Biological function of long noncoding RNA snaR in HER2-positive breast cancer cells

Abstract

Purpose:

Long noncoding RNA, snaR (small NF90-associated RNA), has been reported to be upregulated in various cancer cell lines. We evaluated the additional role of snaR in HER2-positive breast cancer cell lines.

Methods:

We explored changes of expression of snaR among the selected long noncoding RNAs which have a potential in cancer proliferation or progression. The proliferation, migration, and invasion of HER2-positive breast cancer cells (SK-BR3) were evaluated by snaR with RNA interruption in 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide, wound-healing assay, and Transwell assay.

Results:

The expression of snaR was remarkably upregulated in SK-BR3 cell lines together with ANRIL, while the SFMBT2 was downregulated in SK-BR3 cell lines. Although Nespas, 7SK, PSF inhibiting RNA, mascRNA, Hoxa11as, NRON, AK023948, MER11C, p53 mRNA, CAR Intergenic 10, HUC 1 and 2, ZFAS1, SCA8, and SNHG5 were also upregulated and UCA1 was downregulated, the differences were not dominent. Based on the expression result, we explored the functional role of snaR in HER2-positive breast cancer. Downregulation of snaR with small interfering RNA was identified to significanlty inhibit migration as well as proliferation of SK-BR3 cells.

Conclusion:

In this study, snaR was identified as upregulated and to play a role in cancer progression of HER2-positive breast cancer cells. These results suggest snaR as a potential biomarker for HER2-positive breast cancer.

Introduction

The human epidermal growth factor receptor 2 (HER2) is a transmembrane receptor-like tyrosine kinase and can be overexpressed in breast, ovary, prostate, and stomach of human.^1,2 Overexpression of HER2 can be detected in 20%–30% of breast cancers and is strongly associated with poor oncologic outcomes, and therefore, the HER2-overexpressing breast cancer had been considered as an aggressive subtype with unfavorable survival outcomes.³ However, several effective treatments with a mechanism for blocking ErbB receptor families have been proven alone or combined with chemotherapeutic agents or endocrine treatment. In particular, using trastuzumab, a monoclonal antibody that binds to the extracellular domain of HER2 receptor and thereby inhibits its signaling cascade, is regarded as an innovative treatment for HER2-positive breast cancer based on the results that it reduced about 50% of tumor recurrence and improved more than 30% of overall survival after surgery in HER2-positive early breast cancer in combination with systemic chemotherapy.^4–6

Despite the significant benefit of trastuzumab for HER2-positive breast cancer, clinical resistance is ultimately recognized.⁷ Approximately 65% of HER2-positive breast cancer shows poor response to trastuzumab-related treatment, and although HER2-positive breast cancer had responded to initial trastuzumab treatment, 70% of them experienced progression of cancer within a year after initial treatment led to worse oncologic outcome and no definitive therapeutic option has been identified yet.⁸ To overcome these problems for HER2-positive breast cancer, physicians paid attention to find other biomarkers with completely different mechanisms from HER2-related signaling.

Long noncoding RNA (lncRNA) is a heterogeneous class of transcript which is composed of more than 200 nucleotides.⁹ Recently, changes in expression of numerous lncRNAs have been identified in various malignancies, and their mechanisms and roles have been proven in specific cancers. Similarly, several lncRNAs such as antisense noncoding RNA in the INK4 locus (ANRIL), urothelial carcinoma–associated 1 (UCA1), zinc finger antisense 1 (ZFAS1), and small NF90-associated RNA (snaR) were identified as upregulated or downregulated in breast cancer, suggesting possible role in cancer progression.^10–12 In our recent study, we explored the expression of lncRNAs in various breast cancer cell lines based on molecular subtypes and demonstrated the role of snaR in proliferation, migration, and invasion of triple-negative breast cancer cells, where upregulation of snaR was also shown in HER2 overexpressing breast cancer cell line as well.¹³ Therefore, in this study, we aimed to identify the role of snaR in HER2-positive breast cancer cells.

Materials and methods

Human breast cancer cell lines

The normal breast epithelial cells MCF10A and the breast cancer cell lines MDA-MB-231 (basal type, TNBC), MCF7 (luminal-A type, estrogen receptor–positive breast cancer), SK-BR3 (HER2-positive breast cancer), T47D (luminal-A type, estrogen receptor–positive breast cancer), BT474 (estrogen receptor–positive breast cancer), and BT20 (TNBC) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA; 15-6, 15-7). MCF10A was maintained in Dulbecco’s modified Eagle’s medium (DMEM)/F-12 (1:1) medium (Lonza, Walkersville, MD, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA), 10 ng/mL epidermal growth factor, 0.5 µg/mL hydrocortisone, 100 ng/mL cholera toxin, and 10 µg/mL insulin. SK-BR3 was maintained in DMEM (Gibco) supplemented with 10% FBS.

