Knock Down of LINC00504 Represses Proliferation and Invasion via Regulation of miR-140-5p in Breast Cancer

16 Background: Breast cancer is one of the most common cancer in the world. Emerging evidence 17 has demonstrated the critical role of long noncoding RNAs (lncRNAs) in the development of breast 18 cancer. In this study, we aimed to investigate the role of LINC00504 in breast cancer progression. 19 Methods: Quantification real-time PCR was used to analyzed the expression levels of LINC00504 20 and miR‐140-5p in breast cancer tissues and cell lines. Cell proliferation, migration and invasion 21 were assessed by Cell Counting Kit‐8, transwell assay and Immunofluorescence. Dual-luciferase 22


Background
Breast cancer is the most common type of gynecological tumor in China [1].Despite efforts by diagnostic techniques and patient management, there has been little progress in improving the overall survival of breast carcinoma patients.In addition, it has been reported that there is an apparent trend in incidence and mortality rates of breast cancer [2,3].Development of suitable therapy to increase patient survival rate has been limited because the pathophysiological mechanisms contributing to breast carcinoma are largely unknown [4].Therefore, uncovering the molecular mechanisms for development and progression of breast carcinoma is necessary for developing effective therapies.
The long noncoding RNAs (lncRNAs) are a new category of noncoding RNAs with over 200 nucleotides and are deficient in protein coding ability [5][6][7].More and more evidence has shown that LncRNAs play important role in various human cancers, including breast cancer [8], liver cancer [9], gastric cancer [10] and so on.LncRNAs have been linked to every stage of cell life, including cell proliferation, differentiation, apoptosis, and motility.For instance, Knockdown of Long Noncoding RNA GHET1 Inhibits Cell Proliferation and Invasion of Colorectal Cancer [11].
Therefore, identifying the mechanism of regulation of LncRNA is essential for tumor diagnosis and therapy.A long noncoding RNA，called LINC00504，is a newly identified lncRNA.Feng J et al firstly reported that A noncoding RNA LINC00504 interacts with c-Myc to regulate tumor metabolism in colon cancer [12].However, the expression of LINC00504 in breast cancer and its biological effects has not been reported.
In this study, we explored the expression of LINC00504 in breast cancer tissues and cells by using qRT-PCR, CCK-8 and other molecular biology experiments at the breast cancer tissue and cell level, we demonstrated that LINC00504 is upregulated in human breast cancer tissues and cell lines.Cells were cultured in Dulbecco's modified Eagle's medium (DMEM; GIBCO, Grand Island, NY) supplemented with 10% of fetal bovine serum (FBS), 100U/ml penicillin and 100mg/ml streptomycin (Invitrogen, Carlsbad, CA, USA).The cells were maintained in a humidified incubator at 37°C and 5% CO2.

Cell cycle analysis
After transfection, cells were harvested and adjusted to the concentration of 1×106 cell/mL.Then pre-cold ethanol (75%) was used to dissolve the cells, followed by incubation at 4°C for 4h.After that, cells were washed with cold PBS again.Next, cells were stained with BD Pharmingen TM PI/RNase for 30 min at room temperature, followed by flow cytometer at different cell cycle phase (G1, S, and G2).10000 cells were measured for each sample.

Real-Time Quantitative PCR (qPCR) Analysis
TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was used to extract total RNA from the tissues and cell lines according to the manufacturer's instructions.RNA was reverse-transcribed to cDNA by means of the PrimeScript RT Reagent Kit (TaKaRa, Dalian, China).SYBR Premix Ex Taq (TaKaRa) was used to detect LINC00504 and miR-140-5p expression.PCR was carried out at least in triplicate, and the results were analyzed on an ABI 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA).LINC00504 and miR-140-5p expression levels were quantified by the expression of GAPDH and U6, respectively.The relative expression was calculated using the 2 −ΔΔCT method.

