Introduction
There is a rapid rising trend in the incidence of breast cancer (BC) with more than 1 million new-diagnosed cases each year [
1]. According to molecular characteristics of HER-2, estrogen receptor (ER), ki-67, and progesterone receptor (PR), BC can be divided into diverse subtypes, such as HER-2 overexpression, Lumina A, Lumina B, ‘normal-like’ breast tumors and basal-like tumors. As defined, triple-negative breast cancer (TNBC) is a tumor that is negative for HER-2, ER and PR [
2]. Currently, surgical resection and chemotherapy are the major systemic therapies for patients with TNBC due to the lack of effective biomarker [
3]. Compared with patients with other BC subtypes, TNBC patients have significantly higher rates of metastasis and recurrence [
4,
5]. Thus, it is essential to explore more effective biomarkers for early diagnosis and advanced treatment of TNBC.
It was commonly accepted that non-coding RNAs (ncRNAs) act as critical factors in the regulation of gene expression, thereby exerting functions on tumor or non-tumor cell phenotypes [
6,
7]. Long non-coding RNAs (lncRNAs) are generally regarded as genomic transcripts exceeding 200 nucleotides in length [
8]. Lacking open reading structure of indispensable length, lncRNAs are limited in protein-coding [
9]. It has been highlighted by diverse researches that lncRNAs are associated with molecular mechanisms underlying cancer development and can be used as promising biomarkers and therapeutic targets for cancers [
10]. Previously, many studies have emphasized the important roles of lncRNAs in regulating various cell biological behaviors including cell growth, apoptosis, migration and invasion [
11,
12]. Increasing lncRNAs, such as NEAT1 [
13], BORG [
14] and LINC01638 [
15], have been identified as oncogenes in TNBC.
LncRNAs are implicated in tumorigenesis and progression of cancers via multiple mechanisms [
16]. Importantly, lncRNA can function as a competing endogenous RNA (ceRNA) to upregulate mRNA expression by sponging microRNA (miRNA). For example, lncRNA EPB41L4A-AS2 upregulates FOXL1 expression to suppress hepatocellular carcinoma progression via sponging miR-301a-5p [
17]. LncRNA NORAD facilitates cervical cancer cell proliferation and invasion by targeting the miR-590-3p/SIP1 axis [
18]. In addition, lncRNAs can modulate tumor initiation by binding with RNA-binding proteins (RBPs) to maintain mRNA stability. For instance, LINC00324 positively regulates FAM83B mRNA stability to promote cell proliferation in gastric cancer through binding with HuR protein [
19]. LBX2-AS1 stabilizes LBX2 mRNA expression to drive gastric cancer progression via recruiting FUS protein [
20]. LncRNA PITPNA antisense RNA 1 (PITPNA-AS1) has been previously reported as an oncogene in hepatocellular carcinoma by acting as a ceRNA [
21]. However, its functional role and molecular mechanism in TNBC remain unclear.
In this research, we investigated the function and regulatory mechanism of PITPNA-AS1 in TNBC cellular processes. Our findings revealed that MYBL2-activated PITPNA-AS1 sponged miR-520d-5p and recruited DDX54 protein to increase SIK2 expression, thereby promoting cellular activities in TNBC. This finding implicated PITPNA-AS1 as a promising biomarker for TNBC treatment.
Materials and methods
Tissue samples
Total 56 pairs of TNBC tissues and their adjacent non-tumor tissues were collected from patients who were diagnosed with TNBC at the Second Hospital of Shanxi Medical University (Shanxi, China). Before the surgery, all patients have signed informed consents and none of them received any anti-cancer treatments. The specimens collected from the patients were snap-frozen in liquid nitrogen and subsequently preserved at − 80 °C. Gene expression analysis using 56 pairs of TNBC tissues and corresponding non-tumor tissues was conducted, which was approved by Institutional Ethics Committees of the Second Hospital of Shanxi Medical University.
Cell lines
One normal breast epithelial cell, MCF10A, and four human TNBC cell lines (HCC1937, MDA-MB-468, MDA-MB-231, MDA-MB-436) were bought from ATCC (Manassas, VA). Cells were incubated in DMEM (Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin in a humidified air at 37 °C with 5% CO2.
