High serum exosomal long non‐coding RNA DANCR expression confers poor prognosis in patients with breast cancer

Abstract Background Exosomal long non‐coding RNAs (lncRNAs) serve as excellent candidate biomarkers for clinical applications. The expression of differentiation antagonizing non‐protein coding RNA (DANCR) has been shown to be decreased in breast cancer (BC) tissues and cell lines. However, the clinical value of circulating exosomal DANCR in BC has not been explored. Methods A total of 120 BC patients, 70 benign breast disease (BBD) patients, and 105 healthy women were recruited in this study. Total RNA was extracted from serum samples, and the level of serum exosomal lncRNA DANCR was evaluated by quantitative real‐time reverse transcription‐polymerase chain reaction (qRT‐PCR). Results Serum exosomal lncRNA DANCR levels were significantly higher in BC patients than in BBD patients and normal controls. The diagnostic performance of serum exosomal lncRNA DANCR was good, and the combination of serum exosomal lncRNA DANCR, CA153, and CEA greatly improved the diagnostic accuracy for BC. High serum exosomal lncRNA DANCR level was associated with various clinicopathological variables including lymph node metastasis, ER status, HER2 status, and TNM stage. In addition, the BC patients in the high serum exosomal lncRNA DANCR expression group had significantly shorter 5‐year overall survival time. Multivariate analysis demonstrated that serum exosomal lncRNA DANCR was an independent risk factor for BC. Conclusion Serum exosomal lncRNA DANCR may be a useful non‐invasive biomarker for the clinical diagnosis and prognosis of BC.

accuracy of CA153 and CEA is poor. Therefore, it is urgent to identify novel, reliable biomarkers for GC diagnosis and prognosis evaluation.
Long non-coding RNAs (lncRNAs) are a class of non-coding RNAs with a length of longer than 200 nucleotides. 6,7 Dysregulation of ln-cRNAs has been shown to play a critical role in regulating the initiation and progression of BC. 8,9 For instance, OLBC15 was highly expressed in triple-negative BC, and OLBC15 promoted the BC tumorigenesis through destabilizing ZNF326. 10 Exosomes are 30-100 nm nanovesicles containing many molecules, such as lncRNAs, proteins, DNA, RNA, and miRNAs. [11][12][13] Exosomal lncRNAs are correlated with cancer tumorigenesis and progression and can be used as effective biomarkers in various types of cancer including BC. For example, compared to benign breast disease (BBD) and healthy controls, serum exosomal H19 was significantly increased in BC patients, and high serum exosomal H19 expression was strongly associated with worse clinical variables. 14 Similarly, patients with BC exhibited higher serum exosomal HOTAIR levels, and overexpression of serum exosomal HOTAIR was correlated with poorer prognosis. 15 LncRNA differentiation antagonizing non-protein coding RNA (DANCR or ANCR), located on human chromosome 4, has been previously found to be involved in the initiation and the progression of BC. 16,17 However, the diagnostic and prognostic value of circulating exosomal DANCR in BC has not yet been explored. Thus, this study aimed to detect the serum exosomal lncRNA DANCR levels in BC patients, BBD patients, and normal controls and analyzed its role as a potential novel biomarker for BC diagnosis and prognosis.

| Patients and samples
The current study was approved by the Ethics Committee of Taizhou Municipal Hospital. Written informed consent was obtained from all patients and healthy subjects. In this study, blood samples were obtained from 120 BC patients, 70 BBD patients, and 105 healthy volunteers. All recruited BC patients had not received any chemotherapy or radiotherapy prior to the sampling. We also collected the blood samples from all BC cases one month after their surgery. The blood samples were collected in anti-coagulative tubes and centrifuged at 1600 g for 10 min, followed by centrifugation at 16,000 g for 10 min at 4°C.
The supernatant was stored at −80°C until further use. The clinical characteristics of all 120 patients with BC are summarized in Table 1.

