LncRNA EPB41L4A-AS1 Regulates Cell Proliferation, Apoptosis and Metastasis in Breast Cancer

Background: Breast cancer is a leading global public health problem. In our previous study, we identied that lncRNA EPB41L4A antisense RNA 1 (EPB41L4A-AS1) was signicantly downregulated in breast cancer. However, the functional role of EPB41L4A-AS1 in breast cancer has not been claried. Here, we further conrmed the expression and biological function of EPB41L4A-AS1 in breast cancer. Methods: To demonstrate the role of EPB41L4A-AS1 in breast cancer, we transfected breast cancer lines with pcDNA3.1-EPB41L4A-AS1 expression vector to induce ectopic overexpression of EPB41L4A-AS1. Then, to explore the role of EPB41L4A-AS1 overexpression in breast cancer cell growth, cell cycle, apoptosis, invasion and migration capacity, we performed CCK-8 assay, colony formation assay, ow cytometry analysis, wound recovery and transwell assay, respectively. We also constructed a co-expression network to explore the potential effect mechanism of EPB41L4A-AS1. Results: Our research showed EPB41L4A-AS1 expression was signicantly lower in tumor tissues than in adjacent non-cancerous tissues. overexpression of EPB41L4A-AS1 signicantly reduced the proliferation of breast cancer cells. Flow cytometric analysis showed that forced expression of EPB41L4A-AS1 signicantly increased the apoptosis rate of breast cancer cells. In addition, we found that upregulated EPB41L4A-AS1 signicantly inhibited the migration and invasive ability of breast cancer. Functional analysis of co-expressed mRNAs suggested that EPB41L4A-AS1 may be involved in ribosomal, cell cycle, spliceosomal and p53 signaling pathways. Conclusions: Our ndings suggest that EPB41L4A-AS1 is a tumor suppressor gene in breast cancer. co-expression networks on the preliminary sequencing function analysis of co-expression shown participated in Cell cycle, Spliceosome, p53 signaling pathway, etc. We will investigate the correlation of with these pathways in subsequent experiments to clarify the role and of EPB41L4A-AS1 in breast cancer.λ


Background
Breast cancer is a leading global public healthcare problem, having been second cause of leading death in US. New breast cancer accounts for 30% in all women cancer diagnoses 1 . Breast cancer is also a leading cancer cause of mortality in both low-and middle-income nations 2 . Despite the continuous development of new drugs to treat breast cancer, the outcome of breast cancer treatment remains to be improved. In addition, the treatment outcome of breast cancer patients with similar stage grading varies greatly. Therefore, to explore the deeper mechanism and nd effective molecular markers of breast cancer are signi cant.
Long non-coding RNAs (lncRNAs) are a category of transcripts that are lengths longer than 200bp and lack the capacity to encode proteins 3 . A growing number of studies have shown that lncRNAs are involved in a wide variety of bio-processes, including epigenetics, translational, post-transcriptional and translation 4 . Deregulation of lncRNAs has also been demonstrated to be involved in the development and progression of breast cancer 5,6 . Identi cation of cancer-related lncRNAs and investigation of the biological functions of them can provide more insights into the occurrence and progression of cancer.
In our previous study, we explored the pattern of lncRNAs expression in breast cancer utilizing RNA sequencing [7][8][9] . We discovered numerous differentially expressed lncRNAs among breast cancer and nontumorigenic breast tissue, among which a lncRNA, EPB41L4A antisense RNA 1 (EPB41L4A-AS1), was remarkably down-regulated in breast cancer. Nevertheless, the functional role of EPB41L4A-AS1 in breast cancer has not been clari ed. Herein, we further con rmed the expression and the biological functions of EPB41L4A-AS1 in breast cancer.

Tissue specimens and ethics statement
The date of EPB41L4A-AS1 expression levels of breast cancer was download from The Cancer Genome Atlas date portal(TCGA). To validate whether EPB41L4A-AS1 was a lncRNA, the Coding Potential Calculator 2 Tool (CPC2, http://cpc2.cbi.pku.edu.cn.) was employed to predict its protein-coding potential 10 . All breast cancer tissue specimens and paired was collected from the Department of Breast Surgery of the First A liated Hospital of Wenzhou Medical University. After surgical resection, the tissues were immediately transferred to Liquid nitrogen tank and stored at the − 80℃ refrigerator. All processes were authorized by the Ethics Committee of the First A liated Hospital of Wenzhou Medical University.