Cell proliferation，Cell migration and invasion assays
Cell proliferation was detected by Cell Counting Kit-8 (CCK-8; Dojindo, Tokyo, Japan).Cells were seeded in 96-well plates with density 5×103 cells/well and incubated in 37°C with 5% CO2.Next, the CCK-8 assay solution (10μl) was added to each well at indicated time.The absorbance at 450nm was measured with an enzyme immunoassay analyzer (Thermo Fisher Scientific, Shanghai, China).
A transwell chamber (8-μm pore size, Corning, Cambridge, MA, USA) was used to perform cell migration and invasion assays.Transfected cells (2 × 10 5 cells/mL) were resuspended in 200 μL of the serum-free medium and seeded in the upper chamber.Next, the cells were placed on the top side of the membrane (without Matrigel for the migration assay) or placed on the top side of the membrane precoated with Matrigel (BD Biosciences) (for the invasion assay).After incubation at 37°C for 48 hours, the cells migrated or invaded to the lower side of the membrane were fixed in 20% methanol and stained with 0.1% crystal violet for 15min.The cells was counted in five randomly selected visual fields under an inverted Phase-contrast Microscope (Olympus).

Mice experiments
For xenograft mouse experiments, SKBR3 or MDA-MB-231 (5 × 10 6 cells) under different treatments were subcutaneously injected into 8-week-old athymic nude mice (Bikai).Tumor size and weight was measured every 6 days, and the tumor volume was calculated as 0.5 × L× W 2 , with L representing length and w representing width.

Immunofluorescence
SKBR3 and MDA-MB-231 were cultured on glass slides and fixed with 4% paraformaldehyde for 15 min.Then, the cells were washed three times with phosphate-buffered saline (PBS) for 5 min and incubated with blocking buffer (PBS solution containing 3% fetal bovine serum (FBS), 1% goat serum, and 0.1% Triton X-100) for 2h at room temperature.Next, cells were incubated with the primary antibody (anti-VIM, anti-CDH1) diluted in PBS at 4℃ overnight.The slides were then washed with PBS three times prior to being incubated with Alexa Fluor®-488/555 fluorescent conjugated secondary antibody for 1 h in the dark.Afterward, the slides were washed three times in PBS prior to being mounted with Pro-Long® Gold Antifade Reagent with 4′,6-diamidino-2-phenylindole (DAPI: Molecular Probes, Eugene, OR, USA).The slides were observed using an LSM 800 Confocal Microscope (Carl Zeiss AG, Oberkochen, Germany).

The luciferase reporter assay
MiR-140-5p was found to be directly regulated target by LINC00504 using miRcode bioinformatics tools (http://www.mircode.org/).The theoretical binding sequence for miR-140-5p in the LINC00504 gene and its mutant sequence were cloned into the psiCHECK-2 vector (Promega, Madison, WI, USA) to construct a dual luciferase reporter plasmid.The wild-type (wt) 3′-UTR fragment of LINC00504 and its mutant (mut) of the miR-140-5p binding site were cloned into a the psiCHECK-2 vector to form the reporter vector ，which named as Wt-LINC00504 and Mut-LINC00504, respectively.SKBR3 and MDA-MB-231 cells were transfected with Wt (or Mut) reporter plasmid and an NC mimic or miR-140-5p mimic for 48 hours.The luciferase activity was detected using a Dual Luciferase Reporter Gene Assay Kit (Beyotime Institute of Biotechnology, Shanghai, China) according to the manufacturer's protocol.The relative luciferase activity was normalized to Renilla luciferase activity.

RNA immunoprecipitation (RIP) assay
RIP was performed using a Magna RIP RNA-Binding Protein Immunoprecipitation kit (Millipore, Bedford, MA) according to the manufacturer's instructions.Briefly, cells were transfected with miR-140-5p mimics, miR-140-5p NC mimics, and LINC00504, then were lysed in lysis buffer.
Subsequently, cell lysates were incubated with anti-Ago2 (Abcam) or anti-IgG (Abcam) and protein A/G magnetic beads.The magnetic bead-bound complexes were purified by Dnase and Proteinase K (Applied Biosystems).Lastly, qRT-PCR assays were used to determine the relative enrichment of LINC00504 mRNA, with a LightCycler 480 Probes Master Kit on a LightCycler 480 instrument.

