qRT-PCR analyzed by REST software and presented in Table 3. The results showed the information on fold change of the down-regulated lncRNAs in the blood samples of breast cancer patients comparing to the healthy women as (P < 0.05) MEG3 (0.216 ± 0.026), NBAT1 (0.233 ± 0.051), NKILA (0.453 ± 0.087), GAS5 (0.188 ± 0.051), Z38 (0.487 ± 0.113), AK058003 (0.455 ± 0.1), MVIH (0.502 ± 0.122), EPB41L4A-AS2 (0.256 ± 0.057), BC040587 (0.260 ± 0.038). On the other hand, the fold change of over-expressed lncRNAs (P < 0.05) are as follows: H19 (25.35 ± 3.152), SPRY4-IT1 (9.062 ± 1.076), CCAT1 (3.371 ± 0.512), UCA1 (2.817 ± 0.461), AC026904.1 (2.171 ± 0.359).
The expression level of twenty lncRNAs in blood samples of breast cancer patients showed in Fig. 1 and their expression compared with healthy normal women in Fig. 2.
Assessments of lncRNAs profiles association with clinicopathological features of breast cancer patients showed that the expression level of NKILA, NBAT1, and ZFAS1 lncRNAs were related to the tumor size. However, BC040587expression level was related to age, node metastasis, tumor size, and grade of breast cancer patients, and NBAT1 lncRNA expression was also correlated with the patients’ age (P < 0.05). Furthermore, there is a statistical association between SPRY4-IT1 (P = 0.03) and H19 (P = 0.04) expression levels and HER-2 in cancer patients’ blood samples. The correlation between other lncRNAs expression levels and clinicopathological features was not significant (P > 0.05).
The lncRNAs biomarker characterization could be beneficial for early detection and treatment in breast cancer.35 Increasing evidence represented the association of lncRNAs expressed in tumor tissues with cancer development or metastasis.7 At the same time, there are few reports of circulating lncRNAs in blood samples of cancer patients as shown in Table 4. We investigate and compare the expression levels of twenty 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.
Researchers revealed that the lncRNAs Z38 and MVIH expression levels increased in breast cancer tissues, and they showed a relation with stage and lymph node metastasis. 10, 13 However, the present study indicated that circulating Z38 (0.487 ± 0.113) and MVIH (0.502 ± 0.122) expression levels declined in breast cancer patients’ blood samples. The expression levels of Z38 and MVIH in patients’ blood and cancer tissue was different. The possible explanation for this phenomenon might be the different disease stages among various studies. Besides, the low circulating lncRNA levels compared to lncRNA levels in tissue specimens might be due to both the technical and biological determinants that impact circulating lncRNA levels. 50
According to our results, there was a down-regulation in MEG3 expression level (0.216 ± 0.026) in blood samples of breast cancer patients compared to healthy individuals. Similarly, Sun et al. (2016) reported the MEG3 down-regulation in cancer tissues compared with adjacent normal tissues.51 They also reported that MEG3 expression level was significantly associated with stage and lymph node metastasis. The circulating NBAT1 level (0.233 ± 0.051) in breast cancer patients was also significantly decreased, which is in agreement with the Hu et al. (2015) report.20 Although the NBAT1 expression pattern significantly correlated with tumor size and patient age (p < 0.05), other researchers represented its association with lymph node metastasis.20
The down-regulation of circulating EPB41L4A-AS2 (0.256 ± 0.057) proved to involve in tumorigenesis. Nevertheless, Xu et al. (2016) reported the relationship between expression levels and tumor grade, size, and disease stage.26 we did not observe any significant correlation between these characteristics and EPB41L4A-AS2 expression level. There was a report of lncRNA GAS5 down-regulation in breast cancer samples compared to adjacent healthy breast tissue. In the current study, we do not observe the association between GAS5 expression (0.188 ± 0.051) and lymph node metastasis, tumor size, and the histological grade was not significantly associated, which is in agreement with the study performed by Li et al. (2018).16
LncRNAs ZFAS1 and NKILA as tumor suppressors down- regulated in human breast tumors.33, 36 In the current study, low ZFAS1 (0.0497 ± 0.066) and NKILA (0.453 ± 0.087) expression levels were significantly correlated with tumor size (p < 0.05).
The expression level of tumor suppressor BC040587 (0.26 ± 0.038) gene in breast cancer patients’ blood samples was revealed. The same results from breast cancer tissue showed by Chi et al. (2014). 22 It can be used as a prognostic indicator to assess tumor progression due to strongly associated BC040587 expression with tumor size, grade, and node.
A dramatic rise in the circulating H19-LncRNA expression level is shown in breast cancer patients compared to healthy individuals (25.350 ± 3.152). Our findings approved the results of Jiao et al. (2018), who investigated the H19 expression levels in the plasma of breast cancer patients compared with healthy controls. 38 Besides, Dugimont and Adriaenssens (2005)46 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. 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.
Several studies demonstrated that SPRY4-IT1 promotes cell growth, invasion and inhibits apoptosis in several types of cancer, including breast cancer. 13 This statement confirms by our finding that the expression levels of SPRY4-IT1 were significantly overexpressed in blood samples of breast cancer patients (9.062 ± 1.07) in comparison to healthy individuals. In contrast, Jiao et al. (2018) showed down-regulation of lncRNA SPRY4-IT1 in breast cancer patients’ plasma compared to healthy female controls. They used different primer pairs for SPRY4-IT1 amplification, and there is no information about patients’ pathological traits. The results of Shi et al. (2015) investigated the expression levels of lncRNA SPRY4-IT1 in breast cancer tissues compared to adjacent normal tissues.13 According to Xiang et al. (2019), there is no significant relation between SPRY4-IT1 expression in BC tissues and molecular subtypes (ER/PR/HER2) of breast cancer47, found a significant relation between SPRY4-IT1 and HER-2.
In the same way, high expression of lncRNA CCAT1 (3.371 ± 0.512) was not correlated with other clinicopathological factors of breast cancer patients such as differentiation grade, age, tumor size, stage, and lymph node metastases (P > 0.05). Zhang et al. (2015) presented the same results and suggested that CCAT1 might function as an oncogenic lncRNA and serve as a potential prognostic marker in breast cancer. 10
The UCA1 gene encodes for a lncRNA highly expressed in various carcinomas, including bladder, colorectal, and breast cancer, suggesting that UCA1 might serve as a potential biomarker for diagnostic purposes in the future. 43 Meanwhile, in the present study, UCA1 was highly expressed in the blood samples of breast cancer patients (2.817 ± 0.461) and was not associated with other clinicopathological factors of breast cancer patients.
Similarly, Li et al. (2018) reported that AC026904.1 was highly expressed in metastatic breast cancer and closely associated with poor prognosis.48 In our results, the up-regulation with a high expression level (2.171 ± 0.359) was also shown.
According to the Heatmap (Fig. 2), the expression level of ITGB2-AS1 upregulated; however, a significant up-regulation was not shown in the REST results. ITGB2-AS1 could promote the invasion of breast cancer cells, and because the majority of our patients are in the initiation stages of cancer development, significant up-regulation could be suppressed.3