LINC00514 upregulates CCDC71L to promote cell proliferation, migration and invasion in triple‐negative breast cancer by sponging miR-6504-5p and miR-3139

Long noncoding RNAs (lncRNAs) have recently identified as essential gene modulators in numerous cancers. Previous studies have confirmed the oncogenic role of long intergenic nonprotein-coding RNA 00514 (LINC00514) in some cancers. Nevertheless, its biological function and mechanism remain unclear in triple-negative breast cancer (TNBC). Herein, we detected LINC00514 expression level in TNBC tissues and cells using RT-qPCR. The function of LINC00514 in TNBC cellular activities was assessed by colony formation, EdU, wound healing, transwell assays and flow cytometry analysis. The binding between miR-6504-5p/miR-3139 and LINC00514/CCDC71L was validated by luciferase reporter assay. The results indicated that LINC00514 expression was upregulated in TNBC tissues and cells. Furthermore, it was manifested that silenced LINC00514 restrained cell proliferative, migratory and invasive abilities and promoted cell apoptosis. In mechanism, LINC00514 was revealed to sequester miR-6504-5p and miR-3139 in TNBC cells. Furthermore, the low level of miR-6504-5p and miR-3139 was identified in TNBC tissues and cells. Overexpression of miR-6504-5p or miR-3139 inhibited cell growth and migration in TNBC. CCDC71L was recognized as a common downstream gene of miR-6504-5p and miR-3139. Rescue assay verified that overexpressed CCDC71L countervailed the anti-tumor influence of LINC00514 knockdown on TNBC cell proliferation, migration, invasion and apoptosis. LINC00514 promote cell proliferation, migration and invasion in triple-negative breast cancer by targeting the miR-6504-5p/miR-3139/CCDC71L axis in TNBC.


Introduction
Breast cancer (BC) has been commonly acknowledged as a cancer prevalent among women globally [1]. It has various subtypes with multiple clinical outcomes and diverse biological behaviors because of the heterogeneous property [2]. Triple-negative breast cancer (TNBC) is an aggressive subtype losing Her2 amplification, progesterone receptor and estrogen receptor, and accounts for about 10-25% of total BC cases [3,4]. Additionally, TNBC patients present worse clinical outcome, higher incidence and higher risk of distant metastasis [5,6]. Thus, it is quite urgent to probe the novel biomarkers and potential molecular mechanisms underlying TNBC progression. Luo and Wang Cancer Cell Int (2021) 21:180 As a group of noncoding RNAs, long noncoding RNA (lncRNA) lacks protein translation abilities and comprises over 200 nucleotides [7]. Abnormally expressed lncRNAs are commonly discovered in multiple cancer types and their dysregulation is widely reported in numerous cellular activities, such as cancer initiation, apoptosis, migration and metastasis [8]. LncRNA SNHG1 is highly expressed in non-small-cell lung cancer and aggravates proliferative and invasive capacities of cells [9]. Downregulation of BLACAT1 suppresses cell proliferation and arrests cell cycle at G0/G1 phase in cervical cancer [10]. Besides, some lncRNAs have been recognized to play critical roles in TNBC [11,12]. For example, lncRNA MIR100HG is identified as oncogenic gene that promotes cell proliferation in TNBC [13]. LncRNA linc-ZNF469-3 is upregulated and enhanced lung metastasis in TNBC [14]. Therefore, exploration for the new biomarkers from lncRNAs may provide a promising choice for the therapy of TNBC.
LncRNAs have been revealed to act as biological regulators through a variety of mechanisms [15], such as regulating alternative splicing [16], modifying chromosome [17], and sponging microRNA (miRNA) [18]. Long intergenic nonprotein-coding RNA 00514 (LINC00514) is a newly identified lncRNA, which exerts functions in cancers. Nowadays, great attention has been attracted on LINC00514 due to its critical role in the malignancy of papillary thyroid cancer [19] and the progression of osteosarcoma [20]. Nevertheless, the detail function and regulatory mechanism of LINC00514 in TNBC remain unclear. In this study, the high expression of LINC00514 was identified in TNBC tissues and cells, and LINC00514 played oncogenic role in TNBC via miR-6504-5p/miR-3139/CCDC71L axis.

Tissue samples
TNBC samples and corresponding adjacent samples were collected from fifty-two TNBC patients at China-Japan Union Hospital of Jilin University (Jilin, China). Informed consents were signed by each patient. Prior to operation, no patients underwent radiotherapy or chemotherapy. After collecting, the samples were instantly preserved at − 80 °C in liquid nitrogen for further use. The use of tissues has obtained the approval from the Ethical Review Board of China-Japan Union Hospital of Jilin University (Jilin, China).

