miR-217 inhibits triple-negative breast cancer cell growth, migration, and invasion through targeting KLF5

Triple negative breast cancer (TNBC) is one of the most aggressive breast cancers without effective targeted therapies. Numerous studies have implied that KLF5 plays an important roles in TNBC. How is KLF5 regulated by microRNAs has not been well studied. Here, we demonstrated that miR-217 down-regulates the expression of KLF5 and KLF5’s downstream target gene FGF-BP and Cyclin D1 in TNBC cell lines HCC1806 and HCC1937. Consequently, miR-217 suppresses TNBC cell growth, migration, and invasion. MiR-217 suppresses TNBC, at least partially, through down-regulating the KLF5 expression. These results suggest that the miR-217-KLF5 axis might serve as a potential target for treatment of TNBC.


Introduction
Breast cancer is still the main cause of female cancer-related death in United States [1]. Triplenegative breast cancer (TNBC), which lacks expression of estrogen receptor α (ERα) and progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) gene, accounts for approximately 15% of breast cancers [2]. TNBC is more aggressive, has higher rates of relapse and shorter overall survival than other subtypes of breast cancers. The median survival of women with metastatic TNBC is less than 12 months [3]. Up to now, there are still no effective targeted therapies for TNBC. Therefore, it's urgent to identify effective therapeutic targets for TNBC.
Krüppel-like factor 5 (KLF5), a member of KLF family, is highly expressed in the epithelial crypt cells of the gastrointestinal tract and involved in diverse cellular functions [4][5][6]. In breast cancer, KLF5 plays an important role in tumorigenesis and high expression level of PLOS  KLF5 is positively correlated with poor survival rate of breast cancer patients [7,8]. We previously found that KLF5 promotes breast cancer cell proliferation, migration and invasion by upregulating the expression of FGF-BP, mPGES1, and TNFAIP2 and down-regulating the expression of p27 [9][10][11][12]. KLF5 depletion leads to breast cancer cell apoptosis and suppresses xenograft tumor growth in vivo [13,14]. Importantly, pharmacological inhibition of KLF5 by mifepristone leads to suppression of TNBC stem cells [15]. In conclusion, KLF5 is a potential therapeutic target for TNBC.
MicroRNAs are a class of endogenous 21-23-nucleotide non-coding RNAs, usually function through inhibiting translation or inducing degradation of target mRNAs via binding to the specific sites on the 3'UTR of target mRNAs and are involved in diverse cellular functions [16,17]. Numerous studies demonstrated that microRNAs could serve as either tumor suppressor or oncogene depending on the target genes' functions [18]. Both microRNAs and anti-microRNA constructs are now under investigation as potential therapeutic agents for cancer. At present, the first microRNA mimics clinical trial is in progress [19].
In this study, we found that miR-217 downregulates the KLF5 expression via binding to its 3'UTR and suppresses TNBC cell growth, migration, and invasion. Furthermore, we showed that miR-217 inhibits TNBC, at least in part, through targeting KLF5. Our findings support that miR-217 functions as a tumor suppressor in TNBC.

Cell viability assay
The cell viability was measured by SRB (sulforhodamine B) assays. Briefly, cells were seeded in 48-well plates at a density of 8.0×10 3 cells per well. The cells were fixed with 10% TCA at 4℃ for 60 minutes at indicated time, followed by incubating with 0.4% SRB (W/V) solution in 1% acetic acid for 5 minutes at room temperature. At last, the SRB was dissolved with 10 mM unbuffered Tris base, and the absorbance was measured at a single wavelength of 490-530 nm on plate reader (Bio Tek, VT, USA). All experiments were performed in triplicates at least twice independently.

Wound healing, migration, and matrigel invasion assays
The wound healing assay was performed in 6-well plates. 1.2×10 6 HCC1806 cells or 1×10 6 HCC1937 cells were seeded in each well of a 6-well plate. Twenty four hours after cell plating, wounds were made using a pipette tip, and the wounds were recorded under a microscope every 4 hours. The cell migration and matrigel invasion assays were evaluated using 24-well chemotaxis chambers (Corning cell culture inserts, 8 μm pore size) and chemotaxis matrigel chambers (BD BIOCOAT Matrigel chambers, 8 μm pore size) according to published protocol [10].

Real-time PCR
Total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA). Reverse transcription was performed using the TaqMan PCR MicroRNA Reverse Transcription Kit (Applied Biosystems, Austin, TX, USA). For quantitative PCR, SYBR PCR Master Mix (Applied Biosystems) was used to quantify the expression level of RNA on a 7900HT Fast Real-Time PCR System (Applied Biosystems). The miR-217 expression level was detected using a special Bulge-Loop TM miRNAs qPCR Primer Set (Guangzhou RiboBio Co., LTD). U6 was used as the endogenous loading control.

