Background
The morbidity and mortality rates of breast cancer (BC) worldwide are more than 20% and 10%, respectively [
1]. Currently, treatment for BC includes surgery, chemotherapy, radiotherapy, endocrine therapy and targeted therapy however, the therapeutic effect often remains unsatisfactory. Long-term continuous chemoradiotherapy can lead to toxic side effects and weaken the patient, while the cytotoxic effect on cancer cells is also poor [
2,
3]. Current targeted therapies for BC mainly include targeted endocrine therapy for hormone receptor-positive BC and epidermal growth factor receptor (HER)-2-positive BC. However, the use of trastuzumab in the clinical treatment of early HER2-positive and metastatic BCs benefits less than 35% of patients [
4], and the development of drug resistance is an additional problem that needs to be urgently solved.
Human genomics research has often focused on RNAs that encode proteins. RNAs that cannot encode proteins are considered useless RNAs or transcriptional noise. However, these RNAs play an indispensable role in important activities such as cell proliferation, differentiation, senescence and apoptosis. In mammalian genomics, only a small number of transcripts encode proteins, and the vast majority are noncoding RNAs (ncRNAs), accounting for approximately 80% of the human genome. According to the size of the transcript, ncRNAs can be divided into miRNAs and lncRNAs. NcRNAs greater than 200 nt in length are called lncRNAs [
5]. Ponting et al. [
6] studied the evolution of lncRNAs and their role in transcriptional regulation and epigenetic gene regulation. They speculated that lncRNAs may have five different origins: generated by (1) protein-coding gene sequence disruption; (2) chromatin rearrangement; (3) noncoding RNA reverse transcriptional translocation; (4) noncoding genes containing adjacent repeats; and (5) insertion of a transposable element into the genome. However, there is still no definitive theory about the origin of lncRNAs. The biological functions of lncRNA are mainly accomplished through signaling molecules, decoy molecules, guide molecules, scaffold molecules and chromatin modification complexes, occurring at both the transcriptional level and the posttranscriptional level, and by structural RNAs in cell proliferation, differentiation, senescence, death, carcinogenesis, and the occurrence and development of disease [
7].
The expression of lncRNAs in BC tissues varies considerably. Different types of lncRNAs affect BC by interfering with mRNA splicing and inducing apoptosis, X chromosome silencing and genomic imprinting [
8]. Genomics research provides a new feasible solution for the detection, diagnosis, target selection and prognostication of BC. In this study, differentially expressed lncRNAs in BC and para-carcinoma tissues were screened by gene chip technology; from these analyses, the lncRNA IGBP1-AS1 was identified, and its function was elucidated to reveal its clinical value. IGBP1-AS1 has not been studied before and therefore, its biological function as well as regulatory mechanism remained unclear. Furthermore, in this research we also confirmed the downstream targets of IGBP1-AS1. We found that miR-24-1 may be sponged by IGBP1-AS1. miR-24-1 has been reported to be involved in tumorigenesis or chemoprevention of colorectal cancer, ovarian cancer, pediatric pilocytic astrocytomas and ependymomas [
9‐
11]. Zic family member 3 (ZIC3) is a transcription factor that has widely studied. Its function varies in several cancers [
12,
13], however, in BC, it still remains unknown.
Methods
Microarray profiling
The LncRNA microarray expression profiling was carried out strictly based on the protocol provided by the manufacturer to screen the LncRNAs with varied expression levels in BC tissues and adjacent tissues (fold change > 1.5 and Padj< 0.05). At first the target cDNA was synthesized, labeled and purified, after which the Cyanine-3-CTP labeled cRNA was hybridized with the lncRNA microarray chip. The samples were analyzed by microarray after washing. R project was applied for result analysis and clustering.
Culture of cell lines
The BC cell lines (HCC70 and UACC-812) and human mammary cells (76 N-F2V) used in this study were gained from American Type Culture Collection (ATCC, Manassas, VA, USA). These cells were incubated in RPMI-1640 medium with 10% fetal bovine serum (FBS) and a supplement of 100 μg/L streptomycin and 100 μg/L penicillin under the conditions of 37 °C and 5% CO2.
Real-time quantitative polymerase chain reaction (RT-qPCR)
The TRIzol reagent (Invitrogen, USA) was applied for total RNA extraction, and the SYBR Green Mix (Promega) was adopted for primer amplification (forward primer: AGCAGCATTTTCCTGGCTAC; reverse primer: GGTGGAGGGGGAACCCATAG). The synthesis work of the primers was provided by Zhejiang East Genecreate Biological Engineering Co., Ltd. Data analysis was conducted by using the2−ΔΔCt method. The miR-24-1 expression was determined with the TaqMan MicroRNA Assays Kit (Applied Biosystems, USA). U6 and GAPDH were adopted as the endogenous controls for miR-24-1 and IGBP1-AS1. All the experiment procedures were conducted in triplicate.
