Dysregulation of lncRNA LINC00968 may contribute to the tumorigenesis of estrogen receptor-positive (ER+) breast cancer

doi:10.21203/rs.3.rs-59534/v3

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Research Article

Dysregulation of lncRNA LINC00968 may contribute to the tumorigenesis of estrogen receptor-positive (ER⁺) breast cancer

https://doi.org/10.21203/rs.3.rs-59534/v3

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Background: Estrogen receptor-positive (ER⁺) breast cancer (BC) (luminal A and B subtypes) comprises up to 70% of all BC patients. LncRNAs can affect many biological and pathological processes, and dysregulation of them is related to human cancers. The potential role of lncRNA LINC00968 in ER⁺ BC is still unclear.

Methods: We analyzed the LINC00968 expression across 44 paired luminal BC tissues from the TCGA-BRCA RNA sequencing dataset, and we used the GEPIA2 web server and GENEVESTIGATOR software, as well. The qRT-PCR assay was performed to confirm the LINC00968 expression in 71 paired luminal BC tissues and two luminal A cell lines (MCF7 and T47D). Moreover, to better understand the potential function of LINC00968 in luminal BC, multiple in silico data analyses have been done including co-expression, functional enrichment, and genetic alteration analyses.

Results: The results of data analyses retrieved from BRCA dataset and databases revealed the significant downregulation of LINC00968 in luminal A and B BC. Also, the results of qRT-PCR in luminal BC tissues and cell lines confirmed the earlier data. LINC00968 expression was negatively associated with tumor stage and lymph node metastasis. Additionally, functional annotation analyses revealed that LINC00968 might be involved in vascular development and angiogenesis, extracellular matrix organization, and cell motility and migration. LINC00968 might have functions in some cancer-related signaling pathways, like the PPAR signaling pathway, small ligand GPCRs, etc.

Conclusions: Our study found that LINC00968 may be a potential tumor suppressor gene in luminal BC. Downregulation of LINC00968 might promote tumorigenesis, invasion, and metastasis of luminal BC.

Cancer Biology

Bioinformatics

Breast cancer (BC)

in silico analysis

Luminal subtypes

LINC00968

Long non-coding RNAs

Breast Cancer (BC) is a heterogeneous disease and the most frequent malignant disease among women around the globe (1). Also, this type of cancer is the leading cause of cancer mortality in female patients (2). The incidence of BC is increasing in many developing countries (3). Microarray-based gene expression profiling analysis is used to classify BC into five intrinsic/molecular subtypes based on the expression pattern of estrogen receptor [ER], progesterone receptor [PR], human epidermal growth factor receptor 2 [HER2], and a proliferation index marker, Ki67 (1, 4). These subtypes are included in luminal A, luminal B, HER2 enriched, triple-negative (basal-like), and normal-like that display different biological behavior and progression (1). Luminal A is the most frequent molecular subtype, which expresses ER and PR but not HER2, and shows the low expression of Ki67 (1, 4). Luminal B is defined with ER⁺, PR⁺/PR^-, HER2⁺/HER2^-, and high expression of Ki67 (5). The two types of luminal B (with HER2⁺ or HER2^-) have a worse prognosis and higher proliferation rate than luminal A subtype and are more aggressive (5). Luminal A and B constitute approximately 70% of all breast cancers and show a better prognosis than hormone receptor-negative BC (3). The molecular mechanisms of BC have been broadly studied, but some problems still exist, such as delayed diagnosis, recurrence, and metastasis (6). Currently, molecular markers can improve the diagnosis, prognosis, and guidance of new therapies for BC.

Recent advances in sequencing technologies have revealed that roughly 85% of the human genome is transcribed actively (7). Most of the human RNA transcripts are non-coding RNAs (≥80%) (7, 8) that perform important functions in different biological processes (7). Long non-coding RNAs (lncRNAs) are regulatory non-coding RNAs longer than 200 nucleotides (9). The number of lncRNAs in the mammalian genome is higher than protein-coding mRNAs, but they have lower expression levels and tissue-specific expression (8). Many lncRNAs are associated with a variety of diseases such as cancers (10). Long ncRNAs can interact with DNA, RNAs, and protein macromolecules in cells. LncRNAs regulate tumor suppressor or oncogenic pathways through epigenetic, transcriptional, post-transcriptional, and translational mechanisms that can lead to the cancer phenotype (2, 11). The majority of non-coding RNAs show differential expression between tumor tissues and normal tissues (9). LncRNAs can be categorized into tumor suppressor genes and oncogenes according to their functions and the expression patterns in the tumor tissues (12). Some lncRNAs are differentially expressed in the molecular subtypes of breast cancer (9). Various lncRNAs affect the development, proliferation, apoptosis, drug resistance, invasion, and metastasis of BC cells (13). For example, lncRNAs H19, SRA, LSINCT5, Smad7, and NEAT1 can promote the proliferation of BC cells and inhibit apoptosis of them (13). However, lncRNAs ZFAS1, GAS5 can inhibit the proliferation of BC cells and promote apoptosis (13). Also, lncRNAs UCA1, Lnc-ATB, CLARA, and DSCAM-AS1 play important roles in the drug resistance of BC cells (13). Additionally, several lncRNAs like HOTAIR, MALAT1, and BANCR can promote invasion and metastasis of BC cells (13). Exploring diverse functions of lncRNAs in cancers like BC may lead to new ideas about novel biomarkers and targeted therapies and can be useful in early detection (10).

