ABSTRACT
Breast cancer (BC) is a common malignancy among women, and microRNAs (miRNAs) play a role in its progression. Reportedly, microRNA-4521 (miR-4521) participates in regulating the progression of some carcinomas. In the present work, miR-4521 and hepatoma up-regulated protein (HURP) mRNA expressions in BC tissues and cell lines were examined by qRT-PCR. After miR-4521 was overexpressed or knocked down in BC cells, CCK-8 experiment and EdU experiment were employed to detect BC cell growth. Moreover, the Transwell assay was adopted to detect the migration and invasion. Subsequently, flow cytometry was executed to evaluate the changes in cell cycle progression. KEGG analysis was utilized to predict the signaling pathways related with the target genes of miR-524-5p. The binding relationship between miR-4521 and HURP 3ʹUTR was validated by dual-luciferase reporter gene experiment. HURP and NF-kB p65 protein expression in BC cells was detected by Western blot. It was revealed that, miR-4521 was lowly expressed in BC tissues, and its expression was associated with tumor grade, lymph node metastasis and clinical stage of the patients. Overexpression of miR-4521 suppressed the growth, migration, invasion and cell cycle progression of BC cells and restrained p65 expression; downregulation of miR-4521 in BC cells showed opposite biological effects. Additionally, HURP was a downstream target gene of miR-4521, and overexpression of HURP reversed the above effects of miR-4521 overexpression. In short, miR-4521 can inhibit BC progression by modulating the HURP/NF-κB pathway.
KEYWORDS:
1 Introduction
Breast cancer (BC) is one of the most common malignancies in female across the globe and one of the leading causes of cancer-related death [Citation1,Citation2]. Surgery, chemotherapy, radiotherapy, endocrine therapy, and targeted therapy for BC are now available, and with the continuous improvement of these treatment strategies, the mortality of BC patients is gradually decreasing [Citation3]. Nevertheless, the prognosis of some BC patients is still bad due to metastasis and drug resistance [Citation4]. Hence, it is important to investigate the molecular mechanism of BC to provide novel and effective therapy targets.
MicroRNAs (miRNAs) are short non-coding single-stranded RNAs that are involved in different important life activities by targeting and negatively regulating their target genes [Citation5]. MiR-4521 is a tumor-suppressor in some cancers. For example, it is reported to be down-modulated in clear renal cell carcinoma (ccRCC) tissue, and it inhibits the growth, migration and invasion of ccRCC cells and promotes their apoptosis [Citation6]. MiR-4521 is also downregulated in medulloblastoma; downregulation of miR-4521 significantly inhibits the growth and invasion of medulloblastoma cells and induces apoptosis through activation of caspase-3/7 [Citation7]. Nonetheless, the role of miR-4521 in BC has not been reported.
Hepatoma upregulated protein (HURP), also known as DLG associated protein 5 (DLGAP5), belongs to the DLGAP protein family and is firstly identified in rats [Citation8]. HURP is a regulator of the ubiquitin-proteasome proteolytic pathway, and it is also a regulator of cell cycle progression during carcinogenesis [Citation9]. For example, HURP is remarkably up-regulated in endometrial cancer tissues, and high HURP expression is significantly linked to unfavorable prognosis of the patients with endometrial cancer [Citation10]. Interestingly, it is highly expressed in BC, and HURP overexpression is associated with increased mortality of BC patients [Citation11].
Interestingly, bioinformatics suggested that miR-4521 was lowly expressed in BC tissues, and it could probably target HURP. We hypothesized that the dysregulation of miR-4521/HURP axis may contribute to BC progression. Our work is the first study to investigate the role of miR-4521 in BC.
2 Materials and methods
2.1 Clinical cases
30 BC patients who underwent surgery in Xuzhou Central Hospital were recruited for this study. All tissue samples were pathologically confirmed as BC. Only the patients who were newly diagnosed with BC were enrolled, and the patient who received preoperative radiation or chemotherapy were excluded. After the BC tissues and their matched paracancerous normal tissues were excised, the tissue specimens were instantly preserved in liquid nitrogen and frozen at −196°C. The study was authorized by the Ethics Committee of Xuzhou Central Hospital (approval No. KYLL20190803), and all participants had signed a written informed consent before the sample collection.
