmid acted as the control for pcDNA3.1-HMGB3. Breast cancer cells were seeded into 6-well plates and transfected with siRNA, miRNA mimic, miRNA inhibitor, or plasmid. All transient cell transfections were conducted using Lipofectamine® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.,).

RNA Extraction and Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)

Total RNA was extracted from cultured breast cancer cells or tissue samples using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). To analyze miR-876-3p expression, complementary DNA (cDNA) was generated by reverse transcription using the miScript reverse transcription kit (Qiagen GmbH, Hilden, Germany). Quantitative PCR was done with an ABI 7900HT/7900HT-Fast Real-Time PCR System (Thermo Fisher Scientific, Inc.) using the miScript SYBR Green PCR kit (Qiagen GmbH). For LINC00504 and HMGB3 expression detection, PrimeScript™ RT reagent Kit with gDNA Eraser (Takara Biotechnology Co., Ltd.; Shiga, Japan) was used for the reverse transcription reaction. The cDNA was then amplified with TB Green® Premix Ex Taq™ II (Takara Biotechnology Co., Ltd). GAPDH was employed as the reference gene for LINC00504 and HMGB3, while expression of miR-876-3p was normalized to the expression of U6 small nuclear RNA. All gene expression measurements were determined using the 2−ΔΔCq method.

Cell Counting Kit-8 (CCK-8) Assay

After 24 h transfection, cells were harvested and inoculated into 96-well plates at a density of 2×103 cells/well, with six replicates wells. A total of 10 µL CCK-8 solution (Beyotime Institute of Biotechnology; Shanghai, China) was added to each well and the cells were incubated at 37°C for 2 h. Cell proliferation after incubation for 0, 1, 2 or 3 days was measured by absorbance at 450 nm using a microplate reader. The experiments were repeated three times.

Flow Cytometry

Transfected cells were collected after a 48-hour incubation and apoptosis was determined using an Annexin V-FITC Apoptosis Detection Kit (Beyotime Institute of Biotechnology). After rinsing twice with phosphate-buffered solution (PBS), the transfected cells were collected by centrifugation at 1000 x g for 5 min, resuspended in 195 μL Annexin V-FITC binding buffer, and incubated with 5 μL Annexin V-FITC and 10 μL propidium iodide. The double stained cells were incubated at 20–25°C for 15 min in the dark and analyzed with a FACScan flow cytometer (BD Biosciences, San Jose, CA, USA). Experiments were repeated three times.

Transwell Experiments

For migration assays, the transfected cells were washed twice with PBS and resuspended in FBS-free basal medium. A 200 μL cell suspension containing 5 × 104 cells was added to the upper compartment of a Transwell chamber (8 μm pore diameter; BD Biosciences). The lower chamber was filled with 600 μL medium supplemented with 20% FBS. After 24 h, non-migrating cells that did not pass through the pores were removed with a cotton swab, while the migrated cells were fixed with methanol and stained with 0.1% crystal violet. The migrated cells were photographed under an inverted microscope and six visual fields were randomly chosen for cell count. For the invasion assay, the chambers were coated with Matrigel (BD Biosciences), and the subsequent steps were the same as the migration assay described above.

Tumor Xenograft Model

The animal experiment protocols were approved by the Institutional Animal Care and Use Committee of Jilin Cancer Hospital (2019.0217) and strictly conformed with the NIH guidelines for the care and use of laboratory animals. Short hairpin RNAs (shRNAs) specifically for LINC00504 (sh-LINC00504) and NC shRNA (sh-NC) were designed and synthesized by Genepharma Co., Ltd. and packaged into lentivirus. The lentivirus overexpressing sh-LINC00504 or sh-NC were transfected into MCF-7 cells, and the stably transfected cells were selected using puromycin. A total of 12 female BALB/C nude mice, aged 4–6 weeks, were purchased from the Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences (Beijing, China). Sh-LINC00504 or sh-NC stably-expressing MCF-7 cells (1 × 107) were subcutaneously injected into the flanks of nude mice. Each group contained 6 nude mice. The width and length of tumor xenografts was monitored weekly and recorded. Four weeks after cell injection, the mice were euthanized and the tumor xenografts were removed and weighed. The volume was calculated by the formula: volume = (length × width2)/2.

