Identification of differentially expressed lncRNAs and mRNAs in luminal-B breast cancer by RNA-sequencing

Background Luminal B cancers show much worse outcomes compared to luminal A. This present study aims to screen key lncRNAs and mRNAs correlated with luminal-B breast cancer. Methods Luminal-B breast cancer tissue samples and adjacent tissue samples were obtained from 4 patients with luminal-B breast cancer. To obtain differentially expressed mRNAs (DEmRNAs) and lncRNAs (DElncRNAs) between luminal-B breast cancer tumor tissues and adjacent tissues, RNA-sequencing and bioinformatics analysis were performed. Functional annotation of DEmRNAs and protein-protein interaction networks (PPI) construction were performed. DEmRNAs transcribed within a 100 kb window up- or down-stream of DElncRNAs were searched, which were defined as cis nearby-targeted DEmRNAs of DElncRNAs. DElncRNA-DEmRNA co-expression networks were performed. The mRNA and lncRNA expression profiles were downloaded from The Cancer Genome Atlas (TCGA) database to validate the expression patterns of selected DEmRNAs and DElncRNAs. Results A total of 1178 DEmRNAs and 273 DElncRNAs between luminal-B breast cancer tumor tissues and adjacent tissues were obtained. Hematopoietic cell lineage, Cytokine-cytokine receptor interaction, Cell adhesion molecules (CAMs) and Primary immunodeficiency were significantly enriched KEGG pathways in luminal-B breast cancer. FN1, EGFR, JAK3, TUBB3 and PTPRC were five hub proteins of the PPI networks. A total of 99 DElncRNAs-nearby-targeted DEmRNA pairs and 1878 DElncRNA-DEmRNA co-expression pairs were obtained. Gene expression results validated in TCGA database were consistent with our RNA-sequencing results, generally. Conclusion This study determined key genes and lncRNAs involved in luminal-B breast cancer, which expected to present a new avenue for the diagnosis and treatment of luminal-B breast cancer.


Background
Breast cancer is the leading cause of cancer-related death in women, both overall and in less developed countries (1). It is a heterogeneous disease with regard to molecular alterations, cellular composition, and clinical outcome, both between tumor subtypes and within a single tumor, which were commonly defined by gene expression profiling as four main subtypes including luminal A, luminal B, HER-2 enriched and basal-like, (2)(3)(4). Luminal B breast cancer is unique with regard to somatic point mutations, the profile of gene copy number alterations (CNAs), and DNA methylation (5). Expression profiles and gene sets, with prognostic, predictive functions, or both for patients with breast cancer, have been identified in multiple studies (6). Although both luminal-A and luminal-B breast cancers are ER-positive, luminal-B cancers showed worse outcomes as compared to luminal-A cancers (7,8). Therefore, it is urgent to discover novel biomarkers with prognostic and predictive functions for luminal B breast cancer that can be therapeutically targeted.
With advances in high-throughput technology, it is discovered that human transcriptome mainly consists of non-coding RNAs (ncRNAs) with limited or no protein-coding capacity (9,10). Long non-coding RNAs (lncRNAs), with over 200 nucleotides base long, attracts more attention and has been widely linked with various diseases, including cancers (11)(12)(13)(14). The lncRNAs exert momentous roles in multiple cellular processes at transcriptional and posttranscriptional regulation level through transcriptional interference and histone modifications (15,16). The higher expression of SPRY4-IT1 was reported to modulate apoptosis and invasion in melanoma (17). LncRNA UCA1a (CUDR) may promote proliferation and tumorigenesis in human bladder cancer (18).
In this study, differentially expressed lncRNAs (DElncRNA) and mRNAs (DEmRNAs) in tumor tissues of patients with luminal-B breast cancer were identified by RNA-sequencing. Subsequently, protein-protein interaction (PPI) networks of DEmRNAs were conducted. Identification of cis nearby-targeted DEmRNAs of DElncRNAs and construction of DElncRNA-DEmRNA co-expression networks were performed. In this light, we expect this study could represent a new avenue to improve the understanding of the pathogenesis and be helpful for treatment of luminal B breast cancer.

