The study cohort consisted of 60 women diagnosed with primary breast cancer who underwent surgery at Dunedin hospital, New Zealand. Clinicopathological data for the cohort is provided in Table 1. Blocks of formalin fixed paraffin embedded material (FFPE) from breast tumor and separate blocks of normal tissue adjacent to tumor tissue were available. Ethical approval was obtained from the Lower South Regional Ethics Committee, Ministry of Health, New Zealand (Ethics Committee Approval LRS/10/09/035) and all participating women gave written and informed consent for inclusion in the study. Standard procedures including culturally appropriate tissue handling and disposal protocols were followed. In addition to the breast cohort, commercially available RNA (Takara Bio, Shiga, Japan) supplied on dry ice and stored at −80°C before use from a variety of tumors and normal tissues were also quantified for ANRIL expression. Breast cancers were separated into molecular subtypes (Luminal A, Luminal B, Her 2 positive and triple negative breast cancer) using immunohistochemical surrogates for molecular classifications (23). The luminal A group were classified as ER and PR (≥20%) positive, Her 2 negative and low for Ki67 (<14%); Luminal B were classified as ER and PR (≥20%) positive and high for Ki67 (≥14%), or ER positive with low PR positive cells (<20%); Her 2 positive were all Her 2 positive tumors; and the triple negative breast cancer were classified as all tumors that were negative for ER, PR, and Her 2.

Sections (10 sections at 10 μm) from FFPE breast tumor and normal tissue adjacent to the tumor tissue blocks were subjected to RNA extraction using the RecoverAll™ Total Nucleic Acid Isolation Kit (ThermoFisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions. Eluted RNA was subject to further purification using the RNA Clean-Up and Concentration Kit™ (Norgen Biotek, Thorold, ON, Canada) and incubated at 70°C for 20 min immediately prior to cDNA synthesis (24). High Capacity RNA-to-cDNA Kit (ThermoFisher Scientific, Waltham, MA, USA) was used to convert total RNA to cDNA according to the manufacturer's protocol.

Quantification of ANRIL (assay ID Hs01390879_m1) was performed using the Applied Biosystems TaqMan Non-Coding RNA Assay (ThermoFisher Scientific, Waltham, MA, USA) with hypoxanthine phosphoribosyltransferase (HPRT) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) assays (IDs Hs99999909_m1 and Hs03929097_g, respectively) included as reference genes. Gene expression was quantified in triplicate from single cDNA preparations using the LightCycler 480 (LC480) qPCR machine (Roche, Basel, Switzerland). Relative gene expression was calculated using double delta Ct (ΔΔCT) method and qbase⁺ software (25).

RNAscope 2.5 HD Assay-BROWN manual kit™ (Advanced Cell Diagnostics, Newark, CA, USA) was used to measure ANRIL (Hs-CDKN2B-AS1), IL-6 (Hs-IL6), CCL2 (Hs-CCL2), and POSTN (Hs-POSTN) on FFPE tissue sections. Positive (Ubiquitin C, UBC) and negative control (the bacterial gene DapB) probes were performed on serial tissue sections. Tissue blocks were sectioned at 4 μm thickness and mounted onto Leica BOND™ Plus Slides (Leica Biosystems, Wetzlar, Germany). The assays were performed using the manufacturer's protocol. Stained slides were scanned into the Aperio Scanscope CS digital pathology system slide scanner under 400x magnification (Leica Biosystems, Wetzlar, Germany). The number of dots per cell and the number of positive cells were calculated to provide a score to evaluate the amount of staining present. Expression of ANRIL was scored 0–3 as outlined below. A score of 0 (no staining or <1 dot in a total of ten cells), 1 (1–3 dots per cell with <20% of cells per high powered field (hpf, at x400 magnification) positive), 2 (4–9 dots per cell in at least 20% of cells positive or >20% of cells per hpf with at least 1 dot per cell), 3 (>10 dots per cell and at least 20% of cells positive per hpf or >40% of cells with at least 1 dot per cell). For the correlation between ANRIL and POSTN the average amount of dots per 200 malignant cells were calculated from the average of three fields with 200 cells per field counted. Small clusters of staining were considered to contain ten dots and large clusters 20 dots.