Long Intergenic Non-Protein Coding RNA 968 (LINC00968) is located on chromosome 8q12.1, which includes three exons. Studies have indicated that this lncRNA acts as an oncogene in osteosarcoma (14). Also, this gene is upregulated in lung squamous cell carcinoma (LUSC) and plays key roles in the tumorigenesis of this malignancy (15). Moreover, previous studies have stated that LINC00968 was downregulated in BC (16, 17). This lncRNA plays crucial roles in reducing cell proliferation, migration, angiogenesis, and drug resistance of BC cells through different mechanisms (16, 17). We performed this study to evaluate the potential role of LINC00968 in the most frequent subtypes of BC (luminal A and B).

In this study, The Cancer Genome Atlas (TCGA)-BRCA RNA seq dataset was used to assess the expression level of LINC00968 in tumor tissues compared with adjacent non-tumor tissues of luminal A and B BC. Also, the qRT-PCR analysis evaluated the expression level of LINC00968 in luminal A and B BC tissues and luminal A cell lines. Furthermore, multiple bioinformatic analyses were carried out to explore more about the biological roles of LINC00968 in the luminal subtypes of BC.

2.1 Evaluation of the LINC00968 expression in luminal subtypes using BC datasets

We evaluated the LINC00968 expression and conducted this study on luminal A and B BC subtypes since these two subtypes have the highest frequency among BC patients. To explore the expression status of LINC00968 in luminal BC subtypes, we analyzed 44 paired tumor/ adjacent non-tumor tissue samples (containing 26 and 18 pairs from luminal A and B subtypes, respectively) from TCGA-invasive breast carcinoma (BRCA) dataset obtained from the Genomic Data Commons (GDC) Data Portal (https://portal.gdc.cancer.gov (18)). This RNA seq data was analyzed using the DESeq2 package in R statistical software. Also, we determined the expression level of LINC00968 in luminal A and B subtypes using the Gene Expression Profiling Interactive Analysis 2 (GEPIA2) web server (http://www.GEPIA2.cancer-pku.cn (19)). Additionally, we obtained LINC00968 expression in 11 BC categories (containing 1905 luminal A and B samples) and normal breast tissue (323 samples) using GENEVESTIGATOR software (Affymetrix Human Genome U133 Plus 2.0 Array platform) (http://www.genevestigator.com (20)).

2.2. Patients and tissue samples

A total of seventy-one pairs of luminal A and B BC tissue samples and adjacent non-tumor tissue samples were obtained from Breast Cancer Research Center Bio-Bank (BCRC-BB) (Tehran, Iran) (21). All samples were immediately snap-frozen in liquid nitrogen and after transportation, stored at -80˚C. This study was approved by the Ethics Committee of Tehran University of Medical Sciences (TUMS) (Code of Ethics: IR.TUMS.MEDICINE.REC.1398.659). Written informed consent was obtained from all patients prior to their inclusion in the study. The clinicopathological data of patients was recorded from medical documents.

2.3. Cell culture

The luminal A BC cell lines (MCF7 and T47D) were cultured in DMEM (Sigma-Aldrich, St. Louis, MO, USA) with 10% fetal bovine serum (Gibco, Carlsbad, CA, USA), 100 U/ml penicillin and streptomycin (Sigma-Aldrich, St. Louis, MO, USA) in a cell culture and were incubated at 37˚C, 5% CO2 and 95% humidity. The non-tumorigenic epithelial breast cell line (MCF10A) was cultured in DMEM containing 5% horse serum, 10 μg/ml insulin, 20 ng/ml EGF, 100 ng/ml cholera toxin, and 0.5 μg/ml hydrocortisone.

2.4. RNA isolation andquantitative real-time PCR (qRT-PCR)

The total RNA was extracted from tissue samples and cell lines using RiboEx^TM reagent (GeneAll, Korea). The extracted RNAs were treated with DNase I (EN0521, Thermo Fisher scientific, United States). Then, reverse transcription into cDNA was performed by a 5X All-In-One RT MasterMix kit (Applied Biological Materials, CA). Real-time PCR was performed in duplicate using RealQ Plus 2x master mix (AMPLIQON, Denmark) in LightCycler^® 96 instrument (Roche Diagnostics, Mannheim, Germany). The β2M housekeeping gene was used as a normalizer. The primer sequences of LINC00968 were 5′- CCAGACTCCTCAGCCTGAAAT-3′ (forward) and 5′-GTCCCAAACGCAGACCATTTT-3′ (reverse). Also, the primer sequences of β2M were 5′- AGATGAGTATGCCTGCCGTG-3′ (forward) and 5′- GCGGCATCTTCAAACCTCCA-3′ (reverse). The relative expression was calculated using the 2^-∆∆CT method.