Patients and samples
Luminal-B breast cancer tissue samples and adjacent tissue samples were obtained from 4 patients with luminal-B breast cancer in People's Hospital of Deyang City, which were free of treatment. The detailed characteristics of patients are displayed in Table 1. Written informed consent about the use of these samples was obtained from each patient. All procedures performed in this study was in accordance with the ethical standards of the ethics committee of People's Hospital of Deyang City (2017-045) and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

RNA isolation and sequencing
According to the manufacturer's protocol, RNA was extracted with PAXgene blood RNA kit (PreAnalytiX  Reads with low quality (adaptor sequences, sequences with a quality score < 20, and sequences with an N base rate of raw reads > 10%) were removed to obtain the clean reads.

Functional annotation of DEmRNAs
Functional annotation, including Gene Ontology (GO) function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses was performed with Metascape (http://metascape.org/gp/index. html). A value of p < 0.05 was set as the cut-off for significance.

Cis nearby-targeted DEmRNAs of the DElncRNAs
DEmRNAs transcribed within a 100-kb window upstream or downstream of DElncRNAs were searched, which were defined as cis nearby-targeted DEmRNAs of DElncRNAs, to obtain the targeted DEmRNAs of DElncRNAs with cis-regulatory effects. The networks were visualized by Cytoscape software. Functional annotation of the cis nearby-targeted DEmRNAs of the DElncRNAs was performed with Metascape. A value of p < 0.05 was set as the cut-off for significance.

DElncRNA-DEmRNA co-expression networks
To further examine the potential roles of DElncRNAs and DEmRNAs in luminal-B breast cancer, the DElncRNA-DEmRNA co-expression networks were constructed. DElncRNA-DEmRNA pairs with an absolute value of PCC > 0.95 and p < 0.01 were defined as co-expressed DElncRNA-DEmRNA pairs. By using Cytoscape, the coexpressed DElncRNA-DEmRNA networks were visualized. Functional annotation of the DEmRNAs co-expressed with DElncRNAs was performed with Metascape. A value of p < 0.05 was set as the cut-off for significance.

Validation in the Cancer genome atlas (TCGA) database
The mRNA and lncRNA expression profiles of 171 patients with luminal B breast cancer and 59 normal tissues were downloaded from TCGA database to validate the expression patterns of selected DEmRNAs and DElncRNAs.  Table 2 and  Table 3, respectively. Hierarchical clustering analysis of top 100 10 up-and down-regulated DEmRNAs and DElncRNAs was showed in Fig. 1a and Fig. 1b, respectively. Furthermore, the distribution of DElncRNAs and DEmRNAs on all chromosomes was showed in Fig. 1c.

Validation in TCGA database
With mRNA and lncRNA expression profiles downloaded from TCGA database, the expression patterns of four DEmRNAs, including S100A7, CCL5, MIAT and WT1-AS, were verified. As shown in Fig. 7, compared to normal controls, MIAT was down-regulated in luminal B breast cancer tumor tissues which were inconsistent with our results, while S100A7, CCL5 and WT1-AS were up-regulated in luminal B breast cancer tumor tissues which were consistent with our results.