Statistical analysis
All of the results were shown as Mean±SD, and statistical analysis were performed by Student's t-test using the SPSS program (version 12.0) (SPSS, Chicago, IL, USA). P<0.05 was defined to be statistically significant ( Ã , P<0.05, ÃÃ , P<0.01, ÃÃÃ , P<0.001).
its direct downstream targets FGF-BP and Cyclin D1 protein expression in both cell lines. We then analyzed the 3'UTR of KLF5 and found two possible binding sites for miR-217 (Fig 1B). To further test whether miR-217 inhibits the KLF5 expression through putative binding sites at KLF5 3'UTR, we performed dual luciferase reporter assay. As expected, miR-217 significantly suppressed the luciferase activity when the luciferase reporter gene linked with wild type KLF5 3'-UTR; however, the luciferase activity of the second, but not the first, miR-217 binding site mutated reporters was not inhibited by miR-217 (Fig 1C). These results implicated that miR-217 targets KLF5 through binding to the second putative site on the KLF5 3'UTR.
To determine the functions of miR-217 in TNBC, we over-expressed miR-217 in HCC1937 and HCC1806 cells. The over-expression of miR217 was confirmed by qPCR (Fig 2A). As shown in Fig 2B, we found that miR-217 significantly suppressed cell growth, as determined by the SRB assay in both cell lines. We also investigated the effects of miR-217 on cell migration by the transwell migration assay and found that miR-217 significantly inhibited TNBC cell migration compared to the vector control ( Fig 2C).
Additionally, we tested whether miR-217 targets DACH1 and pTEN in HCC1937 and HCC1806 cells. As shown in Fig 4D, although miRNA-217 significantly decreased the KLF5 protein levels in both cell lines, the protein levels of DACH1 and pTEN were not decreased. These results suggest that miR-217 predominately functions through KLF5 in these two TNBC cell lines.

Discussion
The expression of KLF5 is correlated with different hormone status in different breast cancer subtypes [8]. Accumulated evidence suggests that KLF5 is an oncogene in TNBC [7,10,[12][13][14][15]36]. We previously have reported KLF5 promotes TNBC cell proliferation, survival, migration and invasion via regulating a variety of target genes; however, how KLF5 is regulated in breast cancer needs to be further elucidated. To date, only miR-153 has been reported to target KLF5 in TNBC [15]. In this study, we reported that the protein level of KLF5 is downregulated by miR-217 in TNBC cells. MiR-217 directly binds to a putative sequence (968-974 nt) at the KLF5 gene 3'UTR although there are two candidate sequences. It is possible that the flanking sequences determine the binding specificity. Finally, we demonstrated that miR-217 inhibits TNBC cell growth, migration, and invasion, at least partially through KLF5. The miR-217-KLF5 axis might serve as a new target for TNBC therapy.
A microRNA could function as either a tumor suppressor or promoter in different cancers in a context-dependent manner because it targets numerous different target genes. Although miR-217 is overexpressed in aggressive human B-cell lymphomas and promotes mature B-cell lymphomagenesis [28], most studies suggest that miR-217 functions as a tumor suppressor in miR-217 target KLF5 a variety of cancers. In pancreatic ductal adenocarcinoma, miR-217 inhibits tumor cell growth by targeting KRAS and SIRT1 [22,37]. Additionally, downregulation of miR-217 in Ph(+) leukemia cells causes resistance to ABL tyrosine kinase inhibitors [38]. Furthermore, miR-217 was reported to inhibit invasion of hepatocellular carcinoma cells through targeting E2F3 [26]. In esophageal squamous cell carcinoma cells, miR-217 inhibits proliferation, migration, and invasion by targeting long noncoding RNA MALAT1 and kallikrein 7 (KLK7) [39]. Interestingly, miR-217 is down-regulated by cigarette smoke [40]. miR-217 was suggested to be a tumor suppressor by targeting Wnt5a in osteosarcoma and targeting IGF1R in ovarian cancer [41,42].
In breast cancer, Zhang reported that miR-217 promotes MCF-7 and MDA-MB-231 cell proliferation via targeting DACH1 [30]. Overexpression of miR-217 induces drug resistance and invasion of MCF-7 and SKBR-3 cells through activating AKT by downregulation of PTEN [35]. Furthermore, downregulation of miR-217 in MCF7 and MDA-MB-231 cells inhibits cell The miR-217 level was measured by quantitative PCR. B. miR-217 suppressed TNBC cell growth. The growth of HCC1806 and HCC1937 was measured using the SRB assay. C. miR-217 suppressed TNBC cell migration. miR-217 overexpression or vector control HCC1806 and HCC1937 cells were plated in chemotaxis chambers for 24 or 8 hours, respectively, before being fixed for migration detection. Quantative results are shown in the right panel. proliferation through targeting PGC-1α and DACH1 [29,30]. These studies suggest an oncogenic role of miR-217 in breast cancer. However, we found miR-217 suppressed cell growth, migration, and invasion via targeting KLF5 in both HCC1937 and HCC1806 cell lines. Coincidently, KLF5 is lowly expressed in MCF-7, MDA-MB-231, and SKBR3 but highly expressed in HCC1937 and HCC1806 ( [33] and unpublished results). Additionally, miR-217 did not inhibit the expression of DACH1 and pTEN in both HCC1937 and HCC1806 cell lines. Therefore, miR-217 may has a context-dependent roles in a cell line specific manner in breast cancer.
We recently reported that mifepristone inhibits KLF5 expression and cancer stem cells in basal TNBC [15]. Additionally, Curcumin inhibits KLF5 expression through YAP/TAZ in bladder cancer cell lines [43]. ML264 inhibits KLF5 expression in colorectal cancer cell lines [44,45]. microRNA mimics could be used as drugs for cancer treatment [19]. Therefore, we think that KLF5 pharmacological inhibition and miR-217 may be used for TNBC treatment in the future.
In summary, we conclude that miR-217 inhibits TNBC cell growth, migration, and invasion in vitro through targeting the KLF5 transcription factor. The miR-217-KLF5 axis may be used for TNBC therapy.