Cell transfection with lentivirus
The pBLLV-CMV-IRES-ZsGreen IGBP1-AS1, pCMV-IRES-GFP ZIC3 cDNA lentiviral plasmid for overexpression (oe) transfection and Phblv-u6-ZsGreen-Puro ZIC3 shRNA lentiviral plasmid for knock-down transfection were gained from Genelily BioTech Co., Ltd, (Shanghai, China). The transfection procedure was performed with Lipofectamine 3000 (Invitrogen). The transfected cells were cultured with puromycin (2 μg/mL) for 2 weeks, and the stable cells were selected. The RT-qPCR technique was adopted for transfection efficiency verification.
Cell Counting Kit-8 (CCK8) assay
After a 5-day cell incubation at 37 °C and 5% CO2, CCK8 solution was added to each well of the 96-well plates (2 × 103 cells/well). A microplate reader was applied to detect the cell viability by measuring the absorbance value at 450 nm.
MTT assay
After 24, 48, 72 and 96-h cell incubation, 10 μL MMT (5 mg/mL) was added to each well of the 96-well plate (1 × 104 cells/well), and the transfected BC cells were cultured for another 4 h. After removal of the upper layer, 100 μL DMSO was added. The absorbance value at 490 nm was detected with a microplate reader.
Flow cytometry
A previous developed protocol was applied for the flow cytometry assay [
14]. Cell apoptosis was analyzed with the Annexin V-FITC early apoptosis kit, and the flow cytometer (FACScan; BD Biosciences) was adopted to analyze the IGBP1-AS1overexpressed BC cells and negative control cells, the results of which was calculated by CellQuest software (BD Biosciences).
Transwell assay
Transwell chambers (8-μm pore size; Corning Costar, USA) was used for the evaluation of cell invasion and migration. With the cells inoculated on the upper chamber, 20% serum was used as chemoattractant in the lower chamber. After 48-h incubation, methanol was used for fixation, and 0.1% crystal violet was applied for staining. Due to the slight abrasion on the upper surface of the filter, cells located on the lower surface were counted and photographed by using a microscope. All the procedures were carried out in triplicate.
Wound-healing assay
An equivalent amount of HCC70 and UACC-812 cells that was transfected with IGBP1-AS1 over-expressed plasmid and negative control (NC) plasmid were inoculated into 6-well plates. After the cells adhered to the wall and formed monolayers, a gap was drawn in the middle of the cell layer with a pipette tip. After 24 h, a microscope was used to observe the BC cells that migrated into the gap area.
Samples from tissue
The BC tissues and adjacent normal breast tissues used in this study were gained from the surgeries carried out in the Second Affiliated Hospital of Jiaxing University from Jan 2011 to June 2017 and stored at − 80 °C. Informed consents and complete clinical information was obtained from all the patients. In the end, microarray profiling was conducted with 10 BC specimens and 6 normal breast specimens (6 paired). The survival analysis and immunohistochemical assays were conducted in the other 94 BC specimens. The clinical procedures related to human were approved by the institutional human experiment and ethics committee of the Second Affiliated Hospital of Jiaxing University and the Affiliated Women’s Hospital of Zhejiang University. All the surgical procedures were carried out strictly in line with the provisions of the Helsinki Declaration.
In situ hybridization (ISH)
The ISH assay was conducted in accordance with a previously developed method [
15]. After labeling with the digoxigenin antibody (Roche, 11,093,274, 1:1000), the locked nucleic acid probe with complementary sequences of IGBP1-AS1 (custom LNA detection probe, Exiqon) was synthesized. The staining intensity was reviewed by 2 independent pathologists who were blinded to the study design.
Luciferase reporter assays
The pmirGLO-IGBP1-AS1-wt reporter vector was prepared by cloning the IGBP1-AS1 cDNA with miR-24-1 predictive binding site into the pmirGLO Dual-Luciferase miRNA Target Expression Vector (Promega). A similar procedure was carried out with the IGBP1-AS1 cDNA with point mutations of the miR-24-1 seed region binding site to form the pmirGLO-IGBP1-AS1-Mut reporter vector. Then the miR-24-1 and miR-NC were transfected with the vectors into the HEK-293FT cells by using Lipofectamine 3000 (Invitrogen).
The analyses on the websites UCSC (
http://genome.ucsc.edu/) and JASPAR (
http://jaspar.genereg.net/) indicated that the ZIC3 protein might bind to the DNA sites of IGBP1-AS1. The IGBP1-AS1 recombinant luciferase reporter vector with truncated or mutated binding sites was constructed and co-transfected into the HCC70 cells with the ZIC3 expression vector to verify the specific sites of ZIC3 protein binding to IGBP1-AS1 DNA. After 48 h of transfection, the luciferase reporter assay was then carried out with a luciferase assay kit (K801-200, Biovision, USA) following the manufacturer’s instruction. With renilla luciferase as an internal reference gene, the activation degree of the target reporter gene was compared based on the ratio of the relative luciferase unit (RLU) of firefly luciferase assay divided by the RLU of Renilla luciferase assay.