2.5. Detection of the co-expressed genes with LINC00968 in luminal subtypes of BC based on the bioinformatic data

The TCGA-BRCA data of 420 luminal BC tissue samples containing 230 luminal A and 190 luminal B tissue samples were downloaded as a FPKM file from the GDC data portal. Then, the co-expressed genes with LINC00968 were obtained across the mentioned samples using R software (R ≥ 0.5). Pearson correlation analysis of these co-expressed genes has been computed using a standard method.

2.6. Functional annotation, pathway enrichment analysis, and establishment of protein-protein interaction (PPI) network

Gene Ontology (GO) term enrichment analysis was performed using DAVID version 6.8 (https://david.ncifcrf.gov (22)). This web server was used to provide three GO term categories based on a large list of co-expressed genes with LINC00968, including biological processes (BP), cellular components (CC), and molecular functions (MF). Also, GO terms obtained from DAVID were applied to the REVIGO web server (http://revigo.irb.hr (23)). REVIGO web server can take long lists of GO terms and summarize them by removing redundant GO terms. The XGMML files retrieved from REVIGO were imported into the Cytoscape version 3.8.0 to increase their visualization. Besides, the protein-protein interaction (PPI) network of LINC00968 co-expressed genes was obtained from STRING database (https://string-db.org (24)), and the hub genes were determined based on the degree method in cytoHubba plugin of Cytoscape software (25). Also, the Enrichr database (http://amp.pharm.mssm.edu/Enrichr (26)) was used to perform pathway enrichment analysis of the co-expressed genes with LINC00968 using pathway databases such as KEGG, Reactome, and WikiPathways.

2.7. Genetic alterations analysis

The International Cancer Genome Consortium (ICGC) data portal (https://dcc.icgc.org (27)) provided the number of LINC00968 mutations across three BC projects (BRCA/EU, BRCA/UK, BRCA/FR). The ER⁺/HER2^- project (BRCA/EU) contained luminal A and B subtypes of BC.

We obtained the most common mutations in LINC00968 and mutations which occur in exonic regions of this lncRNA among BC projects. Also, we used two databases, lncRNASNP2 (http://bioinfo.life.hust.edu.cn/lncRNASNP (28)) and DIANA-LncBase v2 (www.microrna.gr/LncBase (29)), to obtain predicted miRNAs interacting with transcript variants of LINC00968. Moreover, we reported miRNAs that may be affected by mutations on binding sites of LINC00968.

2.8. Statistical analysis

The qPCR data analysis was performed using IBM SPSS version 24 software (IBM Co., Armonk, NY, USA). Data were expressed as mean ± standard deviation. Paired samples t-test was done to compare the ∆Ct values of tumoral and adjacent non-tumor tissues which often have a normal distribution. In our work, the -∆Ct values of tumor samples and adjacent non-tumor tissues were -14.1 ± 2.7 and -11.9 ± 3.0, respectively. In addition, the Kolmogorov-Smirnov test approved the normality of distribution as well. The association between LINC00968 expression and the clinicopathological data was performed by χ² test. The Pearson correlation coefficient ≥ 0.5 and p-values <0.05 were considered as significant.

3.1. Downregulation of LINC00968 in luminal subtypes of BC tissues and cell lines

From the TCGA dataset, LINC00968 was significantly decreased in luminal A and B subtypes of BC according to the analysis of 44 pairs of cancer and adjacent non-cancerous tissue samples (log₂ fold change (FC)_{luminal A}= -2.9, adj.p-value = 2.54E-23) (log₂ fold change (FC)_{luminal B}= -4.3, adj.p-value = 6.24E-15). Moreover, the result of the GEPIA2 web server indicated the significant downregulation of LINC00968 (p < 0.05) in luminal A and B tumor tissues compared with normal controls (Fig. 1a). Also, the results of GENEVESTIGATOR software elucidated the downregulation of LINC00968 in 11 BC categories containing luminal samples compared with that in normal breast tissue (Supplementary Fig. 1).

To confirm the data obtained earlier, quantitative real-time PCR was performed on 71 paired luminal BC and adjacent non-tumor tissues. The qRT-PCR results demonstrated that there was a significant downregulation of LINC00968 (p <0.001) in luminal A and B BC tissues compared with adjacent non-tumor tissues (Fig. 1b). LINC00968
expression level was remarkably lower in 79% (56/71) of tumor samples in comparison to that in adjacent non-cancerous tissues (Fig. 1c). Also, the qRT-PCR results in T47D and MCF7 cell lines revealed that LINC00968 expression was remarkably lower (p <0.001) in these luminal A cell lines than that in MCF10A cell line (Fig. 1d). Thus, the results of the experimental assay are similar to the bioinformatic results and concordant with them.

3.2. The association of LINC00968 expression with clinicopathological data

The clinicopathological characteristics of patients with luminal BC are shown in Table 1. These clinicopathological features were included in luminal A and B groups, ER/PR/HER2 status, tumor size, lymph node metastasis, tumor stages and grades, age at diagnosis, and p53 protein expression.