2.2 Cell culture and transfection
Human breast cancer cells (MDA-MB-231, MDA-MB-453, MDA-MB-468 and MCF7) and human mammary epithelial cell line MCF-10A were procured from American Type Culture Collection (ATCC) (Rockville, MD, USA). All cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS) (Gibco, Carlsbad, CA, USA) supplemented with 100 U/mL penicillin and 100 μg/mL streptomycin (Gibco, Carlsbad, CA, USA) and cultured at 37°C in 5% CO2. MiR-4521 mimic (miR-4521 mimic) and its negative control (NC mimic), miR-4521 inhibitor (miR-4521 inhibitor) and its negative control (NC inhibitor), HURP overexpression plasmid (HURP-oe) and its negative control (NC-oe), HURP small interfering RNA (si-HURP) and its negative control (scrambled siRNA, si-NC) were transfected into BC cells using LipofectamineTM 2000 (Invitrogen, Carlsbad, CA, USA), and the transfection efficiency was determined by quantitative real-time polymerase chain reaction (qRT-PCR) after 48 h.
2.3 qRT-PCR
Total RNA was extracted from BC tissues and cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and reverse transcribed into cDNA using a ReverTra Ace qPCR RT Kit (Toyobo, Shanghai, China). The purity of RNA and DNA was detected by measuring OD value. The OD260/ OD280 of RNA was 1.8–2.0 and the OD260/ OD280 of cDNA was 1.7–1.9. Subsequently, qRT-PCR was implemented using a THUNDERBIRD SYBR® qPCR Mix kit (Toyobo, Shanghai, China) in the LightCycler 480 Real-Time PCR system (Roche, Shanghai, China). The reaction system is 25 μL, containing cDNA (1 μL), forward primer (0.5 μL), reverse primer (0.5 μL), ddH2O (10.5 μL) and 2× SYBR Green PCR Master mixture (12.5 μL). The PCR cycling condition is 95°C, 30s; 95°C, 5s; 60°C, 30s; 73°C, 10s; 40 cycles. MiR-4521 relative expression and HURP mRNA relative expression were analyzed using the 2−ΔΔCt method, with U6 as miR-4521’s internal reference and GAPDH as HURP’s internal reference. The primer sequences are as follows:
miR-4521 (Forward): 5ʹ-GCTAAGGAAGTCCTGTGCTCAG-3ʹ;
miR-4521 (Reverse):5ʹ-TCGTATCCAGTGCAGGGTC-3ʹ;
U6 (Forward):5ʹ-CTCGCTTCGGCAGCACA-3ʹ;
U6 (Reverse):5ʹ-AACGCTTCACGAATTTGCGT-3ʹ;
HURP (Forward):5ʹ-AAGTGGGTCGTTATAGACCTGA-3ʹ;
HURP (Reverse):5ʹ-TGCTCGAACATCACTCTCGTTAT-3ʹ;
GAPDH (Forward):5ʹ-ACATCGCTCAGACACCATG-3ʹ;
GAPDH (Reverse):5ʹ-TGTAGTTGAGGTCAATGAAGGG-3ʹ.
2.4 Cell counting kit-8 (CCK-8) experiment
The cell suspension was prepared by trypsinization with 0.25% trypsin, and the cell density was modulated to 1 × 104 cells/mL and then the cells were transferred into 96-well plate at 100 µL/well. At the 24 h, 48 h and 72 h, 10 µL of CCK-8 solution (Dojindo Molecular Technologies, Japan) was added to each well and incubation was continued for 2 h. Next, the optical density (OD) value of each group of cells was measured at 450 nm using a microplate reader.