Bioinformatics Analyses

LINC00504 expression in human breast cancer was evaluated using the GEPIA database (http://gepia.cancer-pku.cn/index.html). The miRDB (http://mirdb.org/) and miRcode (http://www.mircode.org/) databases were used to identify putative miRNAs that bind to LINC00504. The potential target genes of miR-876-3p were predicted by the miRDB, Starbase 3.0 (http://starbase.sysu.edu.cn/) and TargetScan (http://www.targetscan.org/vert_60/) programs.

Subcellular Fractionation

To address the intracellular localization of LINC00504, nuclear and cytoplasmic fractions were prepared from breast cancer cells using the PARIS™ Kit (Invitrogen; Thermo Fisher Scientific, Inc). After the extraction of nuclear and cytoplasmic RNA, qRT-PCR was conducted to measure LINC00504 expression in both fractions. GAPDH and U6 were used as the cytoplasmic and nuclear controls, respectively.

Luciferase Reporter Assay

The fragments of LINC00504 and HMGB3 containing the miR-876-3p binding site were generated and sub-cloned into the pmirGLO Dual-Luciferase miRNA Target Expression Vector (Promega Corporation, Madison, WI, USA), which was referred to LINC00504-wild-type (LINC00504-wt) and HMGB3-wt, respectively. The mutant (mut) fragments of LINC00504 and HMGB3 were produced using the Site-Directed Mutagenesis Kit (Agilent, Santa Clara, USA), and were also inserted into the same vector to obtain LINC00504-mut and HMGB3-mut. For reporter assays, breast cancer cells were transfected with wt or mut reporter vectors and miR-876-3p or miR-NC using Lipofectamine® 2000. Luciferase activity was detected at 48 h post-transfection using a Dual-Luciferase Reporter Assay System (Promega).

RNA Immunoprecipitation (RIP)

A RIP assay was carried out using the Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, Bedford, MA, USA). Breast cancer cells were lysed in RIP lysis buffer. 10% of cell lysate was used as the input, and the other parts were incubated with RIP buffer containing magnetic beads at 4°C. The human anti-Ago2 and negative control IgG antibodies (Millipore) were used for immunoprecipitation. The following day, the beads were harvested, washed, and incubated with proteinase K for protein digestion. The immunoprecipitated RNA was extracted and analyzed by qRT-PCR to measure the relative expression of LINC00504, miR-876-3p and HMGB3. Input served as the positive control, while IgG acted as the negative control.

Western Blot Assay

RIPA lysis buffer supplemented with phenylmethanesulfonyl fluoride (both from Beyotime Institute of Biotechnology) was used for total protein isolation. After protein quantitation using the Bradford protein assay kit (Beyotime Institute of Biotechnology), equal amounts of total protein were separated by sodium-dodecyl-sulfate polyacrylamide gel electrophoresis on 10% gels, followed by transferring to polyvinylidene difluoride membranes. After blocking for 2 h with TBS with Tween-20 (TBST) containing 5% non-fat dry milk, the membranes were incubated overnight at 4°C with primary antibodies against HMGB3 (1:1000 dilution; ab133258, Abcam, Cambridge, MA, USA) or GAPDH (1:1000 dilution; ab181603, Abcam). After washing three times with TBST, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1:5000 dilution; ab205718; Abcam) at room temperature for 2 h, followed by detection of the protein bands using the BeyoECL Plus kit (Beyotime Institute of Biotechnology).

Statistical Analysis

All results are presented as the mean ± standard deviation (SD). A Student’s t-test was used to determine the differences between two groups, and a one-way analysis of variance with Tukey’s test was utilized for multigroup comparisons. The correlations among LINC00504, miR-876-3p and HMGB3 expression in breast cancer tissues were examined by Pearson’s correlation analysis. All statistical analyses were done with SPSS 21.0 software (SPSS, Chicago, IL, USA) and statistical significance was considered for 2-sided P-values < 0.05.

Results

LINC00504 is Abnormally Upregulated in Breast Cancer and LINC00504 Knockdown Inhibits the Malignant Behavior of Breast Cancer Cells

The GEPIA (http://gepia.cancer-pku.cn/) database was used for establishing the expression profile of LINC00504 in breast cancer. LINC00504 was overexpressed in breast cancer tissues than in normal tissues (Figure 1A). Consistently, qRT-PCR analysis confirmed that the expression level of LINC00504 was higher in breast cancer tissues compared with corresponding adjacent normal tissues (Figure 1B). Furthermore, all five tested breast cancer cell lines (BT-474, MCF-7, MDA-MB-231, SKBR3, and T-47D) exhibited relatively higher LINC00504 expression compared with the MCF-10A human immortalized breast epithelial cell line (Figure 1C). The correlation between LINC00504 expression and tumor stage or overall survival in patients with breast cancer was also analyzed by GEPIA. However, a high LINC00504 expression was not significantly associated with either tumor stage (Figure 1D, P = 0.147) or overall survival (Figure 1E, P = 0.76) in breast cancer.