Cell culture
The breast cancer cell lines (MCF-7, MDA-MB-231) were acquired from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). The MDA-MB-231 cells were cultured in RPMI 1640 medium (Invitrogen, USA), while the MCF-7 cells were cultured in DMEM medium (Invitrogen, USA).
All cells were cultured at 37°C and 5% CO2 and nourished by the medium containing 10% FBS (Gibco, Grand Island, NY, USA), as well as 100 UI/ml penicillin with 100 µg/ml of streptomycin, mixed and kept at 4°C. The experiments consisted of three groups. The group design consisted of a non-transfected blank control group (Blank group), an empty vector negative control group (NC group) and EPB41L4A-AS1 overexpression plasmid transfection group (Overexpression group).

Real-Time Polymerase Chain Reaction (qRT-PCR)
Extract RNA from cells and tissues according to the manufacturer's instructions using TRIzol reagent (Invitrogen, USA). Then, quantitative RNA was transcribed into cDNA, using Rever Tra Ace qPCR RT Kit Transfection EPB41L4A-AS1 overexpression plasmid (pcDNA3.1-EPB41L4A-AS1) and a control vector were designed and constructed. The cloned plasmids were transfected into cells according to the manufacturer's instructions using Lipofectamine 2000 (Invitrogen) in Opti-MEM solution (Gibco). The negative control group and the blank group were transfected for the empty vector and without vector, but the same amount of Lipofectamine and Opti-MEM was used. The EPB41L4A-AS1 expression in cells after transfected by plasmids were con rmed by qPCR.

Colony forming assay
Transfected cells were seeded into 6-well plate, include 1500 cells each well, incubated at 37℃ with 5% CO2. Observing its growth state, harvesting when its cluster include more than 50 cells probably (about 7-10 days). At that time, colonies were xed with 4% Paraformaldehyde Fix Solution and stained with 0.01% crystal violet solution.

Cell cycle distribution assay
The transfected cells were collected and washed for three times with PBS. One milliliter of 70% ethanol was used to resuspend cells with soft stirring. The cells were stained with 300 µl of propidium iodide (PI), and then analyzed by Flow Cytometer.

Apoptosis detection analysis
Total cells were collected include free in medium and adhere in bottom. Centrifuging collected cells at 1000 rpm for 5 min and washing with 3ml PBS at 1000 rpm for 5 min, repeated three times. Finally, Using Annexin-V-FITC apoptosis detection kit according to the manufacturer's instructions and analyzing by Flow Cytometer.

Transwell assay
The cells transfected and incubated in 6-well plate were trypsinized with Trypsin-EDTA Solution and ceased by 10%FBS DMEM. 3*10 4 for MCF-7 or MDA-MB-231 resuspended in serum-free medium(300µl)were transferred into the upper chamber of transwell, while the bottom chamber was lled with RPMI DMEM culture medium include 20%FBS , then put into incubator about 24h. After24h, cells adhered to the upper surface were cleaned and then, xed with 4% Paraformaldehyde Fix Solution for 15min, obliterated the exceptional cells with PBS, and at last, stained for 15 min with 0.01% crystal violet solution. The pictures were collected by photomicroscope.
Gene ontology (GO) analysis and pathway analysis GO analysis was performed to de ne the biology of EPB41L4A-AS1. GO annotations were obtained by downloading from NCBI and GO databases 11 . Pathway analysis was used to identify pathways signi cantly enriched for EPB41L4A-AS1 according to the Kyoto Encyclopedia of Genes and Genomes (KEGG) database 12 . Fisher's exact test was applied to identify signi cant GO categories and to select signi cant pathways.

Statistical analysis
Statistical analysis was established using SPSS 23.0 statistical software packages (SPSS, Inc., Chicago, IL, USA). Statistical signi cance between groups was analyzed using student's t-test or a one-way ANOVA. P < 0.05 were considered statistically signi cant.