RNA immunoprecipitation (RIP)
The trial was carried out strictly based on the protocol of EZMagna RIP Kit (Millipore) provided by the manufacturer. In brief, after cell lysis with the complete RNA immunoprecipitation (RIP) lysis buffer, extract of the BC cells was incubated together with anti-argonaute 2 (AGO2) or control anti-IgG antibody conjugated magnetic beads at 4 °C for 6 h. The purified RNA was evaluated with the RT-qPCR after removing the proteins of the beads.
RNA pull-down assay
The 3′end biotinylated miR-24-1 (TGCCTACTGAGCTGATATCAGT) or miR-24-1-mut (TGCCTACTCAGCTGATATCAGT) (20 nmol/L) were transfected into the HCC70 cells. After 24 h of incubation with streptavidin-coated magnetic beads (Life Technologies), pull down assay was conducted in a biotin-coupled RNA complex. Finally, the IGBP1-AS1 abundance was calculated based on the RT-qPCR results.
Western blot assay
The RIPA lysis buffer with PMSF was applied for the extraction of the total protein in tissues and cells, which were incubated in an ice bath for 30 min. The lysate was centrifuged for 10 min under the condition of 4 °C and 8000g to collect the supernatant. The SDS-PAGE gel electrophoresis was performed with all the samples, and the separated protein was transferred to PVDF membrane. 5% skimmed milk was used for membrane blocking. After 1 h room-temperature blocking, the membrane was incubated together with primary sheep anti-ZIC3 (1:1000, ab215063, Abcam, UK) and rabbit anti-GAPDH (1:2500, ab9485, Abcam, UK) at 4 °C overnight, followed by 1-h incubation with HRP-labeled secondary antibody. Afterwards, the membrane was rinsed with TBST, and the proteins were visualized by using the ECL Fluorescence Detection Kit (Cat. No. BB-3501, UK) and photographed by Bio-Rad Image Analysis System (BIO-RAD, USA). The software of Quantity One v4.6.2 was adopted for quantification. The protein expression levels were presented as the relative gray values of the interested protein band and the GAPDH protein band. All the procedures were performed in triplicate and the final results were presented as mean values.
Chromatin immunoprecipitation (ChIP)
Formaldehyde was used for cell fixation for 10 min. The chromatin fragments were obtained by cell sonication, and the supernatant was collected after centrifugation. The negative control rabbit IgG (ab109489, 1:300, Abcam, Shanghai, China) and the target protein-specific antibody ZIC3 (sc-101201, 1:1000, Santa Cruz Biotechnology, Shanghai, China) were added to fully incubate supernatant at 4 °C overnight. The protein agarose/Sepharose were used for DNA–protein complex precipitation, which was centrifugated at 12,000g for 5 min, followed by discard of the supernatant. After washing off the non-specific complex, cross-linking was conducted at 65 °C overnight. The DNA fragment was extracted, purified and recovered by phenol/chloroform. The primer was designed to amplify which contains the site of ZIC3 binding to the IGBP1-AS1 DNA promoter (F: 5′-CTTCATGGTGCAGGGTGCTA-3′, R: 5′-TGCATGTGGTTGTGCTCAGA-3′). The amplified product was 775 bp long. A Distal primer (a primer that amplifies the sequence away from the IGBP1-AS1 DNA promoter region) was designed as a negative control for the site primer (F: 5′-AGCTCATTTCTCCCCTTGCC-3′, R: 5′-TCTCTACTCCCACCAGAGGC-3′) and the amplified product was 384 bp long. Using the recovered DNA fragment as an amplification template, site primers and Distal primers were added respectively to perform RT-qPCR to verify whether the site of IGBP1-AS1DNA was the binding site of transcription factor ZIC3.
Animal trials
The BALB/c-nude mice aged 4 to 5 weeks were obtained from Shanghai SLAC Laboratory Animal Co., Ltd. All the laboratory procedures related to animals were approved by the institutional animal care and use committees of both institutions. The concentration of the stably infected cells was adjusted to 5 × 106 cells/mL after being suspended in 50% Matrigel (BD Biosciences, Bedford, MA). 0.4 mL cell suspension (2 × 106 cells) was administrated to the nude mice by means of subcutaneous injection at the left axilla area. The tumor size was examined every 5 days, and volume of the tumor was calculated with the formula of length × width2 × 0.5. All nude mice were euthanized, and the tumors were weighed 30 days later.