Figure 1 Loss of LINC00504 restricts the malignant behavior of breast cancer cells in vitro. (A) LINC00504 expression in breast cancer was analyzed using the TCGA and GTEx databases. (B) qRT-PCR was used to measure LINC00504 expression in 57 pairs of breast cancer tissues and corresponding adjacent normal tissues. (C) The expression of LINC00504 in five breast cancer cell lines (BT-474, MCF-7, MDA-MB-231, SKBR3 and T-47D) and in a human immortalized breast epithelial cell line (MCF-10A). (D and E) The correlation between LINC00504 expression and tumor stage or overall survival in breast cancer was analyzed using the TCGA and GTEx databases. (F) qRT-PCR was done to assess the transfection efficiency of si-LINC00504 in MCF-7 and MDA-MB-231 cells. (G) CCK-8 assay was applied to measure the proliferation of MCF-7 and MDA-MB-231 cells after LINC00504 silencing. (H) Apoptosis of si-LINC00504- or si-NC-transfected MCF-7 and MDA-MB-231 cells was examined by flow cytometry. (I and J) Transwell experiments were employed to measure the migration and invasion capacities of MCF-7 and MDA-MB-231 cells after si-LINC00504 or si-NC transfection. **P < 0.01.

Next, the role of LINC00504 in breast cancer was investigated. The siRNAs targeting LINC00504 were constructed and transfected into MCF-7 and MDA-MB-231 cells. The knockdown efficiency was determined by qRT-PCR. The si-LINC00504#1 was selected for loss-of-function experiments because it exhibited the highest knockdown efficiency among the constructs (Figure 1F). CCK-8 assay revealed that knocking down LINC00504 inhibited the proliferation of MCF-7 and MDA-MB-231 cells (Figure 1G). In addition, when LINC00504 was silenced, apoptosis was significantly increased in MCF-7 and MDA-MB-231 cells (Figure 1H). Furthermore, downregulation of LINC00504 inhibited the migratory (Figure 1I) and invasive (Figure 1J) capacities of MCF-7 and MDA-MB-231 cells. Our findings indicate that LINC00504 promotes the oncogenic processes in breast cancer cells.

LINC00504 is a miR-876-3p Sponge in Breast Cancer Cells

Because the functions of lncRNAs are largely associated with their subcellular localization, lncLocator (http://www.csbio.sjtu.edu.cn/bioinf/lncLocator/) and lncATLAS (http://lncatlas.crg.eu/) were used to determine the location of LINC00504. LINC00504 was predicted to reside primarily in the cytoplasm (Figure 2A and B). Subcellular fractionation followed by qRT-PCR analysis revealed that LINC00504 was indeed distributed predominantly in the cytoplasm of MCF-7 and MDA-MB-231 cells (Figure 2C). Cytoplasmic lncRNAs can sequester certain miRNAs and thereby regulate their mRNA targets.19 Therefore, we identified the miRNAs which could be theoretically sequestered by LINC00504 using the miRDB and miRcode databases. In total, 35 miRNAs and 137 miRNAs were identified by miRcode and miRDB, respectively. Among these candidates, miR-205-5p, miR-129-5p, and miR-876-3p were predicted by both bioinformatics tools (Figure 2D).

