MiR-664b-3p Inhibited Triple-Negative Breast Cancer Cell Growth Via Targeting BRIP1

Many studies indicate that microRNAs (miRNAs) play a crucial role in modulating the development and progression of triple-negative breast cancer (TNBC). However, miR-664b-3p affections on the TNBC functions and mechanisms are still unknown. The purpose of our study was to clarify the effects of miR-664b-3p in cellular TNBC development and progression. In our study, the expressions of miR-664b-3p in cell lines and tissueswere tested by real-time PCR (RT-PCR), immunouorescence, H&E and immunohistochemistry staining. CCK-8 assay, colony formation, EdU, ow cytometry apoptosis, wound scratch, Transwell assays were applied to explore the cell functions. The targeted relationship between miR-664b-3p and its target BRIP1 was determined by dual-luciferase reporter assay and rescue experiments. identied Moreover, we investigated the mechanisms underlying the effect of miR-664b-3p on that miR-664b-3p inhibited and targeting targeting determine the functional biological roles of miR-664b-3p in TNBC cell proliferation, migration, invasion and apoptosis, we ectopically repressed and raised the expression of miR-664b-3p in two TNBC cell lines (MCF-7 and MDA-MB-231). The results showed that ectopic expression of miR-664b-3p resulted in signicant inhibition of cell proliferation, migration, invasion as well as promotion apoptosis.


Introduction
Breast cancer is one of the most common cancer in women, and the death rate caused by breast cancer is very high [1,2], with an estimated annual diagnosis of 1.38 million new cases worldwide [3][4][5][6]. The molecular biomarkers were used in routine clinical practice in order to help make treatment decisions for breast cancer that include human epidermal growth factor receptor 2 (HER2), progesterone receptor (PR) and estrogen receptor (ER) [7]. Triple-negative breast cancer (TNBC) does not express any of these receptors, and it is di cult to treat because most chemotherapy only targets one of the three receptors. Furthermore, TNBC accounts for about 20% of breast cancer and is a highly invasive breast cancer subtype with a higher metastatic rate and lower survival rate than other breast cancer subtypes [8,9].
Chemotherapy remains the dominating clinical treatment for early and late TNBC. However, due to the lack of recognized molecular targets, global oncologists face enormous challenges in their ght against TNBC [10]. Therefore, it is essential to explore the molecular targets and underlying mechanism of TNBC and provide new biomarkers which can be used for TNBC diagnosis and treatment.
In the present study, we investigated the biological functions of proliferation migration, invasion and apoptosis in TNBC cells and identi ed the target gene. Our study innovatively demonstrated that miR-664b-3p was a potential biomarker and tumor suppressor in TNBC directly targeting oncogene BRIP1.

Cell lines and cell transfection
Normal epithelial cells MCF-10A and two human breast cancer cell lines MCF-7, MDA-MB-231 were obtained from the American Type Culture Collection (ATCC). All cells were maintained in RPMI 1640 medium containing 10% fetal bovine serum (FBS) (Invitrogen) at 37℃ in a humidi ed atmosphere with 5% CO 2 . miR-664b-3p mimic, mimic NC, miR-664b-3p inhibitor, inhibitor NC, pc-BRIP1 and pc-NC were transfected by using Lipofectamine 2000 (Invitrogen, Shanghai, China) reagent in the light of the manufacturer's instructions.

Rna Extraction And Real-time Pcr Analysis
RNA from cell lines was extracted using the mirVana miRNA Isolation Kit (Ambion, USA) according to the manufacture's protocol. For validation of miRNA and Real-time PCR assay, TaqMan microRNA Assay protocol (Applied Biosystems) was performed. The internal control was U6 small nuclear RNA and fold change was determined as 2 −∆∆Ct . All tests were performed in triplicate Western Blot Proteins were extracted from cells using RIPA lysis buffer. Proteins were separated by 10% SDS-PAGE and transferred into PVDF membranes (Millipore Corp., MA). After a block with 5% milk for 1 h at room temperature, the membrane was incubated with a primary antibody at 4℃ overnight. Then, the membranes were incubated with corresponding secondary antibodies for 1 h at room temperature. The fold change was calculated by normalizing with GAPDH levels.

Cell Proliferation Assay
Cell proliferation analysis was carried out by using the Cell Counting Kit-8 (CCK-8) and cell colony formation assay. The cells transfected with miR-664b-3p mimics were plated into 96-well plates, and cell proliferation was determined using CCK-8 assay 12 h, 24 h and 48 h, respectively. The optical density (OD) of the samples was measured with a microplate reader at 450 nm. For the colony formation assay, cells were cultured for 72 h, xed and stained with crystal violet. Finally, the colonies were photographed and counted under a microscope.