Discussion
Breast cancer, as the most common non-cutaneous type of cancer, is the leading cause of cancer-related mortality among female globally (19). As luminal-B cancers showed poorer prognosis as compared to luminal-A cancers, we performed this study and identified abundant S100A7 is a member of the S100 protein family, which have been associated with preinvasive ductal carcinoma in situ (DCIS) (20). During breast tumorigenesis and/or progression, several S100 s, including S100A2, S100A4 and S100A7, exhibit altered expression levels based on molecular analysis of breast tumors (21). Cancemi et al. suggested that S100A7 was involved in critical phases of the breast cancer growth and progression (22). Mayama et al. proposed that S100A7 was linked to an aggressive phenotype of ER-positive breast carcinoma, and was potent marker for distant metastasis of ER-positive breast cancer patients (23). In current study, S100A7 was the most significant up-regulated DEmRNAs in luminal B breast cancer tumor tissues, which may indicated that S100A7 exert momentous roles in luminal B breast cancer.
Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is a highly conserved lncRNA, and its overexpression in multiple cancerous tissues has been linked to the proliferation and metastasis of tumor cells. It was first identified as being up-regulated in lung tumors, and a prognostic marker for metastasis and patient survival in non-small cell lung cancer (NSCLC), specifically in early stages of lung adenocarcinoma (24). Subsequently, MALAT1 was shown to be up-regulated in a broad spectrum of tumor types, such as endometrial stromal sarcoma and hepatocellular carcinomas (25,26). Additional, it has been found that MALAT1 gene mutations frequently occurred in luminal-type breast tumors (27). Besides, MALAT1 was one of the top 10 up-regulated DElncRNAs in this study, and co-expressed with S100A7, which emphasized the critical role the MALAT1 in luminal B breast cancer and suggested that MALAT1 may involve in luminal B breast cancer by regulating S100A7.
Chemokines, small-molecular-weight cytokines involved in the physiological control of immune cell migrationare, were reported to perform a crucial function in breast cancer tumorigenesis and progression (19). Recent years, the chemokine C-C motif ligand 5 (CCL5), also known as RANTES, is a member of the CC subfamily, has been associated with aggressive breast cancer (28). Svensson et al. identified CCL2 and CCL5 as two therapeutic targets for estrogen-dependent breast Zhang et al. found that CCL5-mediated Th2 polarization of CD4 + T cells promotes metastasis in luminal breast cancer (31). In our analysis, CCL5 was significant upregulated in luminal B breast cancer tumor tissues, LncRNA myocardial infarction-associated transcript (MIAT) is primarily expressed in heart and fetal brain tissue (32). Dysregulated MIAT was first reported to correlated with myocardial infarction and involved in cardiac hypertrophy and diabetic cardiomyopathy (32)(33)(34)(35). Recent studies suggested that MIAT promoted gastric cancer growth and metastasis by regulation of miR-141/DDX5 pathway, promoted proliferation and   (39). MIAT was found to be overexpressed in both ER-positive breast cancer tissues and ER-positive breast cancer cell line MCF-7, and play a pivotal role in ER-positive breast cancer cell growth (40). Almnaseer-M et al. suggested that MIAT performs a critical function in breast tumorigenesis (41). MIAT was detected to be one of the top 10 up-regulated DElncRNAs, and coexpressed with CCL5. All these findings suggest that MIAT may involve in luminal B breast cancer by regulating the expression level of CCL5.
The Wilms' tumor 1 (WT1) was first cloned in 1990 as a suppressor in Wilms' tumor, which was located at chromosome 11p13 (42). WT1 gene mutations are linked with a subset of Wilm's tumors, the most common pediatric renal cancer (43). Substantial evidence has linked WT1 with the pathogenesis of breast cancer. WT1 is linked to the progression of breast cancer, including migration, invasion and angiogenesis. Knockdown of WT1 was demonstrated to lead to mitochondrial damage and then inhibit malignant cell growth (44). Highly expressed WT1 was linked to poor prognosis of patients with breast cancer (45). Over expression of WT1 was detected in TNBC (46). In agreement with previous studies, the expression of WT1 was observed significant up-regulated in luminal B breast cancer tumor tissues in present study.
Wilms tumor 1 Antisense RNA (WT1-AS) is located upstream of the WT1 gene, and these two genes are bidirectionally transcribed from the same promoter region. Down-regulation of WT1-AS was related to a poorer prognosis in ovarian clear cell adenocarcinoma (47). Lv et al. suggested that WT1-AS promoted cell apoptosis in hepatocellular carcinoma (HCC) and may function as a tumor suppressor in HCC (48). It is reported that WT1-AS was significantly down-regulated in gastric cancers and may correlates with tumor progression (49). In addition, WT1-AS was detected to be was underexpressed in cervical carcinoma and suppress cervical cancer cell growth and aggressiveness (50,51). In the current study, we found that WT1-AS was a DElncRNA and WT1 was a nearby-targeted DEmRNA of WT1-AS, which reminded us to explore the role of WT1-AS-WT1 in luminal B breast cancer.

Conclusion
In conclusion, a total of 1178 DEmRNAs and 273 DElncRNAs between luminal-B breast cancer tumor tissues and adjacent tissues were obtained. We discussed and emphasized the importance role of three DElncRNA-DEmRNA pairs, including MALAT1-S100A7, MIAT-CCL5 and WT1-AS-WT1, involved in luminal B breast cancer, which expected to provide new insight into understanding the mechanism underlying pathogenesis of luminal B breast cancer. The small sample size was a limitation of our study. Although the validation results in TCGA database indicated that our RNA-sequencing results were generally reliable, larger cohorts of patients and further experimental validation studies are needed to conduct to verify this conclusion.