RNA extraction and lncRNA profiling

Total RNA was isolated from cells using RNAiso Plus (Takara, Otsu, Japan) in accordance with the manufacturer’s instructions. RNA concentration was measured using NanoDrop ND-2000 (Thermo Fisher Scientific, Wilmington, DE, USA). RNA was treated with DNA-free™ DNase Treatment and Removal Reagents (Life Technologies, Carlsbad, CA, USA). For lncRNA expression profiling, 2 µg of total RNA was reverse transcribed to complementary DNA (cDNA) using Human LncProfilers™ qPCR Array Kits (System Biosciences, Mountain View, CA, USA) in accordance with the manufacturer’s recommendations.

Small interfering RNA transfection

Small interfering RNAs (siRNAs) specific to snaR were synthesized by Ambion by Life Technologies Inc. (Carlsbad, CA, USA), and the negative control (NC), not showing homology with the human genome, was purchased from Sigma-Aldrich Co., LLC (St. Louis, MO, USA). The sequence of si-snaR was 5′-GGG CAC GAG UUC GAG GCC Att-3′ and the sequence for the negative control was 5′-CGG UAC GAU CGC GGC GGG AUA UC-3′. Cells were inoculated in a six-well plate (4 × 10⁵ per well) for the wound-healing assay or a 96-well plate (5 × 10³ per well) for the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay. After cultured overnight, cells were transfected using Lipofectamine RNAiMAX reagent (Invitrogen, Carlsbad, CA, USA) with the synthesized specific snaR or control siRNA. At 48 h after transfection, total RNA was extracted and the silencing effect was determined by real-time reverse transcription polymerase chain reaction (RT-PCR).

Quantitative RT-PCR

The total RNA in breast cancer cells was extracted using RNAiso Plus (Takara) and was treated with DNA-free™ DNase Treatment and Removal Reagents (Life Technologies). Reverse transcription was performed using Superscript III RT (Invitrogen) and random hexamer (ELPIS Biotech Inc., Daejeon, South Korea), following the manufacturer’s protocol. Real-time PCR was performed for relative quantification of the expression levels of snaR using Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA). The 2^−ΔΔCT method was used to determine relative gene expression, and the level of expression was normalized to that of β-actin (ACTB).

The primer sequences for real-time PCR were as follows: ANRIL (5′-TTG TTA GAA ACC AGG CTG CAC-3′ and 5′-TTC TCT CTT TCT GTG GTT TCT CAA T-3′), snaR (5′-ATT GTG GCT CAG GCC GGT T-3′ and 5′-TTT TTC CGA CCC ATG TGG ACC-3′), SNHG5 (5′-GAG CAG CTC TGA AGA TGC AA-3′ and 5′-TTT TAA CCA AGC GAT TTT CCA-3′), UCA1 (5′-CCC AAG GAA CAT CTC ACC AA-3′ and 5′-GAT GGT CCA AGG GGC TTC-3′), and ACTB (5′-TTG CCG ACA GGA TGC AGA A-3′ and 5′-GCC GAT CCA CAC GGA GTA CT-3′).

MTT assay

The effect of siRNA interference for snaR on the proliferation of breast cancer cells was determined by the MTT assay (Sigma, St. Louis, MO, USA). Cells at a total volume of 200 µL were inoculated in a 96-well plate, with 5 × 10³ cells in each well. At each time point, 50 µL of MTT (2 mg/mL) was added to the wells and the cells were cultured at 37°C for 4 h. After removal of the medium, 100 µL of dimethyl sulfoxide (DMSO) was added and the mixture was shaken for 10 min. The optical density was then determined at 570 nm. All the experiments were conducted in triplicate.

Wound-healing assay

In order to determine effect of siRNA interference for snaR on cell migration, SK-BR3 cells were seeded in six-well culture plates and transfected with negative control siRNA or si-snaR. Next, a line was scratched into the cell monolayer using a sterile pipette tip, and the cells were further incubated. Images were then captured with the aid of a microscope after 0, 24, 48, and 72 h of incubation. The data are representative of three independent experiments.