2.5 5-Ethynyl-2ʹ-deoxyuridine (EdU) assay
48 h after transfection, BC cells were inoculated in 96-well plates at 6000 cells/well and continued to be cultured for 24 h. Following the directions of the EdU kit (RiboBio, Guangzhou, Guangdong), 50 µmol/L EdU solution was supplemented to each well and the cells were incubated for 12 h. Afterward, the cells were fixed with 4% paraformaldehyde for 30 min and incubated with 5% glycine for 5 min. The membrane was permeabilized by 0.5% Triton X-100. Next, the cells were incubated with the ApolloTM staining solution for 30 min, and then stained with Hoechst 33,342 for 30 min, and observed under a fluorescence microscope and photographed, and the rate of EdU positive cells was calculated for each group.
2.6 Flow cytometry
The density of BC cells in logarithmic growth phase was modulated to 1 × 106 cells/mL and rinsed twice in phosphate buffer saline (PBS), and then the cells were fixed in 70% ethanol for 2 h, centrifuged, and the supernatant was discarded; afterward, the cells were washed once in PBS. Next, propidium iodide (PI) staining solution (BD, Shanghai, China) was supplemented, and the cells were incubated for 10 min in the dark. Then the cell cycle was identified using a flow cytometer (BD LSRFortessa™ X-20, Shanghai, China).
2.7 Transwell experiment
For Transwell migration experiment: BC cells were inoculated in the top compartment of Transwell inserts at 1 × 105 cells/well, and DMEM containing 10% FBS was added into the lower compartment and the cells were cultured for 24 h. The non-migrated cells were swabbed with a cotton swab, and the migrated cells were fixed with 90% ethanol, and stained with 0.1% crystal violet solution for 30 min. For the Transwell invasion experiment: the top compartment of Transwell was pre-wrapped with Matrigel, and the remainder of the stages in the experiment were identical to those in the migration experiment. After staining, the cells were cleaned with tap water and air-dried, and five fields of view were randomly chosen and photographed under a light microscopy (100×).
2.8 Dual-luciferase reporter gene experiment
The binding site between miR-4521 and HURP 3ʹUTR was predicted by bioinformatics, and the HURP 3ʹUTR sequence containing the miR-4521 binding site was amplified by PCR; the amplified fragment was cloned into pmiR-GLO reporter vector (Promega, Madison, WI, USA) to construct a HURP wild-type plasmid (wt-HURP). Subsequently, a mutant plasmid for HURP (mut-HURP) was constructed by mutating some nucleotides using gene mutation techniques. BC cells were transfected with wt-HURP, mut-HURP, and miR-4521 mimics or control mimics using LipofectamineTM 2000 (Invitrogen, Carlsbad, CA, USA), and a luciferase activity assay kit (Promega, Madison, WI, USA) was applied to measure the luciferase activity of the cells in each group.
2.9 Western blot
After 48 h of transfection, the transfected cells were harvested and lysed in RIPA lysis buffer (Beyotime, Shanghai, China) to extract the total protein of the cells. A NE‐PER™ Nuclear and Cytoplasmic Extraction kit (Thermo Fisher, Shanghai, China) was used to isolate the nuclear and cytosolic proteins. A BCA kit (Beyotime, Shanghai, China) was used to determine the protein concentration. 30 µg of protein sample was supplemented to each lane and SDS-PAGE was executed, and then the proteins were electrotransferred to PVDF membrane. Next, the PVDF membrane was blocked with 5% skim milk, and then incubated with primary antibodies overnight at 4°C: anti-IκBα (1:1000; ab76429, Abcam, Shanghai, China), anti-p-IκBα (1:1000; ab92685, Abcam, Shanghai, China), anti-HURP (1:1500; ab70744, Abcam, Shanghai, China), anti-p65 (1:1500; ab16502, Abcam, Shanghai, China), anti-fibrillarin (1:3000; ab166630, Abcam, Shanghai, China) and anti-GAPDH (1:2000; ab9485, Abcam, Shanghai, China). The next day, the PVDF membrane was incubated with secondary antibody: Goat Anti-Rabbit IgG H&L (1:3500; ab6721, Abcam, Shanghai, China). Finally, an ECL kit (Beyotime Biotechnology, Shanghai, China) was adopted to develop the protein bands. Finally, the relative amount of protein was analyzed by Image J software (NIH, Bethesda, Maryland, USA). GAPDH was the cytoplasmic protein reference, and fibrillarin was the nuclear protein reference.