Cell transfection
HCC1937 and MDA-MB-468 cells were transfected with the sh-RNAs (plasmid backbone: pGPU6/Hygro) targeting PITPNA-AS1 (sh-PITPNA-AS1#1/2), DDX54 (sh-DDX54#1/2), MYBL2 (sh-MYBL2) and corresponding negative controls (sh-NC) at the final concentration of 100 nM. Stably transfected cells were selected by detection of Hygromycin B resistance. To overexpress SIK2, E2F6, PAX5, FOXP2, ELK5, MYBL2 and YY1, pcDNA3.1 vector targeting corresponding genes and empty pcDNA3.1 vector (control) were transfected into HCC1937 and MDA-MB-468 cells. For overexpressing or silencing miR-520d-5p, miR-520d-5p mimics or inhibitor was transfected into HCC1937 and MDA-MB-468 cells with NC mimics or inhibitor as a negative control, separately. All above plasmids or oligonucleotides were purchased from GenePharma and were transfected into HCC1937 and MDA-MB-468 cells using Lipofectamine 2000 (Invitrogen) for 2 days.
RT-qPCR analysis
Using TRIzol Reagent (Invitrogen), total RNA was extracted from MCF10A, HCC1937, MDA-MB-468, MDA-MB-231, MDA-MB-436 cells and then reverse-transcribed into cDNA with a Reverse Transcription Kit (Takara). A One Step TB Green
® PrimeScript™ RT-PCR Kit (Takara) was utilized for RT-qPCR on a Bio-Rad CFX96 Real-Time PCR system. The 2
−∆∆Ct method [
22] was used for calculation of relative expression fold changes. GAPDH served as the internal reference for PITPNA-AS1 and mRNAs, while U6 served as the internal reference for miRNAs. The thermo-cycling conditions were set as follows: 95 °C for 5 min followed by 45 cycles at 95 °C for 10 s and 55 °C for 30 s, and a melting curve analysis every 0.2 °C from 55 to 95 °C for 2 min was obtained. Relative primer sequences are listed in Table
1, which were designed using GETprime software and synthesized by RiboBio (Guangzhou, China).
Table 1
Relative primer sequences used for PCR
PITPNA-AS1 | Forward: 5′-GCAGGGTGGATAAAGAGGA-3′ |
Reverse: 5′-CCTACTGACAGGATGTCCT-3′ |
miR-524-5p | Forward: 5′-CTACAAAGGGAAGCACTTTCTCG-3′ |
Reverse: 5′-CTCTACAGCTATATTGCCAGCCAC-3′ |
miR-520d-5p | Forward: 5′-CTACAAAGGGAAGCCCTTTCG-3′ |
Reverse: 5′-CTCTACAGCTATATTGCCAGCCAC-3′ |
U6 | Forward: 5′-CTCGCTTCGGCAGCACA-3′ |
Reverse: 5′-AACGCTTCACGAATTTGCGT-3′ |
DENND1B | Forward: 5′-TGCATCCTTAGTTACCTTCCC-3′ |
Reverse: 5′-CAGTTCCTTAGCCAAGTAATCTG-3′ |
ATL3 | Forward: 5’-CAAGAGGAGCAGTGTAATGTG-3′ |
Reverse: 5′-AAACGTTCCATTGCTTCGT-3′ |
SIK2 | Forward: 5′-GAGGTCCACAACAGGTCTC-3′ |
Reverse: 5′-TGCTACAATTCCCTGGGTG-3′ |
SNRNP200 | Forward: 5′-CTCCAATGCCAAGGATGTG-3′ |
Reverse: 5′-CTGATGTTGAAGCCCTGGA-3′ |
AFF3 | Forward: 5′-TACAACTTCCACACCAGCA-3′ |
Reverse: 5′-ATCTCCAAGCAAGGTCCTG-3′ |
UBE2J1 | Forward: 5′-GTACATCGTACGGACTCCAG-3′ |
Reverse: 5′-TCATGGAGGTATTCTTAGCTACAG-3′ |
DECR1 | Forward: 5′-GGTAAAGGAATGACAACTCTTCTG-3′ |
Reverse: 5′-CATTTCGGCTGGCTATCAC-3′ |
DDX54 | Forward: 5′-TGGTGAGTGATCGAGTGAG-3′ |
Reverse: 5′-CTTCCTGTCCTGACTGTCC-3′ |
MYBL2 | Forward: 5′-TCCTGGATTCCTGTAACAGC-3′ |
