Circulating LncRNAs landscape as potential biomarkers in breast cancer

Abstract Background In Iran, the delay in diagnosis and treatment of breast cancer results in low survival rates. Aim It is essential to characterize new therapeutic targets and prognostic breast cancer biomarkers. The rising evidence suggested that long non‐coding RNAs (lncRNAs) expression levels are deregulated in human cancers and can use as biomarkers for the rapid diagnosis of breast cancer. Methods In the present study, a quantitative real‐time polymerase chain reaction (qRT‐PCR) technique was used to measure 20 oncogenic and tumor suppressor lncRNAs expression levels in whole blood samples of female breast cancer patients and healthy women. Receiver operating characteristic curve (ROC) was used to assess the diagnostic value of each selected lncRNA as a biomarker. Results The results revealed that some circulating lncRNAs (MEG3, NBAT1, NKILA, GAS5, EPB41L4A‐AS2, Z38, and BC040587) were significantly down‐regulated in breast cancer patients compared to healthy women. In contrast, other circulating lncRNAs (H19, SPRY4‐IT1, XIST, UCA1, AC026904.1, CCAT1, CCAT2, ITGB2‐AS, and AK058003) were significantly up‐regulated in breast cancer patients compared to controls. It was shown that the expression levels of NKILA, and NBAT1 lncRNAs were related to tumor size, and BC040587 expression level related to age, node metastasis, tumor size, and grade (p < .05). The association between H19 and SPRY4‐IT1 lncRNAs with HER‐2 was confirmed statistically (p < .05). ROC curves illustrated that the blood levels of SPRY4‐IT1, XIST, and H19 lncRNAs have excellent potential in discriminating breast cancer from the healthy controls, showing an AUC of 1.0 (95% CI 1.0–1.0, p = .00), 0.898 (95% CI 0.815–0.981, p = .00), and 0.848 (95% CI 0.701–0.995, p = .01), respectively. Conclusion In conclusion, the expression levels of circulating H19 and SPRY4‐IT1 lncRNAs in breast cancer patients could consider as the prognostic biomarkers and therapeutic targets in breast cancer, because of their excellent power in discriminating breast cancer from healthy individuals and the significant correlation of H19, and SPRY4‐IT1 lncRNAs with clinicopathological traits. We also suggest the possible application of BC040587 lncRNA as a diagnostic and prognostic indicator to assess tumor progression in case of verification in larger patients' cohorts.

application of BC040587 lncRNA as a diagnostic and prognostic indicator to assess tumor progression in case of verification in larger patients' cohorts.

K E Y W O R D S
breast cancer detection, long non-coding RNAs, real-time PCR, whole blood samples

| INTRODUCTION
Breast cancer is the most common malignant disease, affecting about 2 million women worldwide in 2018. 1 In Iran, breast cancer is the first leading cause of cancer death in females, including 27% of all cancers with an age-standardized rate (ASR) 31 per 100 000. According to the latest statistics in Iran, 13 776 new breast malignancies are identified in 2018. 2 The high level of breast cancer mortality is due to a lack of diagnostic markers for early detection, mammography screening programs, and suitable molecular markers for targeted and effective treatment opportunities. Late diagnosis may lead to cancer metastasis with less than 25% in 5-year survival. 3 Breast cancer lacks biomarkers with high specificity and sensitivity for general screening. Therefore, it is essential to search for novel biomarkers. Recently, the circulating lncRNA levels in cancer patients were nominated as a potential biomarker. 4 Growing evidence has shown that lncRNA expression levels are deregulated in human cancers. Therefore, there is a possibility of using lncRNAs as therapeutic targets or potential biomarkers for the rapid diagnosis of breast cancer. [5][6][7] Besides, evaluation of circulating lncRNA levels in body fluids can be considered as a noninvasive diagnostic biomarker for some cancers. 8 Previous studies have reported that some oncogenic lncRNAs are overexpressed in various types of cancer and can serve as a prognostic marker. Overexpression of colon cancer-associated transcript-1 (CCAT1) indicated its role in malignancies' pathogenesis. [9][10][11] Similarly, the oncogenic role of some lncRNAs confirmed by illustrating their up-regulation in breast cancer tissues compared to adjacent normal tissues. [12][13][14][15][16][17] AC026904.1, Urothelial Carcinoma-associated 1 (UCA1), SPRY4 intronic transcript 1 (SPRY4-IT1), microvascular invasion in hepatocellular carcinoma (MVIH), Colon Cancer Associated Transcript 2 (CCAT2), promoter of CDKN1A antisense DNA damage activated RNA (PANDAR) and zinc finger antisense 1 (ZFAS1) and H19 lncRNA, is overexpressed in 73% of breast cancer tissues compared to healthy tissues. 18 Besides, a series of experiments showed that knockdown of Z38 significantly inhibited tumor growth in breast cancer. 19 We hypothesized that the mentioned lncRNAs might be up-regulated in breast cancer and act as an oncogene.
According to the previous studies, some lncRNAs function as tumor suppressor genes, and their down-regulation may proceed to invasion and metastasis and lessen the effectiveness of chemotherapeutic treatment. Neuroblastoma associated transcript-1 (NBAT1) behaved as a tumor-suppressor and down regulated in invasive breast cancer. 20 Likewise, decreased expression levels of FGF14 antisense RNA 2 (FGF14-AS2), X inactive specific transcript (XIST), BC040587, and MEG3 in breast cancer tissue and cell lines compared with corresponding normal control were associated with unfavorable survival in breast cancer. [21][22][23] Meanwhile, down-regulation of lncRNAs such as NF-κB interacting lncRNA (NKILA), EPB41L4A antisense RNA 2 (EPB41L4A-AS2), and Growth arrest-specific transcript 5 (GAS5) inhibit breast cancer progression and may advance invasion and metastasis of breast cancer. 16,24,25 There is controversy regarding down-regulation or upregulation of lncRNA-AK058003 in literatures. 26,27 There is a crucial necessity to develop an early detection platform in order to increase the breast cancer survivals. In addition, advances in breast cancer research will result in the novel diagnosis and treatment of breast cancer. There are few published reports of lncRNAs expression levels in breast cancer patients' blood. Therefore, in the current study, the expression profiles of 20 lncRNAs (H19, CCAT1,   CCAT2, UCA1, SPRY4-IT1, AK058003, Z38, MVIH, XIST, PANDAR,   GAS5, ITGB2-AS1, MEG3, AC026904  including radiotherapy or chemotherapy. Thirty blood samples of healthy women were collected with an average age of 40 years. We use "healthy normal" as the absence of any apparent disease as defined by Aagaard et al. 27 We first screened volunteer blood donors using criteria based on health history, including the absence of systemic diseases such as cancer, hypertension, diabetes, and autoimmune disorders or immunodeficiency. Exclusion criteria for both groups included a body mass index (BMI) outside the range of 18.5-24.9 kg/m 2 , being pregnant, consuming alcohol, suffering from infectious diseases, and specified chronic diseases. Furthermore, we excluded individuals under certain medical treatments such as corticosteroids, immunosuppressive agents, antibiotics, or probiotics within the last 6 months.

