Introduction
Breast cancer (BC) is the most common cancer among women worldwide, accounting for 30% of all female cancers alone [
1,
2]. It is also the second leading cause of cancer death with 11.6% of the total cancer deaths [
3]. The luminal subtype of BC, expressing both estrogen and progesterone receptors (ER
+/PR
+), accounts for a large percentage of this cancer (more than 70%) [
4,
5]. Luminal type of BC is classified into two groups, luminal A and luminal B, according to human epidermal growth factor receptor 2 (HER2) status and levels of ki-67 [
5]. The leading choice for luminal BC treatment is endocrine therapy. However, resistance to endocrine therapy is a major challenge for clinicians [
6]. Also, similar treatment strategies can provide a variety of responses in luminal subtype patients [
7]. In general, despite the better prognosis, due to the heterogeneity and resistance to hormone therapy of luminal subtype, the treatment effectiveness is still limited [
7]. Therefore, identifying novel biomarkers and molecular mechanisms of BC is required to perform early diagnosis and personalized therapeutic strategies. In addition, the limited number of studies on the dysregulation of long non-coding RNAs (lncRNAs) in luminal BC, as well as the high frequency of this subtype, increase the importance of further research on this subgroup.
The GENCODE project results have indicated that only about 2% of the human genome encodes proteins, whereas the vast majority of it is transcribed into non-coding RNAs [
8]. LncRNAs, a class of non-coding RNAs with more than 200 nucleotides, have been shown to play essential roles in many different cellular processes, such as genetic imprinting, transcriptional responses, development, etc. [
9,
10]. Therefore, the aberrant expression of lncRNAs can lead to the pathogenesis of various diseases including cancers [
11]. They have been identified as crucial molecules involved in cancer proliferation, invasion, and resistance to therapy [
12,
13]. Some lncRNAs are expressed highly specific in different cancer types, and some of them are associated with the survival of patients. These two features make them ideal prognostic and diagnostic tools [
14]. Due to the development of lncRNA-based therapeutic approaches, determining the role of different lncRNAs in the tumorigenic process is of great importance. Hence, in the present study, we aimed to figure out the expression level and the underlying molecular mechanisms of LINC01133, ZEB1-AS1, and ABHD11-AS1 in luminal BC.
LINC01133, a novel lncRNA, is located on chromosome 1q23.2. It was reported that LINC01133 exerted a tumor suppressive role in gastric cancer [
15], ovarian cancer [
16], and esophageal squamous cell carcinoma [
17] progression. It was also found that LINC01133 played oncogenic role in the development of some other cancers [
18‐
20]. These dual roles of LINC01133 could attribute to the tissue-specific expression of this lncRNA. ZEB1-AS1 is associated with the progression and development of several cancers. Its aberrant expression was detected in many tumors [
21], such as gastric cancer [
22], hepatocellular carcinoma [
23], and glioma [
24]. ABHD11-AS1, which is located at 7q11.23, is a newly discovered lncRNA. Previous studies have reported the upregulation of this lncRNA in different tumors, including papillary thyroid carcinoma [
25] and pancreatic cancer [
26]. To the best of our knowledge, the present research is the first study that determines the dysregulation of ABHD11-AS1 in BC so far. Despite all the studies that have been done on these lncRNAs, their biological processes and potential mechanisms, specifically in luminal subtype of BC, are still unclear.
In the present study, we examined the expression level of three lncRNAs in luminal BC by qRT-PCR assay. Furthermore, we performed a correlation analysis between the expression of these lncRNAs and clinicopathological parameters of patients. Finally, to investigate the potential role and mechanism of the lncRNAs in luminal BC, different bioinformatics and systems biology analyses were accomplished using various databases.