Reverse: 5′-AGTTCAGAAACTGGGAGGG-3′ |
E2F6 | Forward: 5ʹ-TAGATGGATAGGATCTGATCTTAGC-3ʹ |
Reverse: 5ʹ-GATAAGTCAGAAAGTTCCTCCTG-3ʹ |
PAX5 | Forward: 5ʹ-TGTTTGCCTGGGAGATCAG-3ʹ |
Reverse: 5ʹ-CGGATGATCCTGTTGATGGA-3ʹ |
FOXP2 | Forward: 5ʹ-GATGCAACAACTCCAGCAG-3ʹ |
Reverse: 5ʹ-AGGACTTAAGCCAGCTTGAG-3ʹ |
ELK4 | Forward: 5ʹ-GCCCACTGGGAATACTGAG-3ʹ |
Reverse: 5ʹ-TCAGTATGATGGGTGTCTGTG-3ʹ |
YY1 | Forward: 5ʹ-GAATTTGCTAGGGCTGCAC-3ʹ |
Reverse: 5ʹ-CACATTCTGCACAGACGTG-3ʹ |
GAPDH | Forward: 5ʹ-GCATCCTGGGCTACACTG-3ʹ |
Reverse: 5ʹ-TGGTCGTTGAGGGCAAT-3ʹ |
Subcellular fractionation assay
Subcellular fractionation assay was conducted to assess subcellular localization of PITPNA-AS1. According to manufacturer’ instructions, nuclear and cytoplasmic fractions extracted from TNBC cells were isolated using a Cytoplasmic and Nuclear RNA Purification Kit (Norgen). RT-qPCR analysis was applied for the detection of PITPNA-AS1, GAPDH or U6 expression. GAPDH was a cytoplasmic control and U6 was a nuclear control.
Fluorescence in situ hybridization (FISH) assay
FISH assay was conducted for detection of the subcellular location of PITPNA-AS1. HCC1937 and MDA-MB-468 cells (5 × 103 cells per well) were cultured in a 24-well plate, and the supernatant was discarded after 24 h. After being washed with PBS and fixed by 4% paraformaldehyde, the cells were permeabilized with PBS containing 0.5% Triton X-100. Next, the cells were blocked with the pre-hybridization solution for 4 h at 37 °C and hybridized with PITPNA-AS1 specific probe (RiboBio) overnight at 37 °C followed by washing with the hybridization solution at 42 °C in the dark. Afterwards, 4′-6-diamidino-2-phenylindole (DAPI) was utilized to stain nucleus for 10 min, and a fluorescence microscope (Olympus) was used to capture the images of the cells.
Cell counting kit-8 (CCK-8) assay
Based on manufacturer’s requirements, cell viability was testified by a CCK-8 kit (Boster Biological Technology, CA, USA). In brief, transfected HCC1937 and MDA-MB-468 cells were plated into 96-well plates at a density of 2 × 103 cells/well and were incubated for 0, 24, 48 and 72 h. Next, 10 μL of CCK-8 solution was added into each well for further incubation in 5% CO2 at 37 °C for 1 h after cell adhesion. The culture medium was then removed, and the plates were washed twice by PBS. Lastly, absorbance at 450 nm was monitored by a microplate reader (EL340; Bio-Tek Instruments, Hopkinton, MA, USA) for detecting cell viability.
Colony formation assay was performed to reveal cell proliferation. In brief, 1 × 103 HCC1937 and MDA-MB-468 cells were seeded in 6-well plates. At 2 weeks after incubation, colonies were fixed by methanol for 15 min and were dyed by crystal violet (Sigma-Aldrich) in PBS for 20 min. After that, crystal violet was slowly washed away with running water. The plates were air-dried in an inverted position and number of stained colonies were manually counted.