| Statistical analysis
The statistical program for SPSS 18.0 (SPSS, Chicago, IL, USA) was employed to analyze all the data. Data are expressed as the mean ± standard deviation. For comparisons between two groups, the Student's t-test was used while one-way analysis (ANOVA) and Bonferroni post hoc test were used to compare multiple groups. The χ2 test was applied to analyze the association between lncRNAs expression and clinicopathological status. We categorize the lncRNA expression into high/low groups and clinopathological traits to different groups (according to

| Expression levels of circulating lncRNAs
The expression level of 20 lncRNAs in blood samples of breast cancer patients showed in Figure 1 and their expression compared with healthy normal women in Figure 2.  Table 3).
Expression levels of circulating lncRNAs during different stages of breast cancer were shown in Figure 3, and the lncRNA expression of each stage was compared to healthy individuals using the ANOVA followed by Bonferroni post hoc test.
Assessments of lncRNAs profiles association with clinicopath-

| Diagnostic accuracy of circulating lncRNAs
The results of ROC curve analysis illustrated that the blood levels of

| Correlation analysis between the levels of circulating lncRNAs in the breast cancer patients
According to the Pearson correlation analysis ( Figure 5), all the circulating levels of lncRNAs in red color were discovered to be positively related to each other in breast cancer patients, except from CCAT1 and CCAT2 that has a negative relation with Z38.

| DISCUSSION
Identifying highly sensitive and specific lncRNAs for the early diagnosis and prognosis of breast cancer invasion and metastasis remain a hard task. Many studies have explored biomarkers in tumor biopsies,  Table 3. We compare the expression levels of 20 lncRNAs in blood samples of breast cancer patients and healthy individuals, and then the correlation between lncRNAs deregulation and clinical characteristics was analyzed in this study. Next, we investigated the sensitivity, specificity, and the potential of circulating lncRNAs in discriminating breast cancer from the healthy controls. Finally, we study the correlation between the levels of different lncRNAs in breast cancer patients.
Considering the results of current study (Table 3) 39,47 According to the results, the blood levels of SPRY4-IT1, XIST, and H19 lncRNAs have excellent potential in discriminating breast cancer from the healthy controls ( Figure 4). There is an association between SPRY4-IT1, and H19 deregulation and clinical characteristics (Table 4).
On the other hand, the dramatic rise in the circulating SPRY4-IT1, and H19 LncRNAs expression levels are shown in breast cancer patients compared to healthy individuals. Therefore, we suggest SPRY4-IT1, and H19 lncRNAs can be used as screening biomarkers for early detection of breast cancer.
Our findings approved the results of Jiao et al, who investigated the H19 expression levels in the plasma of breast cancer patients compared with healthy controls. 39 Dugimont and Adriaenssens illustrated a correlation between H19 expression levels and pathological features such as lymph node metastasis, tumor grades, and the presence of estrogen and progesterone receptors that did not validate in the current research. [48][49][50] On the other hand, we have found a positive correlation between H19 LncRNA expression level and HER-2 that indicated H19 as a potential regulator of proliferation in the HER2 enriched subtype (Table 4). In addition, there is a positive correlation between the levels of H19 and NBAT1 lncRNAs in breast cancer blood samples ( Because of strongly associated BC040587 expression with tumor size, grade, and node (Table 4), we suggest the application of BC040587 lncRNA as a diagnostic and prognostic indicator to assess tumor progression that could be prove in larger patients' cohorts.
In conclusion, there is an essential need to search for novel prognostic biomarkers with high specificity and sensitivity for breast cancer screening. In recent years, lncRNAs have been implicated as having oncogenic and tumor suppressor roles and can be used to develop as biomarkers and prognosis factors. In the present study, the dramatic rise in the circulating SPRY4-IT1, and H19 LncRNAs expression levels are shown in breast cancer patients compared to healthy individuals. Furthermore, because of the significant correlation of H19, and SPRY4-IT1 lncRNAs with F I G U R E 5 Correlation between the circulating levels of 20 lncRNAs were analyzed by Pearson correlation in the breast cancer patients clinicopathological traits and their excellent power in discriminating breast cancer from healthy individuals, they could be considered as the prognostic biomarkers and novel therapeutic targets in breast cancer. We also suggest the possible use of BC040587 lncRNA as a prognostic indicator of breast cancer in case of verification in larger patients' cohorts.