RNAscope was also performed on MCF7 cells treated with siRNA toward ANRIL or a control siRNA. Cells from 6 well-plates were combined and made into a cell clot using HistoGel^TM specimen processing gel (Thermo Fisher Scientific, Massachusetts, USA). Cell clots were fixed in 10% neutral buffered formaldehyde overnight, and processed into paraffin wax. Cell clot sections (5 μm) were subjected to RNAscope for ANRIL, POSTN, FBXO18, TERC, PPIB, UBC, and DapB according to the manufacturer's instructions for cells. Cells were imaged using a (Zeiss LSM510; Carl Zeiss, Thornwood, NY, USA), and images were analyzed by Zeiss LSM Image Examiner software version 3.2.0.115 (Carl Zeiss), or imaged using the Aperio Scanscope CS digital pathology system.

MCF7 and MDA-MB-231 cell lines (American Type Culture Collection, ATCC) were cultured in Dulbecco's Modified Eagle Medium (DMEM) from Life Technologies (Gibco BRL, Grand Island, NY, USA) and supplemented with 10% fetal bovine serum (FBS) in a 37°C humidified incubator at 5% CO₂.

Cells were transfected with Lincode Human CDKN2B-AS1 (R-188105-00-0005) siRNA; Dharmacon, Chicago, IL, USA) using Lipofectamine 2000 (Invitrogen, Burlington, ON, Canada). ON-Target plus Non-targeting Pool (D-001810-10-05) was used as a negative control as suggested by the manufacturer. siRNAs were transfected at a final concentration of 5 or 10 nM using Lipofectamine RNAiMax (Invitrogen). The siRNA and RNAiMax were combined in additional culture media and the resulting lipoplexes were added to the wells with a total of 1 × 10⁴ cells/2 mL for overnight transfection. After 24 h the cultured media was replaced with fresh media and finally harvested at 48 h in total. Each experiment was performed at least in triplicate. ANRIL knockdown was verified by real-time RT-PCR using GAPDH and HPRT as reference genes and tested for ANRIL and POSTN expression as detailed elsewhere.

Cells were seeded at a concentration of 1 × 10⁵ cells/mL into a 96-well plate and assayed using the Vybrant MTT Cell Proliferation Assay kit (ThermoFisher Scientific, Waltham, MA, USA) following the manufacturer's protocol for adherent cells.

Student's t test (two tailed) was used to compare two groups, one- way ANOVA for multiple comparisons was used to compare more than two groups, and Pairwise Spearman correlation was performed between ANRIL and POSTN expression using GraphPad Prism7 software (California, USA). A P value < 0.05 was considered statistically significant.

To determine where ANRIL was located in breast tumors we first determined the expression of ANRIL in 60 breast tumors, and other tissues from a commercially available source using RT-qPCR. Expression of ANRIL was found in all tissues in varying amounts being lowest in lung cancer, kidney, lymph node, and smooth muscle and highest in spleen, thymus, pituitary gland, stomach cancer and leukemia (Figures 1A,B). The presence of ANRIL mRNA in a wide variety of tissues including hemopoietic tissues suggested ANRIL could be expressed in both tumor cells and the tumor microenvironment. In breast tumors ANRIL was increased in RNA extracted from tumor tissue compared to the patient-matched normal tissue adjacent to the tumor ANRIL (Figure 1C, P = 0.0037). An analysis of ANRIL based on breast tumor subtypes compared to the normal associated tissues showed ANRIL was increased in Luminal A (Figure 1D, P = 0.0017) and Luminal B (Figure 1D, P = 0.0008) tumors compared to the normal associated tissue. Although the highest average expression of ANRIL was found in triple negative tumors, there was no significant difference in ANRIL when compared to the normal associated tissue. A comparison of ANRIL amongst tumor tissues showed that ANRIL was increased in triple negative tumors compared to Luminal A (P = 0.011) and B (P = 0.027) with no significance found compared to Her 2 positive tumors (P = 0.35).