Cell Migration And Invasion Assays
We used wound healing assay and transwell assay to examine the capacity of cell migration and invasion. For wound healing assay, cells were plated in 6-well plates and allowed to reach 90-95% con uence. Streaks were created with a 10 µl pipette tip. Then, cells were incubated for 24 h and 48 h. Cells were photographed using microscopy. For transwell assay, MCF-7 and MDA-MB-231 cells were cultured into 6-well plates after transfection for 24 h. Cell migration and invasion activity were detected using a transwell assay. The lower chambers were lled with medium containing 10% FBS. After the cells were incubated for 24 h at 37℃ in 5% CO 2 , the non-migrating or non-invading cells were carefully removed with cotton wool. The cells in the bottom chamber were xed with cold methanol for 10 min, following stained with 0.1% crystal violet, and then the cells were counted and photographed under a light microscope at a magni cation of 200×. All experiments were independently repeated in triplicate independently.

In Vivo Tumor Growth
All animal procedures were approved by the Animal Care Committee of Jiangsu College of Nursing. BALB/c nude mice were purchased from the Nanjing Biomedical Research Institute of Nanjing University. MCF-7 and MDA-MB-231 cells (1×10 6 ) stably transfected with miR-664b-3p mimic, mimic NC, miR-664b-3p inhibitor, or inhibitor NC were subcutaneously injected into nude mice. Tumor volume was measured every 3 days and was calculated by the following formula: volume (mm 3 ) = (length ×width 2 )/2.
After transfection for 48 h, the luciferase activities were assayed using a Dual-Luciferase Reporter Assay System (Promega). Every experiment was performed in triplicate.

Statistical analysis
Statistical analysis of the data was performed using SPSS 19.0 software (SPSS Inc., Chicago, IL, USA).
Data are expressed as the mean ± SD of three independent experiments. Differences between the two groups were assessed using a t-test, and multiple groups of comparisons were performed using analysis of variance. P < 0.05 was considered statistically signi cant.

MiR-664b-3p expression is decreased in TNBC cell lines
To examine the biological relevance of miR-664b-3p, its expression was evaluated in cancer tissues and their corresponding adjacent normal tissues by RT-PCR. Decreased expression of miR-664b-3p was also observed in TNBC cell lines (MDA-MB-231 and MCF-7) compared with normal epithelial cell line MCF-10A (Fig. 1A). These ndings indicated that miR-664b-3p might play a putative tumor suppressor role in TNBC.

Mir-664b-3p Regulates Cell Proliferation
To determine the functional signi cance of miR-664b-3p in TNBC, we transfected miR-664b-3p mimics, miR-664b-3p inhibitor or blank control into TNBC cell lines MCF-7 and MDA-MB-231. The transfected e ciency and GFP assays were assessed using RT-PCR assay ( Fig. 2A). Next, we carried out the CCK-8 analysis to analyze the ability of cell proliferation; the results showed that ectopic expression of miR-664b-3p signi cantly decreased cell proliferation as compared to the control group (Fig. 2C). The contrast result was obtained in the miR-664b-3p inhibitor. Mechanically, similar results were obtained in EdU and colony formation assay (Fig. 2D, E). These results indicate an anti-proliferative effect of miR-664b-3p in TNBC.

Mir-664b-3p Mediates Cell Apoptosis
In order to further explore the in uence of miR-664b-3p on TNBC cell apoptosis, the raised percentage of apoptotic cells was measured by ow cytometry in MCF-7 and MDA-MB-231 cells. The results indicated that a signi cant increase in apoptosis was observed in the cells which transfected with miR-664b-3p mimics and a decrease in the cells that were transfected with miR-664b-3p inhibitor (Fig. 3A). Western blot was used to test apoptotic-related factors. As shown in Fig. 3B, we found that restoration of miR-664b-3p signi cantly suppressed the level of Bcl-2 and increased the levels of Bax, Caspase-3 and Caspase-9. Therefore, these results indicated that miR-664b-3p induces apoptosis in TNBC cells.

Effect Of Mir-664b-3p On Tnbc Cell Migration And Invasion
We performed a wound-healing assay with the cells after transfection for 48 h. The results were shown in Fig. 4A. Next, we carried out transwell assays to evaluate the in uence of miR-664b-3p on cell migration and invasion. We found that the number of migration and invasion cells was obviously decreased in MCF-7 and MDA-MB-231 cells, which transfected with miR-664b-3p mimics compared with control cells (Fig. 4B). Western blot analysis was used to detect related proteins MMP-2 and MMP-9 (Fig. 4C). These ndings mean that ectopic expression of miR-664b-3p inhibits migration and invasion in TNBC cells.
Effect of miR-664b-3p on TNBC tumor growth in vivo.
Subsequently, the photo of subcutaneous tumors in nude mice was observed (Fig. 5A). The results revealed that the subcutaneous tumors from nude mice in the miR-664b-3p mimic group presented a lower volume and weight compared with those in the mimic NC group (Fig. 5B, C). In addition, HE staining assay and IHC analysis showed that overexpression of miR-664b-3p aggravated cell apoptosis and suppressed the expression level of cell proliferation indicator Ki67 (Fig. 5D). These results strongly suggested that miR-664b-3p mimic restrained TNBC tumor growth in vivo.