Statistical analysis
Statistical analyses were performed with SPSS 20.0 (SPSS Inc., Chicago, IL, USA).Data are presented as mean ± standard deviation (SD) of at least three independent experiments.Differences between two groups or more than two groups were evaluated, respectively, by Student's t test or one-way analysis of variance (ANOVA).Spearman rank-correlation was performed to calculate the correlation coefficient between LINC00504 and miR-140-5p expression levels.

LINC00504 is up regulated in breast cancer tissues and cells
Bioinformatics analysis was used to identify the expression of LINC00504 in breast cancer patients and healthy people in The Cancer Genome Atlas (TCGA) database.We found a significant increase of the level of LINC00504 in the breast cancer patients compared to healthy control (Figure .1A).
Furthermore, Real-time qpcr was used to investigate the expression of LINC00504 in breast cancer tissues and adjacent normal tissues.The results showed that LINC00504 was expressed at higher levels in tumor tissues compared with adjacent normal tissues, which also confirmed the results of bioinformatics (Figure .1B).We also measured the LINC00504 expression in five human breast cancer cell lines (BT549, T47D, MCF-7, SKBR3 and MDA-MB-231) and in MCF-10A, the normal breast cell line.Same as before results, LINC00504 expression was obviously higher in five breast cancer cell lines compared with the normal breast cell (Figure .1C).These results demonstrated that LINC00504 was up regulated in breast cancer.

knock down of LINC00504 suppressed breast cancer cell proliferation, migration and invasion in vitro and in vivo
The physiological role of LINC00504 was explored using SKBR3 and MDA-MB-231 cells, which were transfected with sh-control and sh-LINC00504.The significantly decrease of LINC00504 expression in these sh-LINC00504 transfected cells was confirmed by qRT-PCR (Figure 2A).Then we performed CCK-8 assays to analyze the effect of LINC00504 knockdown on proliferation.We found the inhibition of the two cell lines proliferation when compared to their corresponding controls (Figure 2B Next, transwell assays was used to study whether LINC00504 can Influence breast cancer cell migration and invasion.The results showed that LINC00504 knockdown suppressed breast cancer cell migration and invasion (Figure 4A, B).Immunofluorescence was used to detect the variety of VIM and CDH1, which were the characteristic proteins of the epithelial-mesenchymal transition (EMT) [13,14].The results demonstrated that knockdown of LINC00504 inhibited the expression of VIM and enhanced the expression of CDH1 (Figure 4C, D), which also proved that LINC00504 boosts breast cancer cell migration and invasion.Collectively, LINC00504 knockdown impaired further progression of breast cancer cells.

MIR-140-5p was a target of LINC00504 in breast cancer
There has been reported that lncRNA could act as a ceRNA to exert its regulatory functions.To further reveal the underlying mechanism responsible for LINC00504 functions in breast cancer, we examined a set of miRNAs that were predicted to interact with LINC00504 using predication software miRcode and RNA22.MIR-140-5p was found to be a promising target of LINC00504 and the predicted bonding site of miR-140-5p in the LINC00504 sequence is showed in (Figure 5A).
After transfection with sh-LINC00504, qRT-PCR detection revealed that miR-140-5p expression was significantly increased after knocking down of LINC00504(Figure 5B).To further verify the interaction between miR-140-5p and LINC00504, luciferase reporter vectors were constructed which contained a wild-type (wt) or mutated (mut) miR-140-5p-binding site in LINC00504.The results of dual-luciferase reporter assays showed that miR-140-5p suppressed the luciferase activity of the LINC00504-wt reporter vector, whereas barely influenced that of the LINC00504-mut reporter vector, (Figure 5C).Furthermore, RIP assay was used to examine the potentially endogenous interaction between LINC00504 and miR-140-5p.The data presented that LINC00504 was substantially enriched by miR-140-5p overexpression with anti-Ago2 in MDA-MB-231 and SKBR3 cells (Figure 5D and 5E).using Spearman's correlation analysis, we found that the levels of LINC00504 were statistically correlated with that of miR-140-5p among breast cancer tissue samples (Figure 5F).These data indicated that miR-140-5p is a direct target of LINC00504 in breast cancer.