RT-qPCR
The extraction of total RNAs from tissues and cells was conducted by TRIzol reagent (Invitrogen) with a Recov-erAll ™ Total Nucleic Acid Isolation kit (Ambion). Reverse transcription reactions were performed using a Prime Script ™ RT reagent kit (Takara, Dalian, China). Later, RT-qPCR was performed with SYBR Premix Ex Taq (Takara Bio, Shiga, Japan) on StepOnePlus System (Applied Biosystems). Based on the 2 −ΔΔCT method, gene expressions were calculated with normalization to GAPDH or U6.

Colony formation assay
Transfected TNBC cells were seeded to 6-well plates, and each well was filled with 5 × 10 3 cells. After 2 weeks, cells were fixed by 5% paraformaldehyde, and then 0.1% crystal violet solution was used for staining. Finally, the number of colonies (more than 50 cells) was counted and recorded.

EdU assay
For the measurement of cell proliferation, Edu assay kit (RiboBio, China) was used. In brief, TNBC cells in each group with treatment of EdU were stained by DAPI. Under fluorescence microscope (Nikon, Japan), visualized images of EdU-positive cells were obtained.

Flow cytometry analysis
TNBC cells were seeded in 6-well plates and then rinsed in PBS. Later, 1 µL of PI (Invitrogen) and 2.5 µL Annexin V conjugated with FITC were added into binding buffer. Subsequently, the binding buffer was used to resuspend the cells after trypsinization. After 15 min, apoptotic TNBC cells were measured by flow cytometry (BD Biosciences).

Western blot analysis
Proteins in TNBC cells were extracted using RIPA buffer (Thermo Fisher Scientific), and then protein concentration was confirmed by BCA-kit (Beyotime, Shanghai, China). Separated by SDS-PAGE, proteins were transferred to PVDF membranes. Then, the membranes were blocked in 5% skim milk and incubated with primary antibodies (Abcam, Cambridge, MA) overnight at 4 °C. Washed by 0.1% TBST in triple, the membranes were incubated with secondary antibody at 37 °C for an hour. The internal control was GAPDH. The results were analyzed and visualized by ECL detection reagent (GE Healthcare, Chicago, IL).

Wound healing assay
To make cells adhere, TNBC cells were cultured in 96-well plates all night with each well filled by 5 × 10 4 cells. Later, sterile pipette tip was used to scratch the wounds. After 24 h, wounds were imaged following washing with PBS.

Transwell invasion assay
Transfected TNBC cells were reaped and placed into the upper transwell chamber (8 μm pores) coated with Matrigel (BD Biosciences). The serum-free medium was placed in the upper chambers, while the lower chamber was supplemented with medium containing 10% FBS. Incubated for 24 h, cells were fixed and stained. Then, an inverted microscope was used to count invasive cells.

Subcellular fractionation assay
PARIS Kit (Invitrogen) was used to isolated nuclear and cytoplasmic RNA in TNBC cells. Then, the extraction of subcellular fractions was carried out. Later, the fractions were subjected to RT-qPCR with normalization to GAPDH (cytoplasm control) or U6 (nucleus control).

RNA pull down assay
LINC00514 and NC-lncRNA labeled with biotin were transfected into MDA-MB-468 and HCC1937 cells. The lysates of TNBC cells were used to incubate with streptavidin magnetic beads for 4 h at 4 °C. Subsequently, precooled lysis buffer and salt buffer was used to rinse the beads. With the extraction of pull-down RNAs, the levels of miRNAs binding to LINC00514 were detected.

Statistical analysis
The statistical analysis was conducted using GraphPad Prism 6 (GraphPad). Data comparison between or over two groups were statistically analyzed by Student's t test or one-way ANOVA. Three biological repeats were included in all experimental procedures with results presenting as the mean ± SD. The analysis of correlation between genes was conducted by Spearman's correlation analysis. The value of p less than 0.05 was considered as cut-off value.

LINC00514 was an upregulated lncRNA in TNBC
First, we analyzed LINC00514 expression profile in TNBC tissues and cell lines by RT-qPCR. Compared with corresponding adjacent tissues, LINC00514 expression was significantly upregulated in TNBC tissues (Fig. 1a). Importantly, LINC00514 expression was higher in patients at advanced stage (III-IV stage) than that in early stage (I-II stage) (Fig. 1b). In addition, upregulation of LINC00514 was found in TNBC cell lines with comparison of MCF-10 A cell line (Fig. 1c). Taken together, LINC00514 was highly expressed in TNBC tissues and cell lines.