Cell invasion assay

For the invasion assay, Transwell chambers with 8-µm pores were coated with Matrigel (Corning Inc., Tewksbury, MA, USA) and incubated at 37°C for 2 h, allowing it to solidify. After transfection, cells were resuspended in DMEM containing 1% FBS and plated in the upper chamber at a density of 1 × 10⁴ cells. The lower chamber contained complete medium supplemented with 10% FBS. After incubation for 48 h, the cells on the internal surface of the chamber bottom were wiped with a cotton swab, fixed with 2% paraformaldehyde, stained with 0.5% crystal violet, and rinsed with phosphate-buffered saline. The invading cells after staining were finally extracted with DMSO and detected quantitatively using microplate reader at 590 nm.

Statistical analysis

Data are presented as the mean ± standard deviation (SD) of three or more independent experiments. The differences in experimental results between two groups were analyzed using Student’s t-test and a statistically significant difference was defined at p < 0.05.

Results

ANRIL and snaR were significantly upregulated in breast cancer cell lines

To identify lncRNAs of which the expression is deregulated in SK-BR3 cells, we performed the expression profiles of lncRNA comparing SK-BR3 and MCF10A cells using qPCR array analysis and extracted transcripts among 90 types of lncRNAs that were upregulated (expression level > 2-fold) or downregulated (expression level <0.5-fold) compared to MCF10A. The expressions of ANRIL, snaR, neuroendocrine secretory protein (Nesp) antisense (Nespas), 7SK small nuclear RNA (7SK), PSF inhibiting RNA, MALAT1-associated small cytoplasmic RNA (mascRNA), HOXA11 antisense (Hoxa11as), nuclear repressor of NFAT (NRON), AK023948, MER11C, p53 mRNA, chromatin-associated RNA (CAR) Intergenic 10, H19 upstream conserved (HUC) 1 and 2, ZFAS1, spinocerebellar ataxia type 8 (SCA8), and small nucleolar RNA host gene (SNHG5) were significantly increased in SK-BR3 cells. However, the expressions of the Scm-related gene containing four mbt domains (SFMBT2) and UCA1 were downregulated in SK-BR3 cells relative to MCF10A cells (Figure 1 and Table 1).

Figure 1.

The alteration of lncRNA expression levels in SK-BR3 (human HER2-positive) breast cancer cell lines compared to normal breast cell line (MCF10A). 7SK small nuclear RNA (7SK), AK023948, antisense noncoding RNA in the INK4 locus (ANRIL), chromatin-associated RNA (CAR) intergenic 10, H19 upstream conserved (HUC) 1 and 2, HOXA11 antisense (Hoxa11as), MALAT1-associated small cytoplasmic RNA (mascRNA), MER11C, neuroendocrine secretory protein (Nesp) antisense (Nespas), nuclear repressor of NFAT (NRON), p53 mRNA, PSF inhibiting RNA, spinocerebellar ataxia type 8 (SCA8), snaR, small nucleolar RNA host gene (SNHG5), and zinc finger antisense 1 (ZFAS1) were all upregulated in SK-BR3 breast cancer cells. However, the Scm-related gene containing four mbt domains (SFMBT2) and urothelial carcinoma-associated 1 (UCA1) was downregulated.

[Figure omitted. See PDF]

Table 1.

Details of long noncoding RNA expression levels in SK-BR3 breast cancer cells.

NCBI reference sequence	LncRNA	Expression in SK-BR3/MCF10A
DQ485453.1	ANRIL	25.22 ± 9.97
NR_004435.1	snaR	7.29 ± 1.11
AJ251759.1	Nespas	5.02 ± 2.68
N/A	7SK	4.59 ± 1.30
N/A	PSF inhibiting RNA	3.62 ± 2.00
FJ209302.1	mascRNA	3.44 ± 1.43
BC025338.1	Hoxa11as	3.32 ± 2.26
AC006443.1	NRON	3.11 ± 0.79
AK023948.1	AK023948	3.03 ± 1.23
N/A	MER11C	3.03 ± 1.07
DA932053.1	p53 mRNA	2.75 ± 1.15
BD286031.1	CAR Intergenic 10	2.70 ± 0.58
AL546946.3	HUC 1 and 2	2.69 ± 0.45
NM_001081005.1	ZFAS1	2.60 ± 0.72
NR_002717.2	SCA8	2.18 ± 0.29
NR_003038.2	SNHG5	2.14 ± 0.85
NR_015379.3	UCA1	0.45 ± 0.13
N/A	SFMBT2	0.02 ± 0.03

NCBI: National Center for Biotechnology Information; N/A: NCBI reference sequence information is not available; LncRNA: long noncoding RNA.

Next, differentially expressed long intervening noncoding RNAs (lincRNAs) were selected for further analysis and were confirmed in several breast cancer cell lines using real-time RT-PCR. The levels of ANRIL and snaR expression were significantly increased in breast cancer cells, while expression of UCA1 was downregulated in breast cancer cells. However, the expression of SNHG5 was not significantly different in SK-BR3 cells (Figure 2).