2.10 Nude mouse tumorigenicity assay
The in vivo experiment in the present work was approved by the Xuzhou Central Hospital. 30 BALB/c nude mice (5 weeks old, female) were maintained in specific pathogen-free (SPF) conditions, and randomly divided into two groups. 2 × 106 MDA-MB-468 cells transfected with miR-4521 mimics or the NC mimics were suspended in sterile normal saline, and subsequently injected into the tail vein of each mouse. Two weeks later, the mice were killed and dissected, and the lungs of the mice were obtained. The lungs were fixed in paraformaldehyde, then embedded in paraffin. Subsequently, the tissues sections were prepared and hematoxylin and eosin (H&E) staining was performed. Then the severity of the lung metastasis of MDA-MB-468 cells was evaluated by a experienced pathologist.
2.11 Statistical analysis
For data analysis, SPSS 23.0 software (SPSS Inc., Chicago, IL, USA) was utilized, and all data were shown as Mean ± SD. D’Agostino & Pearson test was used to analyze the normality distribution of the data. For comparisons between two groups, t-test was employed; for comparisons among multiple groups, one-way ANOVA was implemented. P < 0.05 signified statistical significance.
3 Results
We found low expression of MiR-4521 in the tissues of BC patients with bioinformatics analysis and qRT-PCR. With in vitro and in vivo models, we subsequently investigated the regulatory effects of miR-4521 on the malignant biological behaviors of BC cells. After that, we verified the relationship between miR-4521 and HURP, and clarified the influence of miR-4521/HURP axis on the biological behaviors of BC cells.
3.1 MiR-4521 expression level is reduced in BC tissues
MiR-4521 expression in BC was firstly analyzed by the UALCAN database and miR-4521 was unveiled to be drastically down-regulated in BC tissues (P < 0.001) (). Additionally, miR-4521 under-expression was significant associated with lymph node metastasis and increased clinical stage of BC patients (P < 0.001) (). Moreover, miR-4521 expression in BC tissues and cell lines was detected by qRT-PCR, and the data showed that miR-4521 expression level was reduced in BC tissues as opposed to that in normal tissues (P < 0.001) (). It was also revealed that miR-4521 under-expression was associated with the lymph node metastasis of the patients (). MiR-4521 expression was also notably down-modulated in BC cell lines [MDA-MB-231 (P < 0.01), MDA-MB-453 (P < 0.01), MDA-MB-468 (P < 0.001), and MCF7 (P < 0.05)] as opposed to that in MCF10A cells ().
3.2 Effect of MiR-4521 on BC cell growth, migration, invasion and cell cycle progression
To elaborate on the biological function of miR-4521 in BC, we transfected miR-4521 mimics and inhibitors into MDA-MB-468 and MCF7 cells, respectively. The results of qRT-PCR showed the effective transfection (P < 0.001) (). Furthermore, BC cell growth was analyzed using the CCK-8 experiment and the EdU experiment, and the data showed that miR-4521 upregulation significantly inhibited the growth of BC cells, while down-regulation of miR-4521 significantly facilitated the growth of BC cells (P < 0.01) (). The data of the Transwell experiment revealed that overexpression of miR-4521 markedly impeded the migration and invasion of BC cells, while inhibition of miR-4521 exerted the opposite effect (P < 0.05) (). Flow cytometry showed that miR-4521 induced the cell cycle arrest of BC cells, while inhibition of miR-4521 promoted the cell cycle progression of BC cells (P < 0.05) (). To further consolidate that miR-4521 suppressed BC progression, MDA-MB-468 cells with miR-4521 overexpression and the cells in the control group were injected into the tail vein of the nude mice, and the nude mouse tumorigenicity model was established. H&E staining showed that miR-4521 overexpression markedly suppressed the lung metastasis of MDA-MB-469 cells (P < 0.01) (), which further support that miR-4521 repressed the progression of BC.