Figure 2 LINC00504 functions as a miR-876-3p sponge in breast cancer. (A and B) lncLocator and lncATLAS were used to predict the subcellular location of LINC00504. (C) The subcellular distribution of LINC00504 in MCF-7 and MDA-MB-231 cells was determined by subcellular fractionation followed by qRT-PCR analysis. (D) Schematic diagram indicating the predicted miRNAs targeting LINC00504. (E) qRT-PCR was used to detect the expression of miR-205-5p, miR-129-5p, and miR-876-3p in MCF-7 and MDA-MB-231 cells after LINC00504 depletion. (F) MiR-876-3p expression in 57 pairs of breast cancer tissues and corresponding adjacent normal tissues as analyzed by qRT-PCR. (G) The relationship between miR-876-3p and LINC00504 expression in 57 breast cancer tissues as determined by Pearson’s correlation analysis. (H) The binding sites between LINC00504 and miR-876-3p were predicted by bioinformatics analysis. (I) Luciferase reporter assay was conducted to study the molecular interaction of LINC00504 and miR-876-3p in breast cancer cells. MCF-7 and MDA-MB-231 cells were co-transfected with LINC00504-wt or LINC00504-mut and miR-876-3p mimic or miR-NC, followed by the evaluation of luciferase activity at 48 h post-transfection. (J) RIP assay was carried out in MCF-7 and MDA-MB-231 cells, followed by qRT-PCR to measure the enrichment of LINC00504 and miR-876-3p associated with Ago2. **P < 0.01.

The effect of LINC00504 depletion on the expression of these miRNAs was examined by qRT-PCR. It was observed that miR-876-3p expression was significantly increased in LINC00504-deficient MCF-7 and MDA-MB-231 cells (Figure 2E). Furthermore, miR-876-3p was minimally expressed in breast cancer tissues (Figure 2F) and inversely correlated with that of miR-876-3p expression (Figure 2G; r = −0.6796, P < 0.0001). The wild-type and mutant binding sites between LINC00504 and miR-876-3p are shown in Figure 2H. Luciferase reporter assay was performed to determine whether a physical association existed between LINC00504 and miR-876-3p in breast cancer cells. Luciferase activity of LINC00504-wt was inhibited by miR-876-3p upregulation in MCF-7 and MDA-MB-231 cells, whereas luciferase activity of LINC00504-mut was unaffected (Figure 2I). Using a RIP assay, LINC00504 and miR-876-3p were enriched by anti-Ago2 antibody precipitation, suggesting the existence of LINC00504 and miR-876-3p in RNA-induced silencing complexes (Figure 2J). Taken together, these results suggest that LINC00504 functions as a miR-876-3p sponge in breast cancer cells.

LINC00504 Upregulates HMGB3 Expression in Breast Cancer Cells by Decoying miR-876-3p

To elucidate the role of miR-876-3p in breast cancer, miR-876-3p mimic or miR-NC was transfected into MCF-7 and MDA-MB-231 cells. After confirming overexpression of the miR-876-3p mimic (Figure 3A), gain-of-function assays were performed. The results of CCK-8 and flow cytometry assays indicated that transfection with miR-876-3p mimic resulted in decreased cell proliferation (Figure 3B) and increased cell apoptosis (Figure 3C and D), respectively, in MCF-7 and MDA-MB-231 cells. In addition, Transwell experiments verified that the suppression of migration (Figure 3E) and invasion (Figure 3F) of MCF-7 and MDA-MB-231 cells following miR-876-3p overexpression.

Figure 3 HMGB3 is a direct target of miR-876-3p and is regulated by LINC00504 in breast cancer cells. (A) Overexpression efficiency of the miR-876-3p mimic in MCF-7 and MDA-MB-231 cells as determined by qRT-PCR. (BD) CCK-8 assay and flow cytometry were utilized to examine the effects of miR-876-3p upregulation on the proliferation and apoptosis of MCF-7 and MDA-MB-231 cells, respectively. (E and F) The migration and invasion of miR-876-3p-overexpressing MCF-7 and MDA-MB-231 cells as determined by Transwell experiments. (G) The wild-type miR-876-3p binding sequences within the 3ʹ-UTR of HMGB3. The mutant binding sequences are also shown. (H) HMGB3-wt or HMGB3-mut and miR-876-3p mimic or miR-NC was transfected into MCF-7 and MDA-MB-231 cells. After 48 h incubation, luciferase activity was detected to confirm the association between miR-876-3p and HMGB3 3ʹ-UTR. (I and J) The influences of miR-876-3p mimic transfection on HMGB3 mRNA and protein levels in MCF-7 and MDA-MB-231 cells were determined by qRT-PCR and Western blotting, respectively. (K) HMGB3 mRNA expression in 57 pairs of breast cancer tissues and corresponding adjacent normal tissues was measured by qRT-PCR. (L) Pearson’s correlation analysis was done to assess the correlation between miR-876-3p and HMGB3 mRNA levels in 57 breast cancer tissues. (M and N) qRT-PCR and Western blot analysis were performed to detect the expression of HMGB3 mRNA and protein in LINC00504-deficient MCF-7 and MDA-MB-231 cells. (O and P) si-LINC00504 in combination with the miR-876-3p inhibitor or NC inhibitor was introduced into MCF-7 and MDA-MB-231 cell. qRT-PCR and Western blot analysis were done to measure HMGB3 mRNA and protein. (Q) RIP assay was used to determine the association of LINC00504, miR-876-3p, and HMGB3 with Ago2 in MCF-7 and MDA-MB-231 cells. (R) Pearson’s correlation analysis was used to demonstrate the correlation between LINC00504 and HMGB3 mRNA expression in 57 breast cancer tissues. **P < 0.01.