According to the median level of LINC00968 expression in luminal A and B BC tissues, patients were classified into high and low expression groups. Low expression of LINC00968 in luminal A and B was significantly associated with advanced tumor stage (p = 0.016) (Fig. 1e) and lymph node metastasis (Fig. 1f) (p = 0.022). LINC00968 expression was lowest in stage III. Other clinicopathological factors like PR±, HER2±, tumor size, grade, etc suggested no significant association with LINC00968 expression (p > 0.05).

3.3. GO functional annotation, pathway enrichment analysis, and PPI network construction for LINC00968 co-expressed genes

To better understand the potential functions of LINC00968 in luminal A and B BC, a co-expression analysis was performed. This analysis indicated that LINC00968 is significantly co-expressed with 306 genes (R ≥ 0.5) according to the luminal A and B subtypes of invasive breast carcinoma dataset (Supplementary File 1).

A list of GO terms for three categories, including BP, CC, and MF was retrieved from the DAVID database, and the top 10 enriched GO terms (p-value< 0.05) for LINC00968 co-expressed genes were illustrated in Figure 2. LINC00968 co-expressed genes were mainly involved in some biological processes such as ‘vasculature and blood vessel development’, ‘cardiovascular system development’, ‘cell motility and migration’. Moreover, some significant GO cellular component terms of LINC00968 co-expressed genes were included in ‘extracellular region and matrix’, ‘proteinaceous extracellular matrix’, and ‘cell surface’. Additionally, LINC00968 co-expressed genes were mainly enriched in some molecular functions like ‘glycosaminoglycan binding’, ‘heparin binding’, ‘sulfur compound binding’, and ‘receptor binding and activity’.

Summarization of Gene Ontology terms and removing redundant GO terms using REVIGO indicated GO terms similar to the data retrieved from DAVID (Supplementary Fig 2a, b).

The protein-protein interaction (PPI) network of the LINC00968 co-expressed genes was retrieved from the STRING database. This network indicated that 204 of 306 co-expressed genes had an interaction score > 0.4 with each other (Fig. 3a). The top 24 hub genes were obtained from the PPI network of the LINC00968 co-expressed genes based on the degree method (node degree ≥ 10), and the sub-PPI network was generated using the cytoHubba plugin in the Cytoscape software (Fig. 3b, Table 2). Approximately 79% of the top mentioned hub genes were significantly downregulated in luminal A and B BC except for APOB, NT5E, LUM, DPP4, and PRRX1 based on the GEPIA2 web server.

According to the results of the pathway enrichment analysis (KEGG, WikiPathways, and Reactome), the potential pathways for the co-expressed genes with LINC00968 in luminal A and B BC were obtained. The co-expressed genes with LINC00968 might have functions in some cancer-associated signaling pathways such as the PPAR signaling pathway, small ligand GPCR, GPCR class B secretion-like (Table 3). Additionally, the human complement system, focal adhesion pathway, proteoglycans in cancer, and so on were included in enriched pathways for the co-expressed genes.

3.4. Genetic alterations of LINC00968 in the luminal subtypes of BC

According to the ICGC data portal, there were 206 mutations in LINC00968 across three BC projects (BRCA-EU, BRCA-FR, BRCA-UK) (Fig. 4a, Supplementary File 2). About 193 of 206 LINC00968 mutations occurred in luminal subtypes of BC (BRCA-EU project). Most of the LINC00968 mutations in luminal BC were substitutions. The majority of LINC00968 mutations took place in the intronic sites (Fig. 4b). The most frequent mutations in LINC00968 were chr8:g.57407070T>- and chr8:g.57426123C>T (2.07% frequency) among 206 mutations on genome assembly GRCh37. These mutations occurred in downstream and intronic regions of two transcript variants are shown in Table 4. Furthermore, three mutations in exons of two mentioned transcript variants were shown in Table 4.

DIANA-LncBase v2 database has predicted 256 miRNAs that interact with seven transcript variants of LINC00968. Among these miRNAs, 107 and 29 miRNAs interacted with ENST00000499425 and ENST00000519144 transcript variants, respectively (Supplementary File 3). Chr8:g.57430931T>G mutation occurred in the binding site of ENST00000499425 transcript variant (exonic region) for hsa-miR-5692a, hsa-miR-5590-3p, and hsa-miR-2115-3p. Moreover, Chr8:g.57408827G>C mutation took place in the binding site of ENST00000519144 for hsa-miR-5197-3p. On the other hand, lncRNASNP2 database has reported 38 miRNAs which bind to the exonic regions of ENST00000519144 transcript variant. Among them, hsa-miR-5004-3p, hsa-miR-6842-3p, hsa-miR-4695-3p, and hsa-miR-6511b-3p interacted with exon three of the mentioned transcript. Chr8:g.57408827G>C mutation in exon three of ENST00000519144 transcript might affect the binding site of hsa-miR-5004-3p. These mutations might impact the binding sites of mentioned miRNAs and regulation of genes.