EPB41L4A-AS1 was a down-regulated lncRNA in breast cancer
In the sequencing cohort, we identi ed that the expression of lncRNA EPB41L4A-AS1 was markedly lower in breast cancer tissues than in adjacent normal tissues (fold change = 0.3, P < 0.001, Fig. 1A). To further verify whether EPB41L4A-AS1 was a lncRNA, we adopted the Coding Potential Calculator 2 tool to predict its protein coding potential because of its high predictive accuracy 10 . The result showed that the sequence of EPB41L4A-AS1 had low coding probability which means it was a bona de lncRNA (Fig. 1B). The classical of lncRNA HOTAIR and the classical of mRNA GAPDH were used as negative and positive controls, separately. To further validate the dysregulation of EPB41L4A-AS1, the expression of EPB41L4A-AS1 in 24 pairs of breast cancer tissues and adjacent non-cancerous tissues was measured by qRT-PCR.
As demonstrated in Fig. 1C, the expression of EPB41L4A-AS1 was remarkably lower in tumor tissues than in adjacent non-cancerous tissues (P < 0.001, Fig. 1C). In additional, we verify the deregulation of EPB41L4A-AS1 in The Cancer Genome Atlas (TCGA) database, which contained 837 breast cancer and 105 non-tumor tissues. Consistently, the expression of EPB41L4A-AS1 also showed a signi cant reduction in the TCGA cohort in breast cancer tissues (P < 0.001, Fig. 1D). Based on the RNAfold web server tool, we predicted secondary structure of EPB41L4A-AS1 13 . As showed in Fig. 1E, the free energy of the thermodynamic ensemble is -440.63 kcal/mol which suggested EPB41L4A-AS1 owned a highly structural stability. In conclusion, these results implied that EPB41L4A-AS1 was a down-regulated lncRNA and may exert a tumor suppressor role in breast cancer.
In uence of EPB41L4A-AS1 on the cell proliferation To demonstrate the effect of EPB41L4A-AS1 in breast cancer, we transfected breast cancer lines with pcDNA3.1-EPB41L4A-AS1 expression vector to induce ectopic overexpression of EPB41L4A-AS1. The post-transfection expression level of EPB41L4A-AS1 was signi cantly increased in the cell lines. (P < 0.001, Fig. 2A). The negative control group and the blank group were transfected for the empty vector and without vector. CCK-8 assays suggested EPB41L4A-AS1 overexpression signi cantly reduced the growth rate of both cell lines (P < 0.001, Fig. 2B). Colony formation assays also indicated that EPB41L4A-AS1 overexpression greatly impaired the number of colonies in both MDA-MB-231 cells and MCF-7 cells breast cancer cells (Fig. 2C).
Enforced expression of EPB41L4A-AS1 promoted breast cancer cells apoptosis To investigate whether the EPB41L4A-AS1 effect on breast cancer cell proliferation re ects cell cycle arrest further, we have examined the cell cycling progression by ow cytometry analysis. The results showed that there was no effect on cell cycle in breast cancer cells transfected with EPB41L4A-AS1 vector (Fig. 3A). To determine if the proliferation of breast cancer cells was in uenced by apoptosis, we conducted a ow cytometric analysis. Flow cytometry analysis showed that enforced expression of EPB41L4A-AS1 remarkably increased the apoptotic rate of MCF-7 cells as well as MDA-MB-231 cells (Fig. 3B). In conclusion, these results suggest that EPB41L4A-AS1 plays a key role in apoptosis of breast cancer cells.