Brip1 Is A Target For Mir-664b-3p
We employed three publicly available miRNA target prediction tools, including records, Targetscan and ncRNA, to predict the targets of miR-664b-3p and searched BRIP1 for further analysis (Fig. 6A). As shown in Fig. 6B, luciferase activity was decreased when the WT BRIP1-3 UTR and miR-664b-3p mimic were cotransfected compared with the control group. Moreover, the cells which co-transfected with mut BRIP1-3 UTR had no effect on luciferase activity. Further, the expression of BRIP1 in cell lines was detected by RT-PCR and western blot. As shown in Fig. 6C and Fig. 6D, we found that BRIP1 was up-regulated in TNBC cell lines compared with the normal epithelial cell line. In addition, the expression of BRIP1 was measured via RT-PCR and western blot analyses (Fig. 6E, F). These results indicated that BRIP1 is a direct target of miR-664b-3p.

miR-664b-3p inhibits cell proliferation, invasion and accelerates apoptosis by targeting BRIP1
Rescue assays were performed to further study whether miR-664b-3p executed its function in GC by targeting BRIP1. We further investigated the effect of overexpressed-BRIP1 in the presence of miR-664b-3p mimic on MCF-7 and MDA-MB-231 cells. The expression of BRIP1 was detected by RT-PCR assay (Fig. 7A). Next, we observed that up-regulation of miR-664b-3p signi cantly inhibited cell proliferation via CCK-8, EdU assay and colony formation. However, pc-BRIP1 in the presence of miR-664b-3p mimic reversed the effect of miR-664b-3p mimic on MCF-7 and MDA-MB-231 cells (Fig. 7B-D). Oppositely, ow cytometry analysis delineated that the promotion of cell apoptosis caused by miR-664b-3p mimic was abolished by pc-BRIP1 (Fig. 7E). Furthermore, the protein expression levels of cleaved-caspase-3, cleavedcaspase-9 and Bax were increased, and Bcl-2 expression was lessened by transfected with miR-664b-3p mimic and subsequently overexpressed BRIP1 renewed these protein expression levels (Fig. 7F).
Wound healing and transwell chamber assay disclosed that cell migration and invasion suppressed by miR-664b-3p mimic was facilitated on account of BRIP1 overexpressed (Fig. 8A, B). In concert with these results, the effect of miR-664b-3p mimic on the expression levels of MMP2 and MMP9 was recovery by inhibition of BRIP1 (Fig. 8C). These data revealed that miR-664b-3p inhibits cell proliferation, invasion and accelerates apoptosis by targeting BRIP1.

Discussion
More and more evidence has demonstrated that aberrant expression of miRNAs has been deeply involved in tumor development and progression [25][26][27][28][29][30][31]. Increasing evidence has shown that miR-664b-3p acted as a tumor suppressor in various cancers, including pancreatic adenocarcinomas, live cancer and head and neck squamous cell carcinoma [32][33][34][35]. However, miR-664b-3p and their biological relevance in TNBC have not yet been determined, and miR-664b-3p was considered as potential speci c biomarkers and played an important role in the diagnosis, progression and prognosis of many types of cancers [36].
Here, we researched the potential role of miR-664b-3p in TNBC progression. In the present study, we initially observed miR-664b-3p was signi cantly down-regulated in TNBC tissues compared with normal tissues, and similar results were shown in cell lines. To further determine the functional biological roles of miR-664b-3p in TNBC cell proliferation, migration, invasion and apoptosis, we ectopically repressed and raised the expression of miR-664b-3p in two TNBC cell lines (MCF-7 and MDA-MB-231). The results showed that ectopic expression of miR-664b-3p resulted in signi cant inhibition of cell proliferation, migration, invasion as well as promotion apoptosis.
Multiple lines of evidence have proven that microRNAs exert their diverse biological functions, such as acting as key signal transduction mediators in tumor signaling pathways and regulating cell activities, mainly by degrading mRNA or suppressing mRNA translation [37,38]. BRCA1-interacting C-terminal helicase 1 (BRIP1) was rst identi ed using tandem mass spectrometry by its physical interaction with BRCA1 and also belongs to the Fanconi anemia (FA) genes family. BRIP1 plays a major role in the development of various cancers, including breast cancer [39,40]. Moreover, target prediction analysis further con rmed that BRIP1 might be a target of miR-664b-3p. Furthermore, we performed luciferase reporter assays and the data presented demonstrated that BRIP1 represents a direct target gene of miR-664b-3p in TNBC cells. BRIP1 was overexpressed in TNBC cells. More importantly, further observations illustrated that ectopic miR-664b-3p inhibits cell proliferation, invasion and accelerates apoptosis by targeting BRIP1.

Conclusions
In conclusion, our ndings indicate that overexpression of miR-664b-3p could inhibit cell proliferation, migration, invasion and induced apoptosis by targeting BRIP1 partially. Generally, our study provides a potential biomarker and miR-664b-3p is expected to become the therapeutic target for the treatment of TNBC. However, the deep-going molecular mechanisms by which miR-664b-3p is involved in the development and progression of TNBC need further investigation. Figure 1 MiR-664b-3p expression in TNBC cell lines. RT-PCR analysis was analyzed the expression of miR-664b-3p