To determine the cell type responsible for ANRIL expression in breast tumors, 25 cases with sufficient tissue available and those with ANRIL expressed >2.5-fold compared to the mean expression of the house keeping genes were selected and subjected to analyses using RNAscope. Cases included 5 Luminal A, 8 Luminal B, 4 triple negative, and 8 Her 2 positive tumors. Tissue sections were first assessed using a positive control probe for UBC to check for sufficient RNA quality and sequential sections stained for ANRIL and a negative control probe. All cases showed strong staining for UBC and variable amounts of ANRIL staining that was absent in the negative control. All cases showed the majority of ANRIL expression in malignant epithelial cells with no expression identified in normal mammary epithelium in the same section. In fifteen (60%) cases, scattered non-malignant cells were also positive for ANRIL and included endothelial cells (1–9 dots in positive cells and up to three cells positive focally) and other stromal cells (3–9 dots positive in individual cells). The RNAscope assay revealed different cellular localization for ANRIL (Figure 2) in malignant cells with staining in the nucleus (Figure 2A) and cytoplasm (Figure 2B). The majority of cases had ANRIL only in the nucleus (n = 14, 56% and Figure 2A top panel), 28% of cases had staining in the nucleus and outside of the nucleus (n = 7), and 16% of cases had ANRIL staining outside of the nucleus (n = 4, and Figure 2C). Although the number of tumors in individual tumor subtypes were limited. The majority of Luminal A and Her 2 and all triple negative breast tumors had ANRIL only in the nucleus. Luminal B tumors showed more of a mixed location for ANRIL with one third of tumors with ANRIL in the nucleus, one third with ANRIL outside the nucleus and one third with ANRIL in both the nucleus and outside the nucleus.

Overall these findings suggest ANRIL was largely present in malignant cells and its presence inside and outside of the nucleus suggests ANRIL is heterogeneously located amongst breast tumors.

To determine if malignant cells that expressed ANRIL also expressed CCL2, IL6 or POSTN, the RNAscope analyses were extended to include each cytokine and the areas with ANRIL staining were correlated with the other markers. Only one tumor had IL6 staining in malignant cells with ANRIL staining and no cases showed CCL2 staining in cells with ANRIL. Areas with ANRIL positivity in malignant cells also showed POSTN staining (Figure 3A). An analysis including all ANRIL positive tumors found no correlation between ANRIL and POSTN (Figure 3B, left panel); however, a positive correlation was found when only tumors with nuclear staining were included (Figure 3B, right panel). These results suggest in breast tumors nuclear ANRIL is correlated with POSTN expression.

To test the correlation between ANRIL and POSTN found in breast tumors siRNA was used to knockdown ANRIL levels in two breast cancer cell lines and the effect on POSTN expression measured using RT-qPCR. Two cell lines were selected MDA-MB-231 and MCF7 cells that expressed ANRIL (26) In both cell lines ANRIL was knocked down relative to cells treated with the negative control siRNA (Figure 4A). Cells with reduced ANRIL also had reduced POSTN expression (Figure 4B). The MTT assay showed no significance difference between MCF7 cells treated with siANRIL and those treated with the control siRNA (Figure 4C). MCF7 cells treated with siRNA for ANRIL and the negative control siRNA were also investigated using RNAscope. There was no difference in expression of genes not expected to be affected by ANRIL (PPIB, FBXO18 and TERC) in MCF7 cells treated with the siANRIL compared to those treated with the control siRNA (Figure 4D). However, RNAscope for ANRIL and POSTN showed that although ANRIL and POSTN expression were low in cells treated with the negative control siRNA it was further reduced in cells treated with siRNA for ANRIL (Figure 5), consistent with the findings using RT-qPCR.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.