LINC00504 promoted the progression of breast cancer by the miR-140-5p-VEGFA axis
Next, we explored the effect of miR-140-5p in LINC00504-driven promotion of breast cancer progression.We knocked down LINC00504 and inhibited miR-140-5p in the same time in SKBR3 and MDA-MB-231 cells.CCK8, transwell migration and invasion assays showed that LINC00504 knockdown significantly inhibited cell proliferation, migration and invasion while miR-140-5p inhibition in the meantime abrogated these effects (Figure 6A,B,C), which demonstrated that miR-140-5p plays a key role in LINC00504-related oncogenic effects on breast cancer cells.It has been reported that MicroRNA-140-5p inhibits invasion and angiogenesis through targeting VEGFA in breast cancer [15].Thus, we supposed that VEGFA is involved in the LINC00504/miR-140-5pdependent malignant progression of breast cancer cell.We knocked down LINC00504 and inhibited miR-140-5p in the same time in SKBR3 and MDA-MB-231 cells.The expression of VEGFA, CDH1 and VIM was detected by Western blotting.The results showed that VEGFA and VIM was downregulated while the expression of cdh1 was increased when knockdown of LINC00504.
However, the converse expression of the proteins was observed when knockdown of LINC00504 and inhibition of miR-140-5p existed simultaneously (Figure 6D).These results suggested that LINC00504 induced tumor development via inhibition of miR-140-5p and by targeting VEGFA.

Discussion
Breast cancer is the most common and mortality-related malignant tumor in female around the world [16].The incidence of breast cancer is increasing rapidly during recent years [17].Early breast cancer often does not have typical symptoms and signs, and it is not easy to attract attention.It is often in the middle and late stages when found [18].However, the underlying mechanism that regulates breast cancer development remains largely unknown.It is crucial to develop novel molecular biomarkers for the diagnosis and prognosis of breast cancer.Here, we found that LINC00504 was significantly upregulated in breast cancer.And LINC00504 was critical for the proliferation, migration and invasion of breast cancer cells, which indicated that LINC00504 may be a new biomarker for breast cancer.
Emerging evidence showed that dysregulation of lncRNAs has been demonstrated to be involved in tumorigenesis and progression of breast cancer [19][20][21], suggesting the possibility of lncRNAs to serve as novel target for breast cancer diagnosis and therapy.LINC00504 is a newly found lncRNA, which was highly expressed in colon cancer.Feng J et al firstly reported that LINC00504 interacts with c-Myc to regulate tumor metabolism in colon cancer.However, the detail function and underlying mechanism of LINC00504 on breast cancer still remain unclear.In this study, we showed that LINC00504 was highly expressed in breast cancer tissues and cell lines.By in vitro and in vivo assays, we showed that knockdown of LINC00504 remarkably inhibited cell proliferation.Similarly, a clear weakening trend of cell migration and invasion was observed with LINC00504 depletion in breast cancer.Moreover, we firstly manifested that LINC00504 knockdown resulted in decreased EMT in breast cancer cells.All these results hinted that LINC00504 might contribute to the metastasis of breast cancer.
Previous evidence showed that lncRNAs can serve as competitive endogenous RNA (ceRNA) to sponge miRNAs [22][23][24].To further explore the underlying molecular mechanism by which LINC00504 regulates breast cancer, we made a predication and found that miR-140-5p was a promising candidate.The gene encoding miRNA-140-5p is located in chromosome 16, which has been proven to function in several cancer cells [25][26][27].For instance, Yu n f e n g e t a l .d e m o n s t r a t e d that miR-140-5p could suppress tumor growth and metastasis of non-small cell lung cancer by targeting IGF1R [28].In hypopharyngeal squamous cell carcinoma, miRNA-140-5p suppresses tumor cell migration and invasion by targeting ADAM10-mediated Notch1 signaling pathway [29].
And miRNA-140-5p inhibits invasion and angiogenesis through targeting VEGF-A in breast cancer [30].Above studies indicate that miRNA-140-5p may be a tumor suppressor.To further investigate the correlation between LINC00504 and miRNA-140-5p in breast cancer tumorigenesis, we performed luciferase reporter assays.Results showed that LINC00504 direc tly combined to miRNA-140-5p in breast cancer cells.We also found that LINC00504 significantly inhibited miRNA-140-5p expression, enhanced VEGFA and VIM expression and reduced CDH1 level.Taken together, our results indicated that the oncogene LINC00504 promoted breast cancer progression by negatively regulating miRNA-140-5p, a tumor suppressor via targeting VEGFA.The LINC00504/miRNA-140-5p may act as a novel therapeutic target for the treatment of breast cancer.