Using three bioinformatics tools, HMGB3 was selected as a target for this study because of its central role in breast cancer progression.23–26 The binding sequences of miR-876-3p that are complementary to sequences within HMGB3 are presented in Figure 3G. The luciferase reporter assay was used to detect an interaction between miR-876-3p and HMGB3 in breast cancer cells. Luciferase activity of the HMGB3-wt reporter plasmid was significantly inhibited following co-transfection with miR-876-3p mimic in MCF-7 and MDA-MB-231 cells compared with HMGB3-mut (Figure 3H). Furthermore, qRT-PCR and Western blot analysis indicated that overexpression of miR-876-3p significantly decreased HMGB3 mRNA (Figure 3I) and protein (Figure 3J) levels, respectively, in MCF-7 and MDA-MB-231 cells. In addition, the highly expressed HMGB3 mRNA (Figure 3K) exhibited an inverse correlation with miR-876-3p expression (Figure 3L; r = −0.6504, P < 0.0001). These results collectively identify HMGB3 as a direct target of miR-876-3p in breast cancer cells.

Next, a series of experiments was done to determine whether LINC00504 could attenuate HMGB3 expression in breast cancer cells by sequestering miR-876-3p. The HMGB3 mRNA (Figure 3M) and protein (Figure 3N) levels were reduced by LINC00504 silencing in MCF-7 and MDA-MB-231 cells. In rescue experiments, the inhibition of miR-876-3p partially restored HMGB3 mRNA (Figure 3O) and protein (Figure 3P) expression that was initially suppressed by LINC00504 knockdown in MCF-7 and MDA-MB-231 cells. In addition, the RIP assay indicated that LINC00504, miR-876-3p, and HMGB3 were all enriched in Ago2 antibody-treated MCF-7 and MDA-MB-231 cells (Figure 3Q), suggesting that all three molecules co-exist in RNA-induced silencing complexes. Finally, Pearson’s correlation analysis revealed a positive correlation between LINC00504 and HMGB3 mRNA in breast cancer tissues (Figure 3R; r = 0.6144, P < 0.0001). Overall, these results indicate that LINC00504 exerts significant regulatory actions in regulating HMGB3 expression by sponging miR-876-3p.

LINC00504 Regulates the Behavior of Breast Cancer Bells Through the miR-876-3p/HMGB3 Axis

Rescue experiments were performed to determine whether LINC00504 regulates breast cancer progression by sponging miR-876-3p and increasing HMGB3 expression. First, the efficiency of miR-876-3p inhibitor in decreasing miR-876-3p expression in MCF-7 and MDA-MB-231 cells was assessed by qRT-PCR (Figure 4A). Then, MCF-7 and MDA-MB-231 cells were cotransfected with the si-LINC00504 and miR-876-3p inhibitor or NC inhibitor. CCK-8 assay and flow cytometry revealed that the reduced cell proliferation (Figure 4B) and increased apoptosis (Figure 4C) resulting from LINC00504 downregulation was reversed by miR-876-3p inhibition. Next, Transwell experiments indicated that inhibition of miR-876-3p partially reversed the effects of LINC00504 depletion on the migration (Figure 4D) and invasion (Figure 4E) of MCF-7 and MDA-MB-231 cells.

Figure 4 MiR-876-3p inhibition reverses the anticancer effects of si-LINC00504 in breast cancer cells. (A) The knockdown efficiency of miR-876-3p inhibitor was determined in MCF-7 and MDA-MB-231 cells by qRT-PCR. (B and C) si-LINC00504 was co-transfected with the miR-876-3p inhibitor or NC inhibitor into MCF-7 and MDA-MB-231 cells. CCK-8 assay and flow cytometry were performed to measure the proliferation and apoptosis in the different groups, respectively. (D and E) Migration and invasion of the aforementioned cells was evaluated in Transwell experiments. *P < 0.05 and **P < 0.01.

