lncRNA AFAP1-AS1 promotes triple negative breast cancer cell proliferation and invasion via targeting miR-145 to regulate MTH1 expression

The actin fiber-associated protein 1-antisense RNA1 (AFAP1-AS1) is upregulated in various cancers and associated with cancer proliferation and metastasis. Several cancer-related pathways have been linked to up-expression of this long non-coding (lnc)RNA, but the underlying mechanisms are yet unknown. In triple negative breast cancer (TNBC), AFAP1-AS1 expression is also significantly overexpressed compared to that in other subtypes of breast cancer from the TCGA dataset. In this study, we performed bioinformatic RNAhybrid analyses and identified that miR-145 is a potential target of AFAP1-AS1 and able to reduce MutT homolog-1 (MTH1) expression. Thus, this study investigated the oncogenic activity of AFAP1-AS1 in TNBC cells and the underlying mechanisms that are yet poorly understood. The results showed that miR-145 expression was low, whereas AFAP1-AS1 and MTH1 expression was high in TNBC cells and that miR-145 mimics reduced TNBC cell proliferation and invasion, whereas miR-145 knockdown exerted the opposite activity in TNBC cells. Moreover, knockdown of AFAP1-AS1 reduced tumor cell proliferation and invasion, but miR-145 co-transfection rescued tumor cell viability and colony formation ability. The dual luciferase reporter assay showed that AFAP1-AS1 could directly target miR-145, while miR-145 could directly target MTH1. After knockdown of ATF6, AFAP1-AS1 was reduced along with AFAP1-AS1 promoter activity. This study revealed that AFAP1-AS1 could promote TNBC cell proliferation and invasion via regulation of MTH1 expression through targeting of miR-145.

Luciferase reporter assay. To predict the target gene of AFAP1-AS1, we first performed a bioinformatics analysis. We identified potential genes and focused on miR-145 based on the RNAhybrid results. We then constructed vectors carrying the wild-type or mutated miR145 3′-untranslated region (3′-UTR), which are referred to as pmirGLO/AFAP1-AS1-3′UTR and pmirGLO/AFAP1-AS1-3′UTR Mut, respectively (the detailed construction protocol is described in the supplementary information). For the Luciferase reporter assay, MDA-MB-231 cells were seeded into 6-well plates at a density of 1 × 10 5 /well and grown in 2 mL DMEM for 24 h to reach 70%-80% confluency. The medium was then replaced with 1 mL DMEM without antibodies, and the cells were transfected with each luciferase reporter gene (20 pmol)   www.nature.com/scientificreports www.nature.com/scientificreports/ at a density of 5 × 10 3 /well and incubated overnight in 100 µL DMEM before transfection with miR-145 mimics or a negative control of miR-145 mimics, ASO-NC, ASO-miR-145, pSilencer-NC plus ASO-NC, pshR-AFAP1-AS1 plus ASO-NC, or pshR-AFAP1-AS1 plus ASO-miR-145 for 48 h. Before the end of each assay, 10 µL (5 mg/mL) of the MTT reagent (Sigma-Aldrich, USA) was added into a final volume of 100 µL DMEM and incubated for additional 4 h. After that, the culture medium was replaced with 100 μL dimethyl sulfoxide (DMSO), and the absorbance of each cell culture solution was measured using a microplate reader (Thermo Scientific, USA) at 570 nm. The experiments were performed in triplicate and repeated at least three times.
Cell colony formation assay. After transfections, cells were reseeded into 12-well plates at a density of 200/ well in 2 mL of complete growth medium and incubated for 2 weeks at 37 °C. The growth medium was replaced every 3 days. At the end of the experiments, cells were washed with ice-cold PBS twice and fixed with fresh-made 4% paraformaldehyde at 4 °C for 30 min. Next, cells were washed with PBS three times and stained with 0.1% crystal violet, and cell colonies with 50 cells or more were counted under an inverted microscope (Olympus, Japan). The experiment was repeated at least three times.
Wound healing assay. MDA-MB-231 cells were seeded into 6-well plates at a density of 3 × 10 5 cells/ well and grown overnight. On the next day, the cells were transfected with miR-145 mimics or negative control, pshR-AFAP1-AS1 or pSilencer-NC, and negative control or pshR-AFAP1-AS1 plus ASO-miR-145 for 48 h. After cell monolayers reached 95%-98% confluency, a cell wound was created using a 200-μL sterile plastic tip, and then the cells were washed three times with PBS. The cells were further cultured in serum-free medium at 37 °C for 48 h and imaged under a phase-contrast microscope. The experiment was repeated at least three times.
Transwell invasion assay. Tumor cell invasion capacity was assessed using a Transwell chamber (Millipore, Billerica, USA) with the filter precoated with 25 μL Matrigel (BD Biosciences, Franklin Lakes, NJ, USA). In brief, cells were seeded into the upper chamber with 200 μL serum-free medium at a density of 1 × 10 5 cells/well, and the bottom chambers were filled with 500 μL DMEM supplemented with 20% FBS. After culture for 72 h, cells on the upper filter surface were removed using a cotton swab, while cells that had invaded the bottom side of the filter were fixed with a mixture of methanol and glacial acetic acid (a ratio of 3:1) for 30 min at room temperature and stained with 0.1% crystal violet for 15 min. The numbers of invading cells in three randomly selected fields on each filter were counted under a light microscope (Olympus, Japan). The assay was repeated at least three times. supplemented with a protease inhibitor cocktail (Thermo Fisher Scientific, USA) and quantified using the BCA protein assay kit (CWBIO, Beijing, China). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis was used to separate proteins, and samples were transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membranes were then blocked for 1 h at room temperature with a blocking buffer (5% skim milk in PBS) and further incubated with the primary antibodies at 4 °C overnight. These primary antibodies were a mouse anti-MTH1 and mouse anti-ATF6 antibodies (Tianjin Biotechnology Co., Ltd. Tianjin, China). On the next day, the membranes were washed with PBS-Tween 20 (PBS-T) three times and then incubated with secondary antibody (Tianjin Biotechnology Co., Ltd. Tianjin, China) for 1 h at room temperature. After washing with PBS-T three times, the protein bands were visualized using an enhanced chemiluminescence kit (Thermo Scientific, USA). GAPDH protein was used as a control. The assay was repeated at least three times.  www.nature.com/scientificreports www.nature.com/scientificreports/ Laboratory Animal Science, Chinese Academy of Medical Sciences (Beijing, China) and maintained in a specific pathogen-free (SPF) "barrier" facility. The mice were housed under controlled temperature and humidity and alternating 12-hour light and dark cycles. The mice received SPF mouse chow and sterile water ad libitum. The mice were randomly divided into 5 groups and each group contained 5 mice. MDA-MB-231 cells transfected with different genes (e.g., miR-145 mimics or negative control, pSilencer-NC or pshR-AFAP1-AS1 or pshR-AFAP1-AS1 plus ASO-miR-145) were grown, and 5 × 10 7 /mL cell suspensions were prepared in 100 μL PBS and subcutaneously injected into the back of each mouse on the left side. Mouse weight and tumor formation and size were monitored daily and recorded, and the tumor volumes were calculated from measurements of the www.nature.com/scientificreports www.nature.com/scientificreports/ longest (L) and shortest (S) tumor dimensions taken every 3 days using the formula: V = (L × S 2 )/2. After 3-5 weeks, the nude mice were anesthetized with intraperitoneal injection of 80 mg/kg of ketamine and 10 mg/kg of xylazine according to standard procedures and photographed. Finally, mice were euthanized by cervical dislocation and the tumor xenografts were removed and weighed.