LINC00514 promoted TNBC cell proliferation, migration, invasion and inhibited cell apoptosis
Considering aberrant LINC00514 expression in TNBC, first we accessed the effect of LINC00514 overexpression in TNBC on MCF10A cells. The result of colony formation assay showed that LINC00514 overexpression had no significant influence on the proliferation of MCF10A cells (Additional file 1: Fig. S1 A). We then explored its biological function in TNBC via conducting loss-of-function assays. MDA-MB-468 and HCC1937 cells, which showed higher LINC00514 expression, was used for the investigation. LINC00514 expression was stably silenced by transfecting sh-LINC00514#1/2 in MDA-MB-468 and HCC1937 cells (Fig. 2a). Then, colony formation assay showed that cell proliferation was repressed by LINC00514 downregulation (Fig. 2b). Consistently, the same result was observed in EdU assay (Fig. 2c). However, the apoptosis rate was elevated in LINC00514-silenced TNBC cells through flow cytometry analysis (Fig. 2d). Western blot manifested the upregulation of Bax protein level and the downregulation of Bcl-2 protein level in sh-LINC00514 group (Fig. 2e, f). According to wound healing assay, cell migration was suppressed by silencing LINC00514 (Fig. 2g, h). Likewise, LINC00514 knockdown inhibited cell invasion via the result of transwell assay (Fig. 2i). Overall, LINC00514 accelerated cell proliferation, migration and invasion, and inhibited cell apoptosis in TNBC.
Luciferase reporter assay demonstrated that the luciferase activity of LINC00514-WT reporter was decreased by the overexpression of miR-6504-5p or miR-3139, while no significant change was found in that of LINC00514-Mut reporter (Fig. 3f ). This revealed that both miR-6504-5p and miR-3139 interacted with LINC00514. Moreover, miR-6504-5p and miR-3139 were validated to be downregulated in TNBC tissues (Fig. 3g). Importantly, the expression of LINC00514 was negatively associated with that of miR-6504-5p or miR-3139 (Fig. 3h). Data above confirmed that LINC00514 served as sponge of miR-6504-5p and miR-3139 in TNBC.

MiR-6504-5p and miR-3139 were lowly expressed in TNBC and inhibited cell proliferation, migration, invasion and promoted cell apoptosis
Subsequently, we explored the expression pattern and biological functions of miR-6504-5p and miR-3139 in TNBC cells. Results of RT-qPCR depicted that miR-6504-5p and miR-3139 both expressed at a low level in TNBC cell lines (Fig. 4a). Subsequently, some functional assays were carried out to identify functional role of miR-6504-5p and miR-3139 in TNBC cell growth and migration. Based on results of colony formation and EdU assays, we found miR-6504-5p mimics and miR-3139 mimics inhibited cell proliferation (Fig. 4b, c). As shown in Fig. 4d, the apoptosis of TNBC cells was enhanced by overexpressed miR-6504-5p or miR-3139. In addition, levels of apoptosisrelevant proteins (Bax and Bl-2) in cells with transfection of miR-6504-5p mimics or miR-3139 mimics were tested. The results indicated that upregulation of miR-6504-5p or miR-3139 increased Bax protein level and reduced Bcl-2 protein level (Fig. 4e, f). Through wound healing assay, overexpression of miR-6504-5p or miR-3139 remarkably suppressed cell migratory capability (Fig. 4g, h). Furthermore, transwell assay confirmed the inhibitive role of miR-6504-5p or miR-3139 overexpression in TNBC cell invasion (Fig. 4i). Conclusively, miR-6504-5p and miR-3139 retarded cell growth and migration in TNBC.

CCDC71L was a common target of miR-6504-5p and miR-3139
To further support ceRNA hypothesis, the downstream genes of miR-6504-5p and miR-3139 were explored. By using starBase, two potential mRNAs were found (Fig. 5a). Then, the expression of these two genes in TNBC cells were testified. As observed, CCDC71L was highly expressed in TNBC cells, while that of AGO1 did not exhibit expression difference (Fig. 5b). Subsequently, we found the binding site between CCDC71L and miR-6504-5p or miR-3139 to construct CCDC71L-WT, and mutated the sites to construct CCDC71L-Mut (Fig. 5c). After miR-6504-5p mimics or miR-3139 mimics transfection, the luciferase activity of CCDC71L-WT reporter was considerably decreased, while that of CCDC71L-Mut reporter remained unchanged (Fig. 5d). Additionally, LINC00514 was verified to be overexpressed in TNBC cells by pcDNA3.1/LINC00514 (Fig. 5e). Characterized by easy transfection, HEK293T cells were used for luciferase reporter assay. Data indicated that overexpressed LINC00514 counteracted the decrease in luciferase activity of CCDC71L-WT induced by overexpressed miR-6504-5p or miR-3139 while CCDC71L-Mut luciferase activity was not affected (Fig. 5f ). Then, we confirmed that miR-6504-5p inhibitor and miR-3139 inhibitor apparently decreased miR-6504-5p and miR-3139 expression, separately, in TNBC cells (Fig. 5g). At last, we found that CCDC71L mRNA and protein levels decreased by silenced LINC00514 were partially restored by inhibiting miR-6504-5p, and the co-transfection of miR-6504-5p inhibitor and miR-3139 inhibitor nearly fully reserved the function of LINC00514 knockdown (Fig. 5h, i). Overall, LINC00514 increased CCDC71L expression via sponging miR-6504-5p and miR-3139 in TNBC.