Conclusion
Taken together, our results demonstrated the mechanism of the LINC00504-miR-140-5p-VEGFA axis in breast cancer cell proliferation and invasion and may lead to new lncRNA-based diagnostics or therapeutics for breast cancer.
The effect of LINC00504 knockdown on the proliferation, cell cycle distribution of breast cancer cells in vitro.SKBR3 and MDA-MB-231 cells were transfected with sh-control or sh-LINC00504.LINC00504 expression was detected by qRT-PCR when transfected in the two cells(A).transfected cells proliferation was detected by CCK-8 and colony formation assays(B-D).Flow cytometer was used to analyze the transfected cells at different cell cycle phase (G1, S, and G2)(E).**P<0.01vs sh-control.

2 Materials and methods 2 . 1
Besides, we uncovered the oncogenic function of LINC00504 via regulating the miR-140-5p-VEGFR pathway during breast cancer development.These results might provide a new insight for the treatment of breast cancer.Tissue Specimen and Cell Culture 28 BC specimens and their matched adjacent normal tissues were collected after surgical resection at Shanghai Tenth People's Hospital.All the patients underwent surgical resection and were diagnosed with breast cancer by rapid pathology.And the tissues were immediately snapfrozen in liquid nitrogen after resection and stored at −80°C.The study protocol was approved by Shanghai Tenth People's Hospital Institutional Review Board (The Certificate Number: SHSY-IEC-KY-4.0/17-23/01), and all patients signed informed consent.Human breast cancer cell lines BT549, T47D, MCF-7, SKBR3 and MDA-MB-231 were purchased from the Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences (Shanghai, China).Human normal breast cell line MCF-10A was purchased from American Type Culture Collection (ATCC) (Manassas, VA, USA).
,2C).Similarly, LINC00504 knockdown dramatically decreased the colony formation by SKBR3 and MDA-MB-231 cells (Figure.2D).Cell cycle analysis was used to explore the cell distribution at different cell phases.The results showed that knockdown of LINC00504 increased the numbers of SKBR3 and MDA-MB-231 cells in G1 phase and decreased the numbers of MDA-MB-231 cells in both S and G2 phase, as for SKBR3, the numbers were reduced in G2 phase (Figure.2E).In xenograft mice models, we found that LINC00504 knockdown significantly inhibited tumor growth (Figure.3),which further supports our previous tests that LINC00504 boosts breast cancer cell proliferation.

Figure legends Figure1.
Figure legendsFigure1.LINC00504 was up regulated in breast cancer tissues and cells.The expression of LINC00504 in breast cancer patients and healthy people was assessed in The Cancer Genome Atlas

Figure2.
Figure2.The effect of LINC00504 knockdown on the proliferation, cell cycle distribution of breast cancer cells in vitro.SKBR3 and MDA-MB-231 cells were transfected with sh-control or sh-LINC00504.LINC00504 expression was detected by qRT-PCR when transfected in the two cells(A).transfected cells proliferation was detected by CCK-8 and colony formation assays(B-D).Flow cytometer was used to analyze the transfected cells at different cell cycle phase (G1, S, and G2)(E).**P<0.01vs sh-control.

Figure3.
Figure3.The effect of LINC00504 knockdown on the proliferation in vivo.Transfected cells were planted in mice, and tumor growth was measured.**P<0.01vs sh-control.

Figure4.
Figure4.The influence of LINC00504 knockdown on invasion, and EMT of breast cancer cells.Transfected cells migration and invasion capacities were assessed by transwell assays(A-B).Immunofluorescence was used to detect the variety of VIM and CDH1 in Transfected cells.**P<0.01vs sh-control.

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Figure 4 The
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