The HMGB3 overexpression plasmid pcDNA3.1-HMGB3 was also used for rescue experiments. Western blot analysis verified the overexpression efficiency of pcDNA3.1-HMGB3 (Figure 5A). Cell proliferation was inhibited in MCF-7 and MDA-MB-231 cells in response to LINC00504 knockdown and this effect was abrogated by HMGB3 upregulation (Figure 5B). Furthermore, HMGB3 overexpression abolished the pro-apoptotic (Figure 5C), anti-migration (Figure 5D), and anti-invasion (Figure 5E) effects of si-LINC00504 in MCF-7 and MDA-MB-231 cells. Collectively, these results suggest that LINC00504 increases the expression of HMGB3 by competitively binding to miR-876-3p, thus promoting the oncogenicity of breast cancer.

Figure 5 Overexpression of HMGB3 abrogates the impacts of LINC00504 knockdown on breast cancer cells. (A) Western blot analysis was performed to detect HMGB3 protein expression in MCF-7 and MDA-MB-231 cells after pcDNA3.1-HMGB3 or pcDNA3.1 transfection. (BE) si-LINC00504 in parallel with pcDNA3.1-HMGB3 or pcDNA3.1 was transfected into MCF-7 and MDA-MB-231 cells. CCK-8 assay, flow cytometry and Transwell experiments were done to determine the proliferation, apoptosis, migration, and invasion in the different groups, respectively. *P < 0.05 and **P < 0.01.

LINC00504 Depletion Inhibits Breast Tumor Growth in vivo

In order to evaluate the role of LINC00504 in tumor growth in vivo, a tumor xenograft model was established by injecting nude mice with lentivirus carrying sh-LINC00504 or sh-NC. The size of tumor xenografts formed by sh-LINC00504-stably depleted-MCF-7 cells was significantly smaller compared with the sh-NC group (Figure 6A). Consistently, the volume (Figure 6B) and weight (Figure 6C) of the tumor xenografts from the sh-LINC00504 group was significantly decreased compared with that of the sh-NC group. Total RNA was isolated from the tumor xenografts and the expression level of LINC00504 and miR-876-3p was measured by qRT-PCR. The results indicated that LINC00504 was downregulated (Figure 6D) and miR-876-3p was increased (Figure 6E) in tumor xenografts following sh-LINC00504 transfection. Western blot analysis also indicated that the expression of HMGB3 protein was significantly lower in the sh-LINC00504 group compared with the sh-NC group (Figure 6F). These results indicate that the loss of LINC00504 decreases the growth of breast cancer cells in vivo.

Figure 6 Knockdown of LINC00504 suppresses tumor growth of breast cancer cells in vivo. (A) Representative photographs of tumor xenografts obtained from the sh-LINC00504 and sh-NC groups. (B) The growth curves for the tumor xenografts after injection with MCF-7 cells overexpressing sh-LINC00504 and sh-NC. (C) The weight of the tumor xenografts from the sh-LINC00504 and sh-NC groups. (D and E) LINC00504 and miR-876-3p in the tumor xenografts as measured by qRT-PCR. (F) Western blot analysis indicating HMGB3 protein expression in the tumor xenografts. **P < 0.01.

Discussion

Previous studies have demonstrated that a variety of lncRNAs are dysregulated in breast cancer and they have an effect on the oncogenesis, progression, and metastasis of breast cancer.27–29 Continued studies on the role of breast cancer-related lncRNAs will lead to an understanding of the mechanisms that underly breast cancer pathogenesis and identity new targets for therapy. In the present study, we explored the role of LINC00504 in regulating oncogenesis in breast cancer. We discovered that LINC00504 was overexpressed in breast cancer and aggravated the oncogenicity of breast cancer cells by targeting miR-876-3p and consequently increasing HMGB3 expression.