Discussion
The luminal subtype of BC, which has shown better prognosis among the other subtypes, accounts for approximately two-thirds of this cancer [
43,
44]. However, it is reported that the luminal B subtype has demonstrated poorer recurrence-free survival in adjuvant treatment categories in comparison to luminal A [
44]. Also, it was proved that a large percentage of ER
+ patients with lymph node positive benefit less from adjuvant chemotherapy [
43]. Due to the heterogeneity of this cancer, the same therapies may demonstrate diverse outcomes in patients. So, distinguishing the underlying mechanisms and specific biomarkers can help the individualized treatment of especially high-risked luminal patients.
In this study, LINC01133 and ZEB1-AS1 were significantly downregulated in luminal A and B BC tissues compared to their adjacent non-tumoral tissues. Also, they were downregulated in luminal cell lines, namely T47D and MCF7, compared to a normal breast cell line (MCF10A). Moreover, ABHD11-AS1 was upregulated considerably in the mentioned tissues and cell lines.
LncRNAs have shown greater expression variability among different individuals and a greater degree of tissue-specificity, compared with coding-genes [
45]. The downregulation of LINC01133, ZEB1-AS1(T1-4), and ZEB1-AS1(T5,6) was detected in 64%, 71% and 80% of patients, respectively. Also, the upregulation of ABHD11-AS1 was found in a large proportion of patients (70% of cases). Song et al. reported that the downregulation of LINC01133 is considerably associated with the poor prognosis of BC patients [
46]. On the other hand, Luo et al. suggested that ZEB1‐AS1 promotes triple-negative breast cancer progression [
47]. So, this lncRNA may also play an important role in luminal BC patients, although further investigation is needed. Also, ZEB1-AS1(T5,6) may be used as a biomarker for luminal BC progression. However, more in-vitro tests are needed for more accurate conclusions.
Limited studies have been performed on the dysregulation of these lncRNAs and their roles in cancers so far. So, the combination of various datasets as well as the analysis of their co-expressed genes could provide better identification of functional roles of the mentioned lncRNAs in luminal BC. According to the result of TCGA data analysis available in TANRIC database, LINC01133, ZEB1-AS1, and ABHD11-AS1 expression were significantly different among PAM50 subtype classification. Besides, the expression pattern of LINC01133 and ZEB1-AS1 across luminal and non-luminal BC cell lines was consistent with the data from TANRIC database and also experimental data of the present study. According to the data obtained from the ICGC data portal, most of the mutations of LINC01133, ZEB1-AS1, and ABHD11-AS1 in ER
+ HER2
– patients are from substitution category. The effect of mutations and variations of lncRNAs have not been extensively studied as they constitute a novel class of RNAs. Ponjavic et al. demonstrated that functional lncRNAs show reduced single substitutions, deletions and insertions in their sequences. Although nucleotide substitutions occur mostly in protein-coding sequences compared with noncoding sequences, their effect on lncRNAs are so significant and should not be overlooked [
48]. Substitution mutations in lncRNAs may contribute to the pathogenesis of various cancers by disrupting the secondary structure of RNAs [
18,
49]. The RNA secondary structure plays important roles in multiple cellular processes such as gene regulation and localization, splicing, stability and also translation [
50]. Also, mutations in the upstream regions of lncRNAs, which constitute a considerable percentage of LINC01133 and ABHD11-AS1 mutations, can have significant effects on them. For example, the presence of single-nucleotide polymorphisms in the promoter of a lncRNA may have an effect on its expression pattern [
49]. So, further studies are recommended to determine the effect of these mutations on the structure of the mentioned lncRNAs.
Remarkably, the protein-coding genes are significantly better annotated than lncRNAs [
51]. So, in this study, an attempt was made to determine the function of the target lncRNAs with the help of the known functional information of some coding and non-coding genes, using “guilt-by-association” principle. According to this principle, genes that are involved in some related and/or similar biological pathways may show similar expression patterns across different experimental conditions [
52].
According to the “guilt-by-association” principle and the GO term enrichment analysis of the co-expressed genes with LINC01133, this lncRNA might be involved in ion transport, response to external stimulus, and positive regulation of phosphorylation, all of which are cancer‐associated biological processes. Also, ion/cation/metal binding and cation transmembrane transporter activity are known as the most crucial molecular functions in which LINC01133 is involved. LINC01133 might be mostly found in extracellular region/exosome and membrane-bounded vesicles.