Figure 2.

Confirmation study of the selected lncRNA in diverse breast cancer cell lines. (a) Antisense noncoding RNA in the INK4 locus (ANRIL), (b) small NF90-associated RNA (snaR), (c) small nucleolar RNA host gene 5 (SNHG5), and (d) urothelial cancer associated 1 (UCA1), which were significantly upregulated or downregulated in SK-BR3 breast cancer cells, were selected for confirmation study. Real-time quantitative reverse transcriptase polymerase chain reaction was performed for relative quantification of their expression levels in MCF10A, MDA-MB-231, MCF7, SK-BR3, and T47D. Data are presented as mean ± SD from three independent experiments.

[Figure omitted. See PDF]

Downregulation of snaR inhibits proliferation and migration of SK-BR3 cells

In previous studies, we found that snaR promotes the proliferation, migration, and invasion of MDA-MD-231 breast cancer cells.¹³ To investigate the functional role of snaR in HER2-positive breast cancer in this study, we treated the SK-BR3 cells with the siRNA which leads knockdown of snaR and confirmed the efficiency of interference by quantitative RT-PCR (qRT-PCR). A specific siRNA was shown to induce as significantly downregulated in SK-BR3 cells (Figure 2(a)). After the snaR knockdown, the proliferation of SK-BR3 cells was significantly decreased which was determined by the MTT assay (Figure 3). Also, in a wound-healing assay, the SK-BR3 cells were significantly less migrated (Figure 4). However, the downregulation of snaR by siRNA showed no significant difference in invasiveness of SK-BR3 cells using Matrigel Transwell assay (Figure 5). Based on these assays, the snaR is associated with cell proliferation and migration, but not with invasiveness of SK-BR3 cancer cells.

Figure 3.

Knockdown of small NF90-associated RNA (snaR) suppresses the proliferation of SK-BR3 breast cancer cells. (a) The level of snaR expression was determined in SK-BR3 breast cancer cells upon transfection with si-snaR or negative control scramble (NC) by quantitative reverse transcriptase polymerase chain reaction. (b) Cell proliferation assay following transfection with si-snaR or NC. Data are presented as mean ± SD; significantly different at *p < 0.005 compared with the NC group.

[Figure omitted. See PDF]

Figure 4.

Knockdown of small NF90-associated RNA (snaR) suppresses the migration of SK-BR3 breast cancer cells, which was confirmed in the wound-healing assay. SK-BR3 breast cancer cells were transfected, cultured to monolayer, and scratched with a pipette tip. After a wound has been formatted, the images were taken at 0, 24, and 48 h.

[Figure omitted. See PDF]

Figure 5.

Knockdown of small NF90-associated RNA (snaR) could not regulate the invasiveness of SK-BR3 breast cancer cells. (a) The cell invasion analysis of SK-BR3 breast cancer cells upon transfection with si-snaR or negative control scramble (NC) was performed by Transwell assays with Matrigel-coated membrane. (b) The number of invasive cells per field was not significantly different in SK-BR3 breast cancer cells compared to NC. Data are presented as mean ± SD.

[Figure omitted. See PDF]

Discussion

LncRNAs have been reported to have a diverse range of biological processes from controlling of cell cycle to cell differentiation through various mechanisms at the transcriptional, post-transcriptional, and epigenetic regulatory levels. Based on these mechanisms, the lncRNAs may serve as oncogenic or tumor-suppressor genes similar to protein-coding genes.

LncRNA, snaR, was identified as upregulated and associated with proliferation of HER2-postive breast cancer cells in this study. Although snaR was also expressed in estrogen receptor–positive breast cancer cell lines, we did not perform further confirmation study with estrogen receptor–positive breast cancer cell lines because we intended to establish snaR as a biomarker in aggressive tumor types. However, after all the analysis, it seems that snaR plays a role as biomarker in most subtypes of breast cancer.