EPB41L4A-AS1 regulated migration and invasion of breast cancer cells
The degree of malignancy was associated not only with proliferation, but also migration and invasion. Wound recovery was signi cantly delayed when overexpression of EPB41L4A-AS1 compared with control groups in MDA-MB-231cells (P < 0.01, Fig. 4A). To con rm further the role for EPB41L4A-AS1 in breast cancer, we next examined the migration and invasion functions by transwell assay. The results revealed that the upregulated EPB41L4A-AS1 signi cantly suppressed the migration and invasive ability of breast cancer cells as compared to the negative control group and blank control group (P < 0.001, Fig. 4B and 4C).
Potential effect mechanism of EPB41L4A-AS1 To further clarify the mechanism by which EPB41L4A-AS1 exerted its biological function, we screened 118 mRNAs whose expression levels were highly correlated with EPB41L4A-AS1 based on the preliminary RNA sequencing data. Then, function analysis of co-expression mRNAs of EPB41L4A-AS1 was shown in Table 1. The results of GO analysis suggested that the co-expressed mRNAs were participating in biological processes such as translation initiation, SRP-dependent co-translation, and viral transcription. The pathway analysis, on the other hand, revealed that these co-expressed mRNAs were involved in multiple signaling pathways involving ribosome, cell cycle, replisome, and p53 signaling pathways. Collectively, our analysis might partly explain its effect mechanism.

Discussion
The latest research shows that breast cancer is still the most common malignant tumor in women 14,15 . In recent years, the treatment options for breast cancer have become more and more abundant, just like preoperative neoadjuvant chemotherapy, endocrine therapy, and targeted drug 16, 17 . Rates of long-term survival of breast cancer patients have grown steadily. All of above can be attributed to unremittingly explore the potential mechanism of pathogenesis. Nevertheless, breast cancer differs in its natural history and response to treatment 18 . Genomic variations are the major cause of breast cancer biodiversity.
Hence, there is an urgent need to generate candidate biomarkers in order to stratify patients and personalize treatment to avoid excessive or inadequate treatment.
With the emergence of investigations associated with lncRNAs, it has been found that lncRNAs play signi cant contributions to the process of tumor initiation, progression, metastasis and recurrence 19 .
Earlier studies have already revealed that the overexpression of LINK-A lncRNA boosted cell growth, inhibited apoptosis, and upregulated survivin expression, while knockdown of LINK-A lncRNA had the reverse effect in triple-negative breast cancer 20 . In addition, other scholars have previously reported that the silencing of LOC101060264 suppressed tumor proliferation in nude mice in vivo and suppressed the migration, invasion and proliferation of colon cancer cells in vitro 21 . Furthermore, lncRNAs are also quite prospering for clinical applications in oncology. For example, lncRNA-D16366 has been shown to be a potential biomarker with high diagnostic and prognostic value for the diagnosis of hepatocellular EPB41L4A-AS1 is located in the 5q22.2 region of the genome, which is strongly linked to oncogenesis due to frequent DNA fragment deletions. Previous investigations have indicated that EPB41L4A-AS1 regulates glycolysis and glutaminolysis as well as inhibited tumor cell proliferation 23,24 . Jie Bin et al. had reported that EPB41L4A-AS1 functions as an oncogene by regulating the Rho/ROCK pathway in colorectal cancer 25 . The role of EPB41L4A-AS1 in the development and progression of breast cancer, however, remains unknown. In this article, we examined EPB41L4A-AS1 expression in breast cancer tissues and demonstrated the function of EPB41L4A-AS1 in breast cancer. To our knowledge, the present study identi ed for the rst time that EPB41L4A is involved in breast cancer. The precise mechanisms of EPB41L4A-AS1 remain unknown. Previous studies have covered the extensive existence of sensenonsense transcripts in mammalian cells and noted that antisense RNA perturbation can alter the expression of sense genes 26 . However, our study showed that the expression level of EPB41L4A was not signi cantly altered after overexpression of EPB41L4A-AS1 (data not shown in the results). To explore the mechanisms of EPB41L4A-AS1, we constructed co-expression networks based on the preliminary RNA sequencing data. The function analysis of co-expression mRNAs had shown that EPB41L4A-AS1 might participated in Ribosome, Cell cycle, Spliceosome, p53 signaling pathway, etc. We will investigate the correlation of EPB41L4A-AS1 with these pathways in subsequent experiments to clarify the role and mechanism of EPB41L4A-AS1 in breast cancer.λ In addition, we also observed a difference in p-value between the two experimental groups

Conclusions
We identi ed a signi cantly decreased expression level of EPB41L4A-AS1 in breast cancer and demonstrated enforced expression of EPB41L4A-AS1 promoted breast cancer cells apoptosis as well as inhibition of the proliferation, migration and invasion.