According to the results obtained from the GO term enrichment analysis of ZEB1-AS1 co-expressed genes, this lncRNA is involved in the regulation of RNA metabolic process, regulation of nucleobase-containing compound metabolic process, nucleic acid-templated transcription, etc. Deregulation of all of these biological processes can lead to the development and progression of various cancers, including BC. In addition, ZEB1-AS1 might be plausibly involved in molecular functions including signal transducer activity, kinase activity and phosphotransferase activity, all of which can be linked to cancer progression. ZEB1-AS1 might be mostly enriched in synapse and Golgi lumen.
ABHD11-AS1 might be involved in transmembrane transport, reproductive process, ion transport, neurological system process, etc. ABHD11-AS1 also might be considerably involved in different molecular functions, like signal transducer activity, transmembrane signaling receptor activity, molecular transducer activity, etc. Dysregulation of all of the mentioned functions can lead to the development of various cancers. Moreover, ABHD11-AS1 is likely to be found in intrinsic component of nuclear membrane part, apicolateral plasma membrane, etc.
Significantly enriched KEGG, Reactome and WikiPathways of the co-expressed genes with LINC01133 include sodium/proton exchanges, mucin-type O-glycan biosynthesis, and NRF2 pathway. Abnormal glycoprotein structure of tumor cells can affect the survival, growth and metastasis of cancer cells [
53]. Nuclear receptors (NR) are known as regulators of physiological processes and play oncogenic or anti-oncogenic roles in cancerous cells [
54]. Growing evidence support the involvement of several NRs in the regulation of various pathways related to the initiation and development of BC [
55]. Overexpression of Nrf2 has been shown to increase the expression of glucose‐6‐phosphate dehydrogenase (G6PD) and Hypoxia‐inducing factor 1α (HIF‐1α) in BC cell lines, including MCF7. Also, overexpression of Nrf2 increases Notch1 expression via the G6PD/HIF-1α pathway. Notch signaling pathway regulates BC cell proliferation and migration by affecting the downstream gene, HES‐1, and the epithelial-to-mesenchymal transition (EMT) pathway, respectively [
56]. All of these evidences suggest that LINC01133 could play an essential role in BC.
Moreover, according to the pathway enrichment analysis, ZEB1-AS1 might be involved in tight junction interactions, estrogen biosynthesis, glycerophospholipid catabolism, and Wnt signaling pathway. All of these pathways can be associated with the progression and development of BC. Steroid hormones increase cell proliferation in BC [
57]. Also, alterations in tight junction complexes could facilitate BC initiation and progression by impairing their control over crucial cellular processes such as cell motility and polarity [
58]. Moreover, the Wnt pathway is significantly activated in breast tumors [
59].
ABHD11-AS1 might also be mainly involved in Wnt ligand biogenesis, FGFR1 mutant receptor activation, mesodermal commitment pathway, and Wnt signaling pathway. The involvement of fibroblast growth factors (FGF) has been found in various cellular processes, including proliferation, drug resistance, anti-apoptosis and angiogenesis. Also, amplification of FGFR1 has been found in ER
+ BC [
60].
Data from STRING database showed that 42, 20, and 14 co-expressed genes with LINC01133, ZEB1-AS1 and ABHD11-AS1 have strong interactions with each other. These data confirmed the involvement of these lncRNAs and their co-expressed genes in similar biological pathways.
Taken together, the dysregulation of the three potential lncRNAs, LINC01133, ZEB1-AS1, and ABHD11-AS1 across luminal A and B subtypes of BC was reported in the present study. The bioinformatics analyses performed in this study helped us better identify the possible role of these lncRNAs in luminal BC. However, more experimental studies are needed to confirm these findings and to verify the exact roles of the mentioned lncRNAs.
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