LINC00514 upregulates CCDC71L to Promote Cellular Process in Triple-Negative Breast Cancer by Sponging miR-6504-5p and miR-3139


 Background: 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 elusive in triple-negative breast cancer (TNBC). Methods: Herein, we detected LINC00514 expression level in TNBC tissues and cells via RT-qPCR. The function of LINC00514 in TNBC cellular activities was assessed via colony formation, EdU, wound healing, transwell assays and flow cytometry analysis. Results: The binding between miR-6504-5p/miR-3139 and LINC00514/CCDC71L was validated by luciferase reporter assay. The data indicated that LINC00514 expression was increased in TNBC tissues and cells. Furthermore, it was manifested that silenced LINC00514 restrained cell proliferative, migratory and invasive abilities and accelerated 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 discovered in TNBC tissues and cells. Accordingly, we discovered overexpression of miR-6504-5p or miR-3139 retarded cell growth and migration in TNBC. Later, CCDC71L was recognized as a common downstream gene of miR-6504-5p and miR-3139. Rescue assay verified that overexpressed CCDC71L countervailed the inhibitive influence of LINC00514 knockdown in TNBC cellular process. Conclusion: MiR-6504-5p and miR-3139 were involved in LINC00514-mediated cellular activities through regulating CCDC71L expression, which provided a novel LINC00514/miR-6504-5p/miR-3139/CCDC71L axis in TNBC.


Introduction
Breast cancer (BC) has been commonly acknowledged as a predominant type of cancer among women globally (1). It has various subtypes with multiple clinical outcomes and diverse biological behaviors because of the heterogeneous property (2). Among which, triple-negative breast cancer (TNBC) was an aggressive subtype losing Her2 ampli cation, progesterone receptor and estrogen receptor, and accounted for about 10-25% of total BC cases (3,4). Additionally, TNBC patients presented worse clinical outcome, higher incidence and higher risk of distant metastasis (5,6). Thus, it was quite urgent to probe the novel biomarkers and potential molecular mechanisms underlying TNBC progression.
As a group of RNAs, long noncoding RNA (lncRNA) is lack of protein translation abilities and comprises over 200 nucleotides (7). Abnormally expressed lncRNAs were commonly discovered in multiple cancer types and their dysregulation were widely reported in numerous cellular activities, like cancer initiation, apoptosis, migration and metastasis (8). Formerly, lncRNA SNHG1 was reported to highly express in nonsmall-cell lung cancer and aggravated proliferative and invasive capacities of cells (9). Downregulation of BLACAT1 suppressed cell proliferation and arrested 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 was identi ed as oncogenic gene by promoting cell proliferation in TNBC (13). LncRNA linc-ZNF469-3 was upregulated and enhanced lung metastasis in TNBC (14). Therefore, looking for the new biomarkers from lncRNAs could be a prospective treatment choice for the therapy of TNBC.
It was well known that lncRNAs acted 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) was a newly identi ed lncRNA, which could exert 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 were highly unclear. In this study, we high expression of LINC00514 was found in TNBC tissues and cells, and LINC00514 played oncogenic role in TNBC via miR-6504-5p/miR-3139/CCDC71L axis.

Materials And Methods
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 suffered radiotherapy or chemotherapy. After collecting, each sample was instantly preserved at -80°C in liquid nitrogen for further use. The use of tissues was

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

Colony formation assay
Transfected TNBC cells were planted to 6-well plates, and each well was lled with 5 × 10 3 cells. After two weeks, cells were xed by 5% paraformaldehyde, and then 0.1% crystal violet solution was supplemented for staining. Finally, colonies (more than 50 cells) were 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 uorescence microscope (Nikon, Japan), visualized images of EdU-positive cells were obtained.
Flow cytometry analysis TNBC cells were planted 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 minutes, apoptotic TNBC cells were identi ed by ow cytometry (BD Biosciences).

Western blot analysis
By RIPA buffer (Thermo Fisher Scienti c), proteins of TNBC cells were lysed, and then protein concentration was con rmed 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 loading 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 lled by 5 × 10 4 cells. Later, sterile pipette tip was used to scratch the wounds. After 24 h, wounds were imaged following washing in 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 xed and stained. Then, an inverted microscope was used for the counting of 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 for conducting an incubation with streptavidin magnetic beads for 4 h at 4 °C. Subsequently, precooled lysis buffer and salt buffer was applied to rinse the beads. With the extraction of pull-down RNAs, the levels of miRNAs binding to LINC00514 were detected.

Statistical analysis
Through 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 mean ± SD. The analysis of correlation between genes was conducted by Spearman's correlation analysis. P less than 0.05 was considered as cut-off value.

Results
LINC00514 was an upregulated lncRNA in TNBC Firstly, we analyzed LINC00514 expression pro le in TNBC tissues and cell lines by RT-qPCR. Compared with corresponding adjacent tissues, LINC00514 expression was signi cantly increased 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-10A cell line (Fig. 1C). Taken together, LINC00514 was highly expressed in TNBC tissues and cell lines.