The mice were submitted toa right lateral flank incision after anesthesia to construct the abdominal metastasis model. 100 μL Hank’s balanced salt solution containing HCC70-Luc-vector and HCC70-Luc cells (5 × 106, transfected with Lv-IGBP1-AS1, miR-24-1 mimic, Lv-IGBP1-AS1 + Si-ZIC3 or Lv-ZIC3 + miR-24-1 mimic) was administrated into the right abdominal cavity by injection. 4 weeks after the operation, the bioluminescence images were collected by the Interactive Video Information System (IVIS).
Statistical methods
All the numerical measurements were presented as mean ± SD. The student’s t test and one-way ANOVA test were applied for the evaluation of group differences according to the data nature. The overall survival time was defined as the period between the diagnosis and death of any cause, and the progression-free time duration started from the beginning of treatment and ended when disease progression occurred. Log-rank test and Cox’s regression analysis were applied for univariate and multivariate survival analyses. P < 0.05 was considered as the standard for statistical significance. All the analyses were conducted with SPSS 22.0 and the imagines were drawn with GraphPad Prism 7.0.
Discussion
Previously, studies have identified some BC-related lncRNAs. HOTAIR is a kind of lncRNA that is found in human fibroblasts and is transcribed in antisense, and its expression is abnormally elevated in breast cancer and its metastases. The expression of HOTAIR results in increased invasion of breast cancer cells, which is closely related to the progression of breast cancer [
16], thus leading to decreased survival and poor prognosis. HOTAIR is also a prognostic indicator for breast cancer. Gokmen-Polar et al. [
17] believed that its use for prognostication is only suitable for breast cancer patients with ER-negative disease and lymph node metastases. Therefore, the significance of HOTAIR as a prognostic indicator for breast cancer is relatively limited. Of course, there are different opinions. Milevskiy et al. [
18] found that HOTAIR was co-expressed with FOXA1 and FOXM1 in HER2 receptor-rich tumors when analyzing breast cancer-related gene expression data. Li et al. [
19] screened a group of lncRNA microarrays involving trastuzumab-resistant SKBR-3/Tr cells and found that the expression of GAS5 is decreased in breast cancer patients and SKBR-3/Tr cells after trastuzumab treatment, demonstrating that trastuzumab can reduce the expression of GAS5. It was found that lapatinib upregulated the expression of GAS5 by inhibiting the PI3K/Akt/mTOR signaling pathway. GAS5 acts as a competitive endogenous RNA (ceRNA) of miR-21, competitively binding to miR-21 to increase PTEN and promote proliferation and metastasis of trastuzumab-resistant breast cancer cells. This process improves the therapeutic resistance of breast cancer to trastuzumab. Finally, it has been shown that GAS5 can improve the therapeutic resistance of HER2-positive breast cancer to trastuzumab, suggesting that GAS5 can be used as a novel prognostic indicator and candidate drug target for HER2-positive breast cancer and can improve the effect of treatment on trastuzumab-resistant patients.
Our findings suggest that expression of the lncRNA IGBP1-AS1 was remarkably lower in BC tissues and cell lines than in adjacent normal tissues. The in vivo imaging system showed that BC cells overexpressing IGBP1-AS1 had less invasive ability in the peritoneal cavity of mice than the control cells, which was also confirmed in vitro. At the same time, a retrospective analysis was performed on tumor tissues from 94 patients with BC. Multivariate analysis showed that IGBP1-AS1 expression was an independent risk factor affecting both OS and PFS and was not affected by disease stage. The median OS and PFS in the IGBP1-AS1high expression group were remarkably higher than those in the IGBP1-AS1low expression group, indicating that IGBP1-AS1 has clinical value in the prognosis of BC. Loss-of-function assays were not performed in this study as the level of IGBP1-AS1 in BC were relatively low. Remarkable changes in phenotypes may not be observed when knock-down was conducted. The rescue experiments have proved that to some extent. Further mechanism research has identified that LncRNA IGBP1-AS1/miR-24-1/ZIC3 axis has significant impact on proliferation and invasion ability of BC cells in vitro and in vivo. miR-24-1 had never been reported in BC before. According to our results, it may act as an oncogene in BC, which was in accord with studies in colorectal cancer and ovarian cancer [
9,
10]. It was not the first time that miR-24-1 was involved in loop-feedback regulation [
20]. Some members of the ZIC family has been reported as tumor suppressor in gastric cancer including ZIC3 [
12]. Interestingly, we found that ZIC3 also binds to IGBP1-AS1 at the promoter region making the axis a ring loop which is rare. It is a favorable feedback regulation in BC which may present another new therapeutic strategy. Although this regulation might be weakened or compensated in the human body. There were also some limitations. Sample size for survival analysis needs to be enlarged greatly to make bigger clinical impact as well as performing a prospective study. The signaling pathways that were affected by the LncRNA IGBP1-AS1/miR-24-1/ZIC3 loop need to be further explored.
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