In vivo
Statistical analysis. All statistical analyses were performed using SPSS version 15.0 software (SPSS, Chicago, IL, USA). All of our experiments were repeated three times, and the data are presented as mean ± standard error. Student's t test was used for comparisons between two groups, and one-way analysis of variance with the Bonferroni post-test was used for comparisons among three or more groups. A two-side value of P < 0.05 was considered statistically significant.

Differential expression of miR-145, AFAP1-AS1, and MTH1 in normal breast cells and different breast cancer cell lines.
In this study, we first analyzed AFAP1-AS1 expression in TNBC and found that AFAP1-AS1 expression was significantly higher in TNBC than in other subtypes of breast cancer using TCGA dataset ( Figure S1) We also found that expression levels of miR-145 and MTH1 in TNBC were obviously lower and higher than those in luminal breast cancer, respectively ( Figures S2 and S3).
We then assayed their expression in breast cancer cell lines and found that the expression level of miR-145 was lower in breast cancer cells compared with that in normal mammary epithelial MCF-10A cells (Fig. 1A). In contrast, the expression levels of AFAP1-AS1 and MTH1 were higher in breast cancer cells compared with those in MCF-10A cells (Fig. 1B).
Differential effects of miR-145 and AFAP1-AS1 on regulation of breast cancer cell viability and invasion. Furthermore, we found that transfection with miR-145 mimics reduced MDA-MB-231 cell viability and colony formation capacity, whereas knockdown of miR-145 using ASO-miR-145 had the opposite effects on breast cancer cell viability and colony formation ( Fig. 2A,B). Moreover, knockdown of AFAP1-AS1 expression by pSilence-AFAP1-AS1 transfection reduced the viability and colony formation capacity of MDA-MB-231 cells (Fig. 2C,D), whereas miR-145 co-transfection rescued tumor cell viability and colony formation ability

Differential effects of miR-145 and AFAP1-AS1 on regulation of breast cancer cell wound healing and invasion in vitro.
We found that miR-145 mimics reduced MDA-MB-231 cell wound healing and invasion in vitro, whereas knockdown of miR-145 using ASO-miR-145 had the opposite effects on breast cancer cell wound healing and invasion in vitro (Fig. 3A-C). Moreover, knockdown of AFAP1-AS1 expression by pSilence-AFAP1-AS1 also reduced the wound healing and invasion capacities of MDA-MB-231 cells in vitro (Fig. 3D-F), whereas ASO-miR-145 rescued tumor cell viability and colony formation ability (Fig. 3D-F).

Interaction of miR-145 with ATF6 and ATF6 feedback with AFAP1-AS1 in breast cancer cells.
We next explored how these genes