Extensive studies have reported that the majority of lncRNAs are abnormally expressed in tumor tissues, and each lncRNA exerts its function through diverse mechanisms in different cancers (30). According to the previous studies, LINC00968 was upregulated in osteosarcoma and lung squamous cell carcinoma and was downregulated in breast cancer (14, 16, 17, 31). LINC00968 increased proliferation of osteosarcoma through PI3K/Akt signaling pathway (14). Reduction of drug resistance in BC can take place through LINC00968 overexpression, which inhibits the activation of the Wnt2/β-catenin signaling pathway by silencing WNT2 (Wnt family member 2) (17). Furthermore, LINC00968 inhibits the progression of BC through suppression of hsa-miR-423-5p and upregulation of PROX1 (prospero homeobox protein 1) (16). The present study evaluated the expression levels and the role of LINC00968 in the most frequent subtypes of BC (luminal A and B). The combination of experimental and bioinformatic analyses can be useful for deciphering the potential role of LINC00968 in luminal BC.

In the present study, we identified LINC00968 as a dysregulated lncRNA in luminal BC. The current study elucidated that LINC00968 was significantly downregulated in luminal A and B BC tissues, and T47D and MCF7 cell lines (luminal A cell lines) compared with adjacent non-tumor tissues and MCF10A cell line, respectively. However, how this lncRNA was regulated in luminal BC remains unknown. Moreover, LINC00968 was downregulated in a great fraction of tumor tissues (79% of patients). One of the features of several lncRNAs is expression variability among individuals and tissue-specific expression (32). The results of the qRT-PCR were concordant with the results of TCGA-BRCA RNA-seq analyses of 44 pairs of tumor and adjacent non-tumor tissues, GEPIA2 web server, and GENEVESTIGATOR software which indicated downregulation of LINC00968 in luminal A and B BC.

The low expression levels of LINC00968 in tumor samples were significantly associated with advanced tumor stage and lymph node metastasis. It was considered that LINC00968 might act as a tumor suppressor gene in the luminal BC, and dysregulation of this lncRNA might lead to the invasion and metastasis of luminal BC. Thus, LINC00968 might be involved in luminal A and B BC carcinogenesis. The low expression of LINC00968 was not significantly correlated with other clinicopathological parameters, including PR, HER2 status, tumor size, grade, etc.

We performed further analyses to elucidate more about the potential role of LINC00968 in luminal BC. First, we got a list of genes mainly co-expressed with LINC00968 based on several databases and bioinformatic tools. Then, we carried out GO and pathway enrichment analyses according to the list of genes. The genes network was extensively utilized for the prediction of the functions of un-annotated genes according to the "guilt by association" (GBA) principle (33). This principle states one gene might show the same regulatory mechanisms and roles with its co-expressed genes in the related processes and functions (34). We can infer the unknown function of a gene through previous information about its co-expressed genes. Thus, considering this principle and according to the gene ontology (GO) terms enrichment analyses, LINC00968 might be involved in multiple biological processes in luminal BC like vasculature development, angiogenesis, extracellular matrix organization, cell motility and migration, and so on. Angiogenesis is an important process in the development of new blood vessels, invasion, and metastasis of tumor cells (35). Migration of cancer cells occurs by degrading the surrounding extracellular matrix (ECM) and intravasation into the blood stream or lymphatic system and transit to distant tissues (36). These results were consistent with our experimental data, which showed the involvement of dysregulated LINC00968 in advanced tumor stage and lymph node metastasis.

Also, the molecular function analysis proposed that LINC00968 was mostly involved in glycosaminoglycan binding, heparin binding, sulfur compound binding, receptor binding and activity, and so on. It has been stated that the improper function of these molecules and processes was associated with cancers. Glycosaminoglycans (GAGs) are components of the extracellular matrix (ECM) and can interact with both ligands and receptors of cancer cells. This interaction can regulate downstream signalings like epidermal growth factor receptor (EGFR), estrogen receptors (ERs), or Wnt members (37). Interaction between molecules expressed by cancer cells and factors in the tumor microenvironment can affect proliferation and survival, adhesion, migration of tumor cells, and tumor invasion and metastasis (38). Heparin is a part of glycosaminoglycan, and derivatives of this molecule can significantly reduce BC cell proliferation and metastasis both in vitro and in vivo (39). Receptors, especially hormone receptors, are important in the biology of BC, and ER/PR are involved in the development and progression of BC.

According to the STRING database, about 66% of the LINC00968 co-expressed genes had significant interaction with each other. Also, considering the PPI network of the co-expressed genes with LINC00968 and GEPIA2 web server, most of the hub genes (79%) were significantly downregulated in luminal A and B BC tissues in comparison to normal breast tissues (data not shown). Thus, these results have confirmed that LINC00968 and its co-expressed genes are involved in common biological processes. Data obtained from Enrichr database has stated that LINC00968 and its co-expressed genes are involved in several cancer-associated signaling pathways like PPAR signaling pathway, small ligand GPCR, GPCR class B secretion-like, focal adhesion pathway, proteoglycans in cancer, and etc. Studies have reported that different members of PPAR family like PPARα and PPARβ/δ play important roles in breast epithelial homeostasis and tumorigenesis (40, 41). PPARs are nuclear hormone receptors and are activated by fatty acids (41). Adipogenesis is associated with the BC recurrence and reduced survival of patients (41). G-protein coupled receptors (GPCRs) are the largest group of cell membrane receptors that are involved in the development and progression of various malignancies like BC (42). Moreover, it is evident that signaling through GPCRs affects some processes in cancer biology, such as vascular remodeling, invasion, and migration (43). Focal adhesions (FAs) provide attachment of cells to the ECM, and focal adhesion kinase (FAK) has significant roles in some human cancers like BC invasion and metastasis (44).