Flow cytometry analysis
Transfected HCC1937 and MDA-MB-468 cells were subjected to the staining of propidium iodide (PI) and FITC-Annexin V for detection of apoptosis rate. HCC1937 and MDA-MB-468 cells were seeded in 6-well plates at a concentration of 1 × 105 cells/well, after which cells were incubated in 5 μL of Annexin V-FITC for 10 min and 10 μL of PI for 15 min at 4 °C in the dark. Later, using CellQuest software (BD Biosciences, San Jose, CA), cells were analyzed by flow cytometer (FACScan; BD Biosciences), and apoptosis rate of TNBC cells was assessed. The early apoptotic cells were distributed in the second quadrant and late apoptotic and necrotic cells were in the third and fourth quadrants. Apoptosis rate (%) was defined as cell percentage in the third quadrant.
Wound healing assay
Wound healing assay was performed to detect cell migration. TNBC cells (1.5 × 106) were plated into the 6-well plates. Then, a pipette tip was utilized to horizontally scratch the wound after cell reached about 80% confluence. After cells were washed twice, cells were incubated in serum-free medium for 24 h. The gap between cells was photographed under a low-magnification phase-contrast microscope (Olympus MK, Tokyo, Japan).
Transwell assay
Transwell assay was conducted to reveal cell migration in vitro. Matrigel-coated upper chamber (8.0 μm pore size, BD Biosciences) was filled with TNBC cells (5 × 104/well) suspended in DMEM. The DMEM supplemented by 10% FBS was placed in the bottom chamber. Forty-eight hours later, cells in the upper chamber were scraped off by cotton swabs. Cells in the lower chamber were fixed with methanol and stained with 0.5% crystal violet. Finally, the average number of stained cells were counted using an inverted microscope (Olympus) under five randomly selected visual fields by Image J software.
Western blot analysis
RIPA lysis buffer containing protease inhibitors was used for the extraction of total protein from HCC1937 and MDA-MB-468 cells. Before being transferred to a polyvinylidene fluoride membrane (Millipore), equivalent proteins were isolated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis. After being blocked with 5% skimmed milk for 1 h, the membranes were incubated with primary antibodies against E-cadherin (ab40772, 1/20000), N-cadherin (ab76011, 1/5000), Vimentin (ab92547, 1/200), Slug (ab27568, 1/500), Twist (ab175430, 1/1000), SIK2 (ab53423, 1/1000), DDX54 (ab76947, 1/2000), and GAPDH (ab9485, 1/2500) overnight at 4 °C. Next, secondary antibody (ab6721, 1/10000) conjugated with HRP was added for further incubation of 1 h at room temperature. All the antibodies were purchased from Abcam (Cambridge, USA). A chemiluminescence detection system was applied to visualize the proteins. The gray value of each protein band was analyzed by the ImageJ software, and the ratio of the gray value of the target protein to GAPDH was calculated.
In vivo experiment
For animal study, a total of 20 female BALB/C nude mice (6-week-old; weighing 20–30 g) were purchased from Shi Laike Company. The animal experiments were performed with ethical approval from the Second Hospital of Shanxi Medical University (Shanxi, China). The nude mice were subcutaneously injected with 0.2 mL suspension of HCC1937 cells (3 × 106) that were stably transfected with sh-PITPNA-AS1 or sh-NC. The tumor volume was measured every 4 days. \({\text{Volume}}\,({\text{mm}}^{3} ) = 1/2\,{\text{L}} \times {\text{D}}^{2} .\) L represents the maximum diameter, and D represents the shortest diameter of the tumor. After 28 days, mice were euthanized, and tumors were excised for weighing.
Immunohistochemistry staining (IHC)
Tumor tissues were fixed in 4% paraformaldehyde, and then dehydrated in ethanol solutions. Next, tissues were embedded in paraffin, and cut into 4 μm sections. The sections were routinely deparaffinized, followed by antigen retrieval. After washing in PBS for 15 min, tissues were blocked with goat serum for 1 h followed by incubation with primary antibody against Ki67 (ab92742, 1/500) overnight at 4 °C. Subsequently, the sections were cultivated with HRP-conjugated secondary antibody (ab6721, 1/1000). The slides were then counterstained with hematoxylin for 1 min and mounted with neutral balsam. The Ki67 positivity was detected using a Histostain™ SP-9000 immunohistochemical staining kit (Zymed Laboratories, South San Francisco, CA, USA). An OLYMPUSBX-41 microscope (Olympus) was used to capture the images.