LINC00514 expedited TNBC cellular process
Considering aberrant LINC00514 expression in TNBC, we then explored its biological function in TNBC via conducting loss-of-function assays. MDA-MB-468 and HCC1937 cells, which presenting higher LINC00514 expression, was used for the investigation. At rst, 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 promoted in LINC00514-silenced TNBC cells through ow 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). According to wound healing assay, cell migration was suppressed by silencing LINC00514 (Fig.   2F). Likewise, LINC00514 knockdown inhibited cell invasion via the result of transwell assay (Fig. 2G).
Overall, LINC00514 accelerated cell proliferation, migration and invasion, and retarded 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 signi cant change was found in that of LINC00514-Mut reporter (Fig. 3F). This revealed that both miR-6504-5p and miR-3139 could interact with LINC00514. Moreover, levels of miR-6504-5p and miR-3139 were validated to be low 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 con rmed 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 cellular process 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 via overexpressed miR-6504-5p or miR-3139. In addition, levels of apoptosis-relevant 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). Through wound healing assay, overexpression of miR-6504-5p or miR-3139 remarkably suppressed cell migratory capability (Fig. 4F). Furthermore, transwell assay con rmed the inhibitive role of miR-6504-5p or miR-3139 overexpression in TNBC cell invasion (Fig. 4G). 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 testi ed. 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 weakened, while that of CCDC71L-Mut reporter remain unchanged (Fig. 5D). Additionally, LINC00514 was veri ed to overexpress in TNBC cells by pcDNA3.1/LINC00514 (Fig. 5E). Characterized by easy transfection, HEK293T cells were used for further conducting luciferase reporter assay. Data indicated that overexpressed LINC00514 counteracted luciferase activity of CCDC71L-WT that was inhibited by overexpressing miR-6504-5p or miR-3139 while CCDC71L-Mut luciferase activity was not affected (Fig. 5F). Then, we con rmed 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). Namely, LINC00514 increased CCDC71L expression via sponging miR-6504-5p and miR-3139 in TNBC.

LINC00514 boosted TNBC cellular activities by upregulating CCDC71L
For further analyzing whether LINC00514 played oncogenic role in TNBC by mediating CCDC71L, some restoration experiments were conducted in MDA-MB-468 and HCC1937 cells. CCDC71L expression was upregulated in TNBC cells (Fig. 6A). As demonstrated, proliferative ability of TNBC cells inhibited by LINC00514 silencing was reversed via overexpressing CCDC71L (Fig. 6B-C). Besides, the elevated cell apoptosis caused by LINC00514 knockdown was counteracted by CCDC71L overexpression (Fig. 6D). Consistently, overexpressed CCDC71L countervailed the effect of LINC00514 de ciency on apoptosisrelated proteins levels (Fig. 6E). Meanwhile, LINC00514 knockdown-mediated inhibition on the migratory ability of TNBC cell was recovered by upregulating CCDC71L (Fig. 6F). At last, upregulated CCDC71L offset the suppressive role of sh-LINC00514 transfection in cell invasion (Fig. 6G). Hence, we validated that LINC00514 expedited cell proliferation, suppressed cell apoptosis, and accelerated cell migration and invasion via increasing CCDC71L expression 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). As an aggressive subtype of BC, the progression of TNBC was attributed to numerous oncogenes and anti-oncogenes (26,27). To promote TNBC therapy e cacies and develop novel TNBC treatments, it was critical to fully understand the molecular events, especially lncRNA (14,28). Previous studies demonstrated that LINC00514 accelerated cell proliferation and invasion in vitro and aggravated tumor growth by targeting miR-204-3p/CDC23 axis in papillary thyroid cancer (19). In osteosarcoma, LINC00514 increased URGCP expression to promote cell cycle and suppress cell apoptosis via sponging miR-708 (20). However, the biological role of LINC00514 was still poorly understood in TNBC. Herein, this study was the rst to reveal that LINC00514 exhibited a high level in TNBC tissues and cells, especially in the tissues of patients at advanced stage.
Loss-of-function assay manifested that LINC00514 knockdown restrained cell proliferation, induced cell apoptosis, weakened cell migration and invasion. These ndings suggested LINC00514 as an oncogenic lncRNA in TNBC.
The mechanisms of lncRNAs have not been fully explored; nevertheless, emerging reports indicated that lncRNA served as ceRNA to release miRNA targets by sequestering miRNA (29). To investigate the underlying regulatory mechanism of LINC00514-mediated cancer development, we conducted in silico studies to research putative miRNA for LINC00514. Through bioinformatics analysis and RNA pull down assay, miR-6504-5p and miR-3139 were predicted to be potential miRNAs. And it was supposed that miR-6504-5p and miR-3139 had the binding sites on LINC00514 sequence. Further, luciferase reporter assay con rmed 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 their expressions were negatively associated with that of LINC00514. Therefore, we supposed that LINC00514 interacted with miR-6504-5p and miR-3139 in TNBC.
As another class of noncoding RNA, miRNAs only contained 20-24 nucleotides and exerted 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 signi cantly inhibited cell proliferation, migration, invasion and contributed to cell apoptosis. This con rmed the anti-oncogenic property of miR-6504-5p and miR-3139 in TNBC. Previously, increasing researched validated that miRNAs could directly bind to mRNA 3′-UTR to post-transcriptionally regulate mRNA translation or degradation (32). Our present study identi ed CCDC71L, a novel mRNA which has not been explored in cancers, as the common target of miR-6504-5p and miR-3139. Importantly, we discovered that the inhibitive effect of silenced LINC00514 on CCDC71L mRNA and protein levels was partially restored by miR-6504-5p inhibition, but fully rescued by co-inhibition of miR-6504-5p and miR-3139. Rescue assays demonstrated that CCDC71L overexpression counteracted LINC00514 silencing-mediated suppression on TNBC cellular process. Therefore, it was suggested that LINC00514 regulate CCDC71L expression via sponging miR-6504-5p and miR-3139.
In conclusion, our study for the rst time uncovered the functional role and molecular mechanism of LINC00514 in TNCB, and discovered that LINC00514 acted as sponge of miR-6504-5p and miR-3139 to increase CCDC71L expression, thereby promoting TNBC cellular process. This nding might be helpful for the further exploration of new TNBC therapy.