3.3 MiR-4521 targets HURP
To decipher the downstream mechanism of miR-4521, miRDB, TargetScan, miRWalk and miRPathDB databases were adopted to screen the downstream target genes of miR-4521, and 222 candidate target genes were obtained (). Enrichment analysis of the downstream target genes of miR-4521 using the KEGG database revealed that the target genes of miR-4521 were closely associated with several cancer-related pathways including HIF-1 pathway, Rap1 pathway, and Ras pathway (). Among the candidates, HURP attracted our attention (). Dual-luciferase reporter gene assay showed that transfection of miR-4521 mimic remarkably impeded the luciferase activity of wt-HURP reporter, while it exerted no significant effect on the luciferase activity of mut-HURP reporter (P < 0.01) (). With qRT-PCR and Western blot assays, it was revealed that miR-4521 overexpression significantly inhibited HURP expression, while inhibition of miR-4521 exerted the opposite effect (P < 0.01) (). Additionally, qRT-PCR revealed that HURP mRNA was down-modulated in BC tissues and negatively correlated with miR-4521 expression (P < 0.01) ().
3.4 Effects of miR-4521/HURP axis on BC cell growth, migration, invasion and cell cycle progression
To probe the function of miR-4521/HURP axis in the biological behaviors of BC cells, miR-4521 mimics and HURP overexpression plasmid were co-transfected into MDA-MB-468 cells, and miR-4521 inhibitor and si-HURP were co-transfected into MCF7 cells. qRT-PCR showed that the transfection was successful (P < 0.05) (). Via CCK-8, EdU, Transwell, and flow cytometry, it was revealed that HURP overexpression facilitated MDA-MB-468 cells’ growth, migration, invasion, and cell cycle progression, counteracting the biological effects of miR-4521 (P < 0.05) (). Compared with the miR-4521 inhibitor group, depletion of HURP remarkably repressed the growth, migration, invasion and cell cycle progression of BC cells (P < 0.05) (). Also, miR-4521 inhibitor and si-HURP were co-transfected into MDA-MB-468 cells, and miR-4521 mimics and HURP overexpression plasmid were co-transfected into MCF7 cells. CCK-8 showed that, compared with the miR-4521 inhibitor group, depletion of HURP repressed the growth of MDA-MB-468 cells; HURP overexpression reversed the biological effects of miR-4521 overexpression on the proliferation of MCF7 cells (Supplementary Figure 1). These data suggested the biological effects of miR-4521 was at least partly dependent on HURP.
3.5 Effects of miR-4521/HURP axis on NF-κB pathway in BC cells
To elucidate the downstream mechanism by which HURP exerts its oncogenic effect in BC, GSEA was employed to predict the downstream pathways of HURP, and the results showed that HURP high expression was probably associated with the activation of NF-κB pathway (). So p-IκBα and p65 expression was detected by Western blot, and the data showed that miR-4521 overexpression remarkably restrained p-IκBα and p65 expression in BC cells, while the restoration of HURP promoted the expression of p-IκBα and p65 (P < 0.01) (); inhibition of miR-4521 induced p-IκBα and p65 expression in BC cells, and knockdown of HURP exerted the opposite effect (P < 0.01) (). These data suggested that miR-4521/HURP axis could probably regulate BC progression via modulating NF-κB signaling.
4 Discussion
MiRNA can negatively modulate various target genes’ expression and thus regulates a series of biological processes, and is considered to be an promising biomarker and target in cancer diagnosis and treatment [Citation12,Citation13]. In this work, miR-4521 was demonstrated to be down-regulated in BC tissues and cells, and miR-4521 under-expression was significantly linked to adverse clinical indicators of BC patients, suggesting that miR-4521 has the potential to improve the diagnosis and therapy of BC.