RNA pull down assay
HCC1937 and MDA-MB-468 cells were transfected with 50 nM biotin-labeled PITPNA-AS1 for 48 h. Cells were then incubated in RIPA lysis buffer for 10 min and then the mixture were centrifuged at 14,000×g to obtain the supernatant. Protein lysate was incubated with M-280 streptavidin beads which were pre-coated with RNase-free bovine serum albumin and yeast tRNA. Next, the beads were incubated at 4 °C for 3 h. The binding miR-520d-5p was purified by TRIzol and its expression was examined by RT-qPCR. As per manufacture’s guidelines, a Pierce Magnetic RNA–Protein Pull-Down Kit (Thermo Fisher Scientific) was employed to detect the proteins binding with PITPNA-AS1. Protein extracts from HCC1937 or MDA-MB-468 cells were subjected to biotinylated PITPNA-AS1 (50 pmol) and M-280 streptavidin magnetic beads (Invitrogen) for 1 h at 4 °C. Finally, the cells were washed five times with RIPA buffer, and incubated with 5× loading buffer at 95 °C for 5 min. The eluted DDX54, FMR1, IGF2BP1, IGF2BP2 proteins were detected by western blotting.
Luciferase reporter assay
For luciferase reporter assay, PITPNA-AS1-WT/Mut or SIK2-WT/Mut reporter was constructed by subcloning wild-type (WT) or mutant (Mut) sequences of PITPNA-AS1 complementary to miR-520d-5p or SIK2 3′UTR into the pmirGLO dual-luciferase vector. Then, these constructed reporters were co-transfected with miR-520d-5p mimics or NC mimics into HCC1937 or MDA-MB-468 cells. For PITPNA-AS1 promoter-luciferase analysis, wild type or mutated PITPNA-AS1 promoter sequences (site 1 and site 2) were subcloned into the pGL3-Basis luciferase vector (Promega, Madison, WI, USA), and these constructs were co-transfected with pcDNA3.1/MYBL2 or pcDNA3.1 into HCC1937 and MDA-MB-468 cells. After 2 days, a Dual-Luciferase Report Assay System (Promega) was used for the measurement of luciferase activity. Relative luciferase activity was defined as the ratio of the relative light unit of firefly luciferase to that of Renilla luciferase.
RNA immunoprecipitation (RIP) assay
Based on the manufacturer’s instruments, an EZ-Magna RIP kit (Millipore) was applied. In brief, HCC1937 and MDA-MB-468 cells were washed with pre-cooled PBS and then lysed in RIPA lysis buffer in an ice bath for 5 min, and centrifuged at 12,000×g for 10 min at 4 °C. Then, cell extract was incubated with magnetic beads coated human anti-Ago2 (Millipore), anti-DDX54 (Millipore) or control anti-IgG (Millipore) at 4 °C overnight. Finally, expression of PITPNA-AS1, miR-520d-5p, SIK2 immunoprecipitated by Ago2 and expression of PITPNA-AS1, SIK2 immunoprecipitated by DDX52 were analyzed by RT-qPCR after being purified by proteinase K.
Actinomycin D assay
Actinomycin D can inhibit the synthesis of mRNAs. After transfection with sh-NC, sh-PITPNA-AS1#1, sh-DDX54#1 for 48 h, HCC1937 and MDA-MB-468 cells were treated with actinomycin D (5 μmol/L) for 0, 4, 8, 12 h. Later, RT-qPCR was used for the evaluation of relative SIK2 expression levels.