The data retrieved from the ICGC data portal reported that a great number of mutations occur in LINC00968 across ER⁺/HER2^- BC project (containing luminal subtypes). Most of the LINC00968 mutations are of substitution type and mostly take place in the intronic regions. The somatic mutations directly or indirectly change gene expression (45) and lncRNA expression profiles in cancers. Some somatic mutations that are located on lncRNA transcription factor binding sites affect the lncRNA expression in cancers (45). Somatic mutations can affect RNA secondary structure and the regulation of gene expression (46). Somatic mutations in introns can disrupt splice sites or intronic splicing regulatory sequences, activate cryptic splice sites, and change transcriptional enhancer or silencer binding sequence. Therefore, the intronic mutations in LINC00968 may affect transcription levels and RNA splicing.

The miRNAs that bind to the mutated regions of LINC00968 in introns and downstream regions have not been reported in the databases. Chr8:g.57430931T>G mutation, located in the exon three of ENST00000499425, may affect the interaction of this transcript with hsa-miR-5692a, hsa-miR-5590-3p, and hsa-miR-2115-3p. Besides, chr8:g.57408827G>C mutation in the exonic region of ENST00000519144 may have an impact on the interaction of mentioned transcript with hsa-miR-5197-3p and hsa-miR-5004-3p. These mutations may alter the specificity of transcript variants of LINC00968 to mentioned miRNAs and can impact miRNA-mediated gene regulation. The lncRNA-miRNA interaction can regulate neovascularization and angiogenesis in cancers (47). Studies have demonstrated that hsa-miR-5590-3p negatively regulates the TGFβ/SMAD signaling pathway, and it can be a potential therapy in various cancers (48). Moreover, the lncRNA MIR100HG can enhance the progression of triple-negative breast cancer (TNBC) through interacting with miR-5590-3p (49). The expression of miR-2115 is associated with HER2⁺BC, whereas luminal samples show a gradient expression of this miRNA (50). Also, exosome miR-5004-3p can regulate AR and PTEN genes in chemoresistance cancer cells (51). Future experiments are required to verify the interaction between each LINC00968 transcript variant and the mentioned miRNAs in BC and the effect of these interactions on mRNAs expression as well.

Since most of the studies face limitations, the lack of any information about the effect of ethnic groups of luminal BC patients on LINC00968 expression is a limitation of the current study.

Based on the data collected from previous research and this study, dysregulation of lncRNAs is thought to be involved in the carcinogenesis. In the current study, downregulation of LINC00968 was observed in luminal A and B tumor tissues and luminal A cell lines. Downregulation of LINC00968 was remarkably associated with lymph node metastasis and advanced tumor stage. Thus, LINC00968 might act as a tumor suppressor gene in luminal BC. Furthermore, this novel lncRNA might play crucial roles in several cancer-associated signaling pathways in luminal BC like the PPAR signaling pathway, small ligand GPCRs, GPCRs class B secretion-like, focal adhesion pathway, etc. Taken together, LINC00968 might impact tumorigenesis of luminal subtypes of BC.

Ethics approval and consent to participate

The present study was approved by the Ethics Committee of Tehran University of Medical Sciences (TUMS), and written informed consent was obtained from all of the participants (Code of Ethics: IR.TUMS.MEDICINE.REC.1398.659).

Consent for publication

Not applicable

Data availability statement

The datasets supporting the conclusions of this article are available in:

[GDC data portal] at (https://portal.gdc.cancer.gov/)

[GEPIA2 web server] at (http://www.gepia.cancer-pku.cn)

[International Cancer Genome Consortium data portal] at (https://dcc.icgc.org)

[DAVID database] at (https://david.ncifcrf.gov)

[REVIGO database] at (http://revigo.irb.hr)

[Cytoscape software] at (http://www.cytoscape.org)

[Enrichr database] at (http://amp.pharm.mssm.edu/Enrichr)

[STRING database] at (https://string-db.org)

[LncRNASNP2] at (http://bioinfo.life.hust.edu.cn/lncRNASNP)

[DIANA-LncBase v2] at (www.microrna.gr/LncBase)

Citation of all data is provided in the references list.

The datasets supporting the conclusions of this article are also included within the article and its additional files.

Conflicts of interest

The authors declare that they have no conflicts of interest.

Funding

This work was supported by the Faculty of Medicine, Tehran University of Medical Sciences (TUMS). The samples were obtained from Breast Cancer Research Center Bio-bank (BCRC-BB).