The miRNA-mRNA regulatory axis is reported to regulate a variety of classical cancer-related pathways in BC, and is important in BC tumorigenesis and progression [Citation14]. For instance, miR-145-5p expression is down-modulated in BC tissues and negatively correlates with the expression of sex-determining region Y-box2 (SOX2); miR-145-5p inhibits the growth of BC cells by targeting SOX2 and exerts the tumor-suppressive effect in BC [Citation15]. MiR-539 is lowly expressed in BC tissues and cell lines, and miR-539 under-expression implies poor prognosis of BC patients; miR-539 can specifically and negatively regulate specificity protein 1 to impede the growth and promote apoptosis of BC cells [Citation16]. MiR-216b-5p is significantly reduced in BC tissues, and miR-216b-5p can target histone deacetylase 8 to restrain BC cell growth and block the cell cycle progression [Citation17]. As a member of miRNAs, miR-4521 is validated to be dysregulated in some cancers and mainly shows tumor-suppressive properties. Specifically, miR-4521 is down-modulated in gastric cancer tissues, and miR-4521 under-expression is linked to poor prognosis of gastric cancer patients, and miR-4521 impedes the metastatic potential of gastric cancer cells both in vitro and in vivo [Citation18]. MiR-4521 is down-regulated in ccRCC, and miR-4521 up-regulation inhibits the growth, migration and invasion of renal cancer cells in vitro by targeting niban apoptosis regulator 1 [Citation6]. In this study, bioinformatics analysis manifested that miR-4521 expression was down-modulated in BC and linked to adverse clinicopathological characteristics of the patients; further experiments manifested that miR-4521 overexpression remarkably impeded the growth, migration and invasion of BC cells and blocked the cell cycle progression, while down-regulation of miR-4521 had the opposite biological effects, suggesting that miR-4521 was also a tumor suppressor in BC.
In the work, HURP was identified as a downstream target of miR-4521. Several studies have shown that HURP is abnormally up-modulated in cancer tissues and correlates with cancer progression. For example, HURP is overexpressed in colorectal cancer tissues, and HURP overexpression is remarkably linked to unfavorable prognosis of the patients; depletion of HURP restrains the migration and invasion of colorectal cancer cells [Citation19]. In lung cancer, HURP is also significantly up-regulated, and high expression of HURP implies shorter overall survival time and recurrence-free survival time in lung cancer patients, and is a promising biomarker for lung cancer diagnosis and prognosis evaluation [Citation20]. Importantly, reportedly, HURP is overexpressed in BC tissues and that depletion of HURP significantly impedes the growth of BC cells and blocks the cell cycle progression [Citation21]. Another study reveals that interference of HURP expression notably impedes BC cell growth, implying that HURP may work as a favorable therapeutic target for BC [Citation22]. The present study showed that miR-4521 negative regulated HURP expression and inactivated NF-κB signaling, and the biological function of miR-4521 in BC was counteracted by HURP overexpression. These demonstrations partly explained the mechanism of BC progression.
5 conclusion
Our study demonstrates that miR-4521 can target HURP to suppress the growth, migration and invasion of BC cells and block the cell cycle progression. Our work suggests miR-4521 is a potential therapeutic target for BC.
Highlights
MiR-4521 is lowly expression in breast cancer tissues.
MiR-4521 represses the malignancy of breast cancer cells.
HURP is identified as a target gene of miR-4521.
MiR-4521 represses HURP/NF-κB pathway to inhibit breast cancer progression.
Ethics statement
Our study was approved by the Ethics Review Board of Xuzhou Centeral Hospital.
Supplemental Material
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The authors declare that they have no competing interests.
Data Availability Statement
The data used to support the findings of this study are available from the corresponding author upon request.
Supplementary material
Supplemental data for this article can be accessed here
Additional information
Funding
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