| Exosome isolation
Total exosomes were isolated from serum with the total exosome isolation reagent (Invitrogen) according to the manufacturer's protocol. The serum was thawed on ice at 25°C. Then, possible residual cell debris were removed by a centrifugation step at 2000 g for 30 min.
Then, serum supernatant was mixed with the exosome isolation reagent, followed by incubation for at 4°C 30 min and centrifugation at 10,000 g for 10 min. The supernatant was discarded, and the exosomal pellet was resuspended in PBS for RNA extraction.

| RNA extraction and quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR)
Total exosomal RNA was extracted from serum samples with an miRNeasy Serum/Plasma Kit (QIAGEN). The RNA was quantified on a NanoDrop ND-1000 Spectrophotometer (Thermo Scientific) and immediately reverse transcribed into cDNA. Before RNA extraction, 25 fmol/ml of synthesized cel-miR-39 (Applied Biosystems) was added as an exogenous spike in control. Then, qRT-PCR was performed with SYBR PrimeScript miRNA RT-PCR kit (Takara Biotechnology Co. Ltd) on the 7500 Real-Time PCR systems (Applied Biosystems). The relative levels of serum exosomal lncRNA DANCR were expressed using the 2 -ΔΔCt method.

| Measurement of CA153 and CEA in serum
The levels of CA153 and CEA were measured using electrochemiluminescence assays with available commercial kits.

| Serum exosomal lncRNA DANCR was dramatically increased in BC patients
qRT-PCR was used to detect the serum exosomal lncRNA DANCR levels in all participants. Serum exosomal lncRNA DANCR levels were significantly higher in BC patients than in BBD subjects and controls (p < 0.001, Figure 1A). In addition, BC patients with positive lymph node metastasis (p = 0.005, Figure 1B), negative ER status (p = 0.012, Figure 1C), negative HER2 status (p < 0.001, Figure 1D), and advanced TNM stage (p < 0.001, Figure 1E) exhibited higher serum exosomal lncRNA DANCR levels compared with their respective controls.

| Increased serum exosomal lncRNA DANCR expression was correlated with clinical variables in BC patients
The median serum exosomal lncRNA DANCR expression was used as a cut-off value, and all 120 BC patients were classified into two groups: high serum exosomal DANCR expression group (n = 60) and low serum exosomal DANCR expression group (n = 60). As

| Alteration of serum exosomal lncRNA DANCR levels following treatments
One month after surgical resection, paired blood samples were collected from all BC subjects. qRT-PCR was used to measure the serum exosomal lncRNA DANCR expression level, and we found the serum exosomal lncRNA DANCR levels were markedly reduced following surgery (p < 0.001, Figure 3).

| Serum exosomal lncRNA DANCR expression was a prognostic biomarker for BC
The Kaplan-Meier curve for OS regarding serum exosomal lncRNA DANCR expression is presented in Figure 4.

| DISCUSS ION
Dysregulation of lncRNAs has been demonstrated to play major roles in breast cancer progression. 18 This study enrolled a total of 120  16 Similarly, DANCR was significantly higher in BC tissues than in paired normal tissues. DANCR upregulation not only promoted tumorigenicity in xenograft animal but also greatly enhanced cell proliferation, invasion, and migration in vitro through regulating miR-216a-5p. 17 Besides BC, the role of lncRNA DANCR was also reported in different types of cancer. LncRNA DANCR was increased both in tumor tissues and serum samples of gastric cancer (GC) patients, and its upregulation was closely associated with malignant phenotypes.
LncRNA DANCR overexpression significantly promoted the carcinogenesis in vitro and in vivo, while inhibition of DANCR showed the opposite effect. 19 In bladder cancer (BC), lncRNA DANCR expression was dramatically higher in BC tissues than in normal con-

CO N FLI C T O F I NTE R E S T S
None.

DATA AVA I L A B I L I T Y S TAT E M E N T
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.