Authors' contributions

M.A. performed experimental study and data analysis, bioinformatic analysis, literature review, and manuscript writing. S.M. contributed to the experimental study and data analysis, and edited the manuscript. K.M. designed the research strategy, performed the literature review, edited the manuscript, and reviewed the manuscript. J.T.B. designed the research strategy, performed literature review, edited the manuscript, and reviewed the manuscript. A.M. performed the data analysis and edited the manuscript. M.M.N. performed the statistical analysis and edited the manuscript. A.S. supervised the whole project and designed the research strategy, performed literature review, edited the manuscript and reviewed the manuscript. All the authors read and approved the final manuscript.

Acknowledgments

We thank Dr. Rezvan Esmaeili (Head of Genetics Department, Breast Cancer Research Center of Motamed Cancer Institute, ACECR, Tehran, Iran), Narges Jafarbeik-Iravani, and Dr. Rasoul Abdollahzadeh for the guidance of the experimental tests and other members of the Genetics Department of Breast Cancer Research Center of Motamed Cancer Institute, ACECR, Tehran, Iran. Also, the authors would like to thank Ms. Marzie Samimifar for proofreading the English language of the manuscript.

BC, breast cancer; BRCA, invasive breast carcinoma; BCRC-BB, Breast Cancer Research Center Bio-Bank; BP, biological process; CC, cellular component; Del, Deletion; ER, estrogen receptor; ECM, extracellular matrix; FAs, Focal adhesions; FAK, focal adhesion kinase, GO, gene ontology; GAGs, Glycosaminoglycans; HER2, human epidermal growth factor receptor 2; LncRNA, long noncoding RNAs; LINC00968, Long Intergenic Non-Protein Coding RNA 968; MF, molecular function; PR, progesterone receptor; PPAR, peroxisome proliferator-activated receptor; PI3K, Phosphatidylinositol 3-kinase; PPI, protein-protein interaction; PROX1, prospero homeobox protein 1; qRT-PCR, quantitative real time reverse transcription-polymerase chain reaction; SBS, Single Base Substitution; TCGA, The Cancer Genome Atlas; WNT2, Wnt family member 2.

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Table 1. The association of LINC00968 expression with clinicopathological characteristics in luminal A and B BC patients

Clinicopathological characteristic

Number of cases

LINC00968 expression

P-value

(χ² test)

Low

N (%)

High

N (%)

Group

Luminal A

Luminal B

32(91.4%)

3(8.6%)

29 (80.6%)

7 (19.4%)

0.188

Estrogen receptor

Negative

Positive

0 (0.0%)

35 (100.0%)

0 (0.0%)

36 (100.0%)

Progesterone receptor

Negative

Positive

4 (11.4%)

31 (88.6%)

3 (8.3%)

33 (91.7%)

0.662

HER2^a

Negative

Positive

34 (97.1%)

1 (2.9%)

32 (88.9%)

4 (11.1%)

0.174

Tumor size

<2cm

2-5cm

>5cm

11 (31.4%)

16 (45.7%)

8 (22.9%)

7 (21.2%)

20 (60.6%)

6 (18.2%)

0.458

Lymph node metastasis

Yes

4 (11.8%)

30 (88.2%)

12 (35.3%)

22 (64.7%)

0.022^*

Tumor stage

I-II

III

14 (45.2%)

17 (54.8%)

26 (74.3%)

9 (25.7%)

0.016^*

Grade

5 (14.3%)

25 (71.4%)

5 (14.3%)

3 (8.3%)

25 (69.4%)

8 (22.2%)

0.555

P53

Negative

Positive

5 (26.3%)

14 (73.7%)

3 (23.1%)

10 (76.9%)

0.835

Age at diagnosis

<40

>40

9 (28.1%)

23 (71.9%)

9 (25.7%)

26 (74.3%)

0.824

^aHER2 = human epidermal growth factor receptor 2

^*P < 0.05

Table 2. The top hub genes in PPI network of the co-expressed genes with LINC00968

Gene name	Node degree
EGFR	36
VWF	32
IGF1	28
CAV1	24
CD34	24
PECAM1	24
APOB	22
DCN	21
CDH5	19
LPL	17
ELN	17
TGFBR2	15
ADIPOQ	15
PDGFRA	15
ANGPT1	14
NT5E	14
LUM	14
ANXA1	12
GNG11	11
FGF7	11
FIGF	11
DPP4	10
FOXO1	10
PRRX1	10