HER2 overexpression in breast cancer referred as HER2-positive breast cancer is identified approximately in 20%–30% of breast cancers.^3,14–16 This subtype is well characterized as an aggressive type revealing rapid progression, early systemic metastasis, early relapse, and lower survival outcomes.^3,17 However, a targeted agent, trastuzumab, pertuzumab, and lapatinib significantly improved oncologic outcome of HER2-positive breast cancer, and the oncologic risks could be reduced as similar as hormone receptor–positive breast cancer.^18,19 Although the activated HER2 initiates intracellular downstream signal involved in diverse biological processes ultimately associated with tumor progression, trastuzumab, one of the monoclonal antibody, can block the HER2 receptor.²⁰ However, even if the trastuzumab had led significant improvement in clinical outcomes, resistance to trastuzumab ultimately occurs causing unfavorable clinical problems. When a resistance to target agent has been detected, it is necessary to approach other therapeutic agents with totally different mechanism rather than receptor inhibition.^21,22 Although some possible mechanisms of resistance against anti-HER2 agents have been suggested, further clarification is still needed.^23,24

LncRNAs have been identified as strongly associated with various malignancies. In particular, the key biological functions of lncRNA to malignancies are possibly oncogenic or tumor suppressive mediated by microRNAs (miRNAs).^2,25 This hypothesis provides a theoretical basis for developing a new treatment of malignancies. By intentionally downregulating lncRNAs, tumor progression or cell growth could be inhibited. Similar in breast cancer, several lncRNAs such as ANRIL or UCA1 and snaR were revealed as upregulated and strongly associated with tumor progression or poor prognosis in previous studies.^{12,13,26–28} However, as breast cancer is an extremely heterogenic disease, these knockdown processes may not result as identical based on tumor subtypes or characteristics. Therefore, the study of biological functions of lncRNA should be conducted differently according to the subtype of breast cancer. Although several lncRNAs had been reported upregulated or downregulated in breast cancer, there can be controversy in other studies.²⁹ Therefore, further researches should be continued.

snaR, one of noncoding RNA, had been discovered in a screening study for binding partners with the double-stranded RNA binding protein nuclear factor 90 (NF90). snaR is highly structured, noncoding RNAs with 117–120 nucleotides and can be classified as two family subsets (snaR-A and snaR-B).^30,31 Usually, snaR is upregulated in several malignancies, and therefore, downregulation of snaR can regulate tumor proliferation, migration, or invasiveness which is related with tumor progression process. In our previous study, the snaR was identified as significantly in higher level in triple-negative breast cancer cells. And based on recent study, the similar results were seen in HER2-positive breast cancer. However, only tumor proliferation and migration were inhibited by knockdown process of snaR, not invasiveness.

Proliferation, migration of tumor cells, invasion, and metastasis of tumor are sequential processes of tumor progression.³² Usually, proliferation and migration of tumor occur in early phase of tumor progression, and in the late phase, the tumor cells are attached on adjacent site with invasion and may lead metastasis to other organs.³³ Tumor cell migration is generally regulated by integrins, matrix-degrading enzymes, cell–cell adhesion molecules, and cell–cell communication.^34,35 Based on our recent studies, the proliferation and migration of tumor cells were inhibited by knockdown of snaR commonly in HER2-positive breast cancer cells and triple-negative breast cancer cells. However, an impactful difference between two cancer cell lines was inhibition of invasiveness of tumor cells. Basically, without considering treatment options, HER2-positive breast cancer would be more aggressive than triple-negative breast cancer, and the invasiveness of tumor cells was not inhibited only with the inhibition of snaR. This means that the knockdown of snaR could be helpful to develop an additional treatment agent for breast cancer, but may be insufficient to be a main treatment agent.

The higher expression level of lncRNA in malignancies means that this affects tumor progression directly or indirectly. Therefore, a process of inhibition of specific lncRNA would be one further step toward cancer treatment. Based on our study, the snaR was significantly upregulated in various subtypes of breast cancer cell lines, including triple-negative breast cancer cells, HER2-positive breast cancer cells, and hormone-receptor-positive breast cancer cells.¹³ However, because of strong heterogeneity of breast cancer, each study should be conducted independently.

This study was performed with HER2-positive breast cancer cells, and the fact that the downregulation of snaR can inhibit tumor proliferation and migration was proven. Because this mechanism involves a completely different pathway, it can be applied for treatment of HER2-positive breast cancer which showed resistance to trastuzumab. Further study with snaR in therapeutic agent–resistant breast cancer cells would provide new strategies for treatment of refractory breast cancers.

Although we confirmed the role of snaR with respect to breast cancer cell lines in this study, we could not confirm in tissue level. And, also, we could not clarify the mechanism of snaR which affects proliferation and migration of HER2-positive breast cancer cell lines. Further study with HER2-positive breast cancer tissue would be necessary to get more information.

In conclusion, snaR was identified as upregulated in HER2-positive breast cancer cell lines, and the knockdown of snaR was strongly associated with suppression of tumor proliferation and migration. This result would provide an important theoretical basis for treatment of HER2-positive breast cancer as a targeted agent.

J.H.J. and Y.S.C. have contributed equally to this work.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by a grant from Biomedical Research Institute, Kyungpook National University Hospital (2015).