LINC00504 is upregulated in non-small cell lung cancer and is significantly associated with TNM stage and lymph node metastasis.20 Patients with non-small cell lung cancer harboring a high LINC00504 expression exhibit shorter overall survival compared with patients with low LINC00504 expression.20 In addition, LINC00504 is identified as an independent prognostic marker for patients with non-small cell lung cancer.20 LINC00504 is also highly expressed in ovarian21 and colon22 cancers. Functionally, LINC00504 performs an oncogenic role in ovarian21 and colon22 cancers; however, the expression profile and functions of LINC00504 in breast cancer have yet to be determined. In this study, we demonstrated that LINC00504 was highly expressed in breast cancer tissues and cell lines. Functional in vitro assays revealed that knocking down LINC00504 with siRNA attenuated breast cancer cell proliferation, migration, and invasion, while inducing apoptosis. Furthermore, the tumor growth in mouse xenografts was markedly inhibited by LINC00504 depletion. These results suggest that LINC00504 may represent a novel target for anticancer therapy in breast cancer.

Similar to proteins, the subcellular distribution of lncRNAs is important to their function.30 Therefore, we investigated the localization of LINC00504 in breast cancer cells and confirmed that it was primarily localized to the cytoplasm. Recently, a ceRNA network has been proposed in which lncRNAs maintain the expression and integrity of target mRNAs by sequestering certain miRNAs.31 To identify the mechanism through which LINC00504 affects the malignant characteristics of breast cancer cells, bioinformatics analysis was done to identify putative target miRNAs. Three miRNAs downstream of LINC00504 were selected and their expression was assessed in breast cancer cells following LINC00504 silencing. MiR-876-3p expression was increased after LINC00504 depletion, whereas knocking down LIN00504 did not influence the expression of the other two miRNAs. A correlation analysis revealed that LIN00504 exhibited a significant inverse association with miR-876-3p expression in breast cancer tissues. Furthermore, RIP and luciferase reporter assays verified that LIN00504 and miR-876-3p directly bind to one another. Overall, these findings suggested that miR-876-3p is a molecular sponge for LIN00504 in breast cancer cells.

MiRNAs can directly interact with the 3ʹ-UTR of target genes and disrupt their normal function.32 HMGB3 was identified as a downstream target of miR-876-3p in breast cancer by several methods including a luciferase reporter assay, qRT-PCR, Western blot, and correlation analyses. We also explored the interaction between LIN00504, miR-876-3p, and HMGB3 in breast cancer cells. Our results indicated that inhibiting LIN00504 expression reduced HMGB3 mRNA and protein levels in breast cancer cells, whereas the expression of HMGB3 was restored after miR-876-3p inhibition. Further analysis revealed a positive correlation between LIN00504 and HMGB3 in breast cancer tissues. Moreover, LIN00504, miR-876-3p, and HMGB3 were found to co-exist in RNA-induced silencing complexes. To sum up, all these observations identified a new ceRNA regulatory model in breast cancer, in which LIN00504 could work a ceRNA in breast cancer by sponging miR-876-3p to increase the expression of HMGB3.

MiR-876-3p is aberrantly expressed in multiple human cancers;33–35 however, its role in breast cancer etiology and progression is poorly understood. Our studies have shown that miR-876-3p is weakly expressed in breast cancer. Further investigation into miR-876-3p’s role revealed that its upregulation inhibited cell proliferation, migration, invasion, and promoted apoptosis, indicating that it exerts anti-oncogenic and -malignant effects in breast cancer. HMGB3, a member of the high mobility group box subfamily, is directly targeted by miR-876-3p in breast cancer cells. It exhibits strong expression in breast cancer and contributes significantly to cancer progression by influencing a number of tumor-related processes.23–26 Finally, our data confirmed that HMGB3 expression was positively regulated by LIN00504 and negatively regulated by miR-876-3p in breast cancer cells. The results of our rescue experiments indicated that increasing activity of the miR-876-3p/HMGB3 axis effectively reversed the inhibitory effects of LIN00504 deficiency on breast cancer cells. Our findings are the first to demonstrate that the LIN00504-miR-876-3p-HMGB3 pathway has a significant impact on the malignant phenotype of breast cancer.

Conclusion

In summary, our study showed that LIN00504 was upregulated in breast cancer and exerted tumorigenic effects during breast cancer progression. LIN00504 upregulated HMGB3 expression by sequestering miR-876-3p, thereby aggravating the aggressiveness of breast cancer cells. Furthermore, a novel regulatory pathway, LIN00504-miR-876-3p-HMGB3, was discovered which may provide new targets for breast cancer diagnosis and therapy.

Disclosure

The authors declare that they have no competing interests.

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