Discussion
In recent decades, extensive documents have strongly supported the participation of lncRNAs in the pathogenesis and development of human cancers [23][24][25]. The progression of TNBC was attributed to numerous oncogenes and anti-oncogenes [26,27]. To promote TNBC therapy efficacies and develop novel TNBC treatments, it is critical to fully understand the molecular events, especially lncRNAs [14,28]. Previous studies have demonstrated that LINC00514 accelerates cell proliferation and invasion in vitro and aggravates tumor growth by targeting the miR-204-3p/CDC23 axis in papillary thyroid cancer [19]. In osteosarcoma, LINC00514 increases URGCP expression to promote cell cycle and suppress cell apoptosis via sponging miR-708 [20]. However, the biological role of LINC00514 in TNBC remains unknown. Herein, this study was the first to reveal that LINC00514 exhibited a high level in TNBC tissues and cells, especially in the tissues of patients at advanced stage. Loss-offunction assay revealed that LINC00514 knockdown inhibited cell proliferation, induced cell apoptosis, suppressed cell migration and invasion, while in the human normal breast epithelial cell MCF-10 A, LINC00514 showed overexpression showed no evident influence on cell proliferation in TNBC. These findings suggested that LINC00514 served as an oncogenic lncRNA in TNBC. Increasing reports indicated that lncRNAs serve as ceRNA to release miRNA targets by sequestering miR-NAs [29]. To investigate the underlying regulatory mechanism of LINC00514-mediated cancer development, we conducted in silico studies to research putative miR-NAs for LINC00514. Through bioinformatics analysis and RNA pull down assay, miR-6504-5p and miR-3139 were predicted to be potential miRNAs binding with LINC00514. It was supposed that miR-6504-5p and miR-3139 have the binding sites on LINC00514 sequences. Further, luciferase reporter assay confirmed the binding of LINC00514 to miR-6504-5p and miR-3139. In addition, we found that miR-6504-5p and miR-3139 were both lowly expressed in TNBC tissues and cells and their expression levels were negatively associated with that of LINC00514 in TNBC tissues. Therefore, we supposed that LINC00514 interacted with miR-6504-5p and miR-3139 in TNBC.
As another class of noncoding RNA, miRNAs only contain 20-24 nucleotides and exert critical effects on tumorigenesis and progression [30,31]. MiR-6504-5p and miR-3139 are novel miRNAs whose functions and mechanism have not been depicted in cancers, especially in TNBC. In this study, miR-6504-5p and miR-3139 were both found to be downregulated in TNBC cell lines, and overexpression of miR-6504-5p or miR-3139 significantly inhibited cell proliferation, migration, invasion and contributed to cell apoptosis in TNBC. This confirmed the anti-oncogenic property of miR-6504-5p and miR-3139 in TNBC. Previously, increasing studies validated that miRNAs can directly bind to mRNA 3′-UTR to post-transcriptionally regulate mRNA translation or degradation [32]. Our present study identified CCDC71L, a novel mRNA that has not been explored in cancers, as the target of miR-6504-5p and miR-3139. CCDC71L was revealed to be highly expressed in TNBC cell lines. The binding between CCDC71L and miR-6504-5p and miR-3139 was verified by the luciferase reporter assay. MiR-6504-5p and miR-3139 target the 3'UTR of CCDC71L to suppress its expression. Importantly, we discovered that the inhibitive effect of silenced LINC00514 on CCDC71L mRNA and protein levels was partially restored by miR-6504-5p inhibition, and fully rescued by co-inhibition of miR-6504-5p and miR-3139. Rescue assays demonstrated that CCDC71L overexpression or miR-6504-5p and miR-3139 co-inhibition counteracted LINC00514 silencing-mediated suppression on TNBC cell proliferation, migration and invasion. Therefore, it was suggested that LINC00514 regulate CCDC71L expression via sponging miR-6504-5p and miR-3139.
LINC00514 is of clinical value as a promising therapeutic target of TNBC. The synthesized sh-LINC00514 provides a treatment option for TNBC patients at advanced stage. However, the relationship between LINC00514 and the prognosis in TNBC patients remains unclear. In the future, more studies need to be conducted to explore the association between the level of LINC00514 and TNBC prognosis.
In conclusion, our study for the first time revealed the functional role and molecular mechanism of LINC00514 in TNCB, and discovered that LINC00514