Chromatin immunoprecipitation (ChIP) assay
With a Magna ChIP Kit (Millipore), ChIP was performed to explore which site is responsible for the binding between MYBL2 and PITPNA-AS1 promoter. Briefly, crosslinked chromatin DNA was separated into fragments of 200–2000 bp through sonication. Next, lysates were immunoprecipitated with anti-MYBL2 or anti-IgG (internal control) at 4 °C overnight. Subsequently, the mixture was centrifuged, and the precipitate was washed with the low salt buffer, the high salt buffer, the LiCl solution, and the trace element solution. The protein-DNA complex was eluted with 250 μL of ChIP Wash Buffer and de-crosslinked with 20 μL of 5 M NaCl. Quantity of immunoprecipitated DNA was detected by RT-qPCR.
Statistical analysis
Data were expressed as mean ± standard deviation and were analyzed by SPSS 22.0 (SPSS, Chicago, USA) from three biological and technical replications. The variance significance was evaluated by Student’s t test for difference between two group or ANOVA for that among three groups. P < 0.05 was set as the threshold of statistical significance.
Discussion
Increasing literatures reported that lncRNAs are involved in occurrence and development of various human cancers by sponging miRNA or recruiting RBPs [
23,
24]. Although the essential roles of abnormally expressed lncRNAs have been highlighted in TNBC progression [
14,
25], the potential role and mechanism of lncRNA PITPNA-AS1 underlying TNBC remain obscure and deserve to be explored. Our study suggested that PITPNA-AS1 expression was higher in TNBC tissues and cells than in control tissues and cells, and mainly distributed in the cytoplasm of TNBC cells. Moreover, decreased PITPNA-AS1 repressed TNBC cell proliferation, facilitated cell apoptosis, and suppressed cell migration and invasion in vitro. Furthermore, PITPNA-AS1 silencing inhibited xenograft tumor growth in vivo. Collectively, PITPNA-AS1 exhibited oncogenic properties in TNBC.
MiRNAs are another class of ncRNAs with about 22–24 nucleotides in length and played important roles in cancer progression [
26,
27]. Existing evidence has depicted that lncRNAs can combine with specific miRNA to facilitate or suppress the initiation or progression of tumors [
28,
29]. In this study, miR-520d-5p was identified for further exploration via bioinformatics analysis and a series of molecular mechanism experiments. Previously, miR-520d-5p was found to inhibit cell proliferation and cell cycle via targeting PTTG1 in glioma [
30]. In gastric cancer, miR-520d-5p is an important regulator in cell proliferation and survival [
31]. In addition, miR-520d-5p functions as an anti-oncogene in colorectal cancer and suppresses tumor growth and metastasis via regulating CTHRC1 [
32]. Herein, we found that miR-520d-5p had binding capacity with PITPNA-AS1 in TNBC. Furthermore, miR-520d-5p expression was at a low level in TNBC tissues and cell lines. These findings suggested that PITPNA-AS1 sequestered miR-520d-5p in TNBC.
Salt inducible kinase 2 (SIK2) has been validated to exert tumor-promoting functions in a variety of cancers, including TNBC [
33,
34]. Nevertheless, the relationship between SIK2 and miR-520d-5p (or PITPNA-AS1) in TNBC cells needs investigation. In our study, it was verified that SIK2 was directly targeted by miR-520d-5p in TNBC. More importantly, results in our study indicated that PITPNA-AS1 positively modulated SIK2 expression not merely via sponging miR-520d-5p.
DEAD-box helicase 54 (DDX54) was recognized as a member of RBPs and reported as an oncogene in some cancers [
35,
36]. In this study, SIK2 mRNA stability was inhibited by PITPNA-AS1 knockdown. DDX54 was identified to interact with PITPNA-AS1 (or SIK2) in TNBC cells. In addition, restoration experiments suggested that TNBC cellular processes inhibited by silenced PITPNA-AS1 was rescued by SIK2 overexpression or co-effect of miR-520d-5p inhibition and DDX54 upregulation.
Emerging investigations have implied that transcriptional regulation mediated by transcription factor was a major reason for the aberrant expression of lncRNAs [
37,
38]. MYB proto-oncogene Like 2 (MYBL2) was known as a transcription factor in lung adenocarcinoma [
39]. Our study revealed that MYBL2 positively regulated PITPNA-AS1 expression and bound to PITPNA-AS1 promoter, which indicated that the upregulation of PITPNA-AS1 in TNBC was transcriptionally induced by MYBL2.
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