Table 3. Enriched pathways for the co-expressed genes with LINC00968

Pathway	P-value	Genes
KEGG Human
Complement and coagulation cascades	2.36E-05	VWF; CFH; C1S; F10; C1R; PROS1; SERPING1; MASP1
ABC^a transporters	5.41E-04	ABCA6; ABCB5; ABCA9; ABCC9; ABCA8
Renin secretion	5.78E-04	PTGER4; ADCYAP1R1; NPR1; PDE1B; GNAI1; AQP1
Focal adhesion	0.003138	PDGFRA; TNXB; VWF; LAMA2; CAV2; CAV1; VEGFD; IGF1; EGFR
Renin-angiotensin system	0.004693	MME; CMA1; CTSG
PPAR^b signaling pathway	0.005019	ADIPOQ; AQP7; LPL; ACSL4; PLIN1
Regulation of lipolysis in adipocytes	0.009182	NPR1; AQP7; PLIN1; GNAI1
Proteoglycans in cancer	0.010948	CAV2; LUM; CAV1; TWIST2; ANK2; IGF1; EGFR; DCN
Protein digestion and absorption	0.011302	DPP4; MME; COL14A1; XPNPEP2; ELN
Neuroactive ligand-receptor interaction	0.013212	PTGER4; PTGFR; ADCYAP1R1; PENK; SCTR; LEPR; HTR2B; S1PR1; CTSG; AVPR1A; TACR1
WikiPathways Human
Human Complement System	1.92E-06	CD93; CFH; C1S; F10; C1R; PROS1; ADIPOQ; SERPING1; MASP1; DCN
Complement and Coagulation Cascades	2.28E-06	VWF; CFH; C1S; F10; C1R; PROS1; SERPING1; MASP1
Adipogenesis	1.50E-04	GDF10; SFRP4; ADIPOQ; EBF1; LPL; LIFR; IGF1; PLIN1; FOXO1
Genes targeted by miRNAs in adipocytes	8.47E-04	HAND2; IGF1; ERG
Small Ligand GPCRs^c	0.002683	PTGER4; PTGFR; S1PR1
Focal Adhesion	0.003035	PDGFRA; TNXB; VWF; LAMA2; CAV2; CAV1; VEGFD; IGF1; EGFR
PPAR signaling pathway	0.003274	ADIPOQ; AQP7; LPL; ACSL4; PLIN1
Complement Activation	0.004126	C1S; C1R; MASP1
Angiogenesis	0.005305	PDGFRA; ANGPT1; TIMP2
GPCRs, Class B Secretin-like	0.005305	ADCYAP1R1; SCTR; ADGRL4
Reactome Human
Extracellular matrix organization	1.37E-06	TNXB; LAMA2; COL14A1; COL25A1; LUM; CMA1; ELN; FBLN5; DCN; MFAP5; MFAP4; TIMP2; PECAM1; MMP19; CTSG; JAM2; DDR2
ABC-family proteins mediated transport	5.41E-04	ABCA6; ABCB5; ABCA9; ABCC9; ABCA8
Degradation of the extracellular matrix	0.001073	COL14A1; COL25A1; CMA1; TIMP2; MMP19; CTSG; DCN
Metabolism of Angiotensinogen to Angiotensins	0.001603	MME; CMA1; CTSG
Formation of Fibrin Clot (Clotting Cascade)	0.002667	VWF; F10; PROS1; SERPING1
Scavenging by Class A Receptors	0.002683	SCARA5; MASP1; APOB
Elastic fibre formation	0.003208	MFAP5; MFAP4; ELN; FBLN5
Cell surface interactions at the vascular wall	0.004107	ANGPT1; PROS1; CAV1; PECAM1; APOB; JAM2
Intrinsic Pathway of Fibrin Clot Formation	0.004126	VWF; F10; SERPING1

^aABC= ATP binding cassette;

^bPPAR= Peroxisome proliferator-activated receptors;

^cGPCRs = G protein coupled receptors

Table 4. List of two most common mutations and mutations in exonic regions of LINC00968

DNA Change	Mutation Type	Transcript Variant	Consequence	Frequency
chr8:g.57407070T>-	Del^a of <=200bp	ENST00000519144.5	Downstream	2.07%
chr8:g.57426123C>T	SBS^b	ENST00000519144.5	Intron	2.07%
chr8:g.57426123C>T	SBS	ENST00000499425.1	Downstream	2.07%
chr8:g.57431702C>T	SBS	ENST00000499425.1	Exon	0.69%
chr8:g.57430931T>G	SBS	ENST00000499425.1	Exon	0.69%
chr8:g.57408827G>C	SBS	ENST00000519144.5	Exon	0.69%

^aDel= Deletion, ^bSBS= Single Base Substitution

SupplementaryFig.1.pdf
Supplementary Fig 1: PDF file: The expression level of LINC00968 cross 12 anatomical/ cancer categories including 11 luminal BC categories and normal breast tissue retrieved from GENEVESTIGATOR.
SupplementaryFig.2.pdf
Supplementary Fig. 2: PDF file: The summarization of gene ontology (GO) terms related to LINC00968 co-expressed genes. The summarization of (a) GO cellular component terms and (b) GO molecular function terms associated with LINC00968 retrieved from DAVID. The summarization was performed using the REVIGO web server. Node colors is representative of significance of p- value and node size indicates the log size.
SupplementaryFile1.xlsx
Supplementary File 1: xlsx file: The genes were co-expressed with LINC00968 across 420 luminal breast cancer samples.
SupplementaryFile2.xlsx
Supplementary File 2: xlsx file: The mutations in LINC00968 across three breast cancer projects obtained from ICGC data portal.
SupplementaryFile3.xlsx
Supplementary File 3: xlsx file: List of 256 predicted miRNAs interacting with transcript variants of LINC00968.

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Dysregulation of lncRNA LINC00968 may contribute to the tumorigenesis of estrogen receptor-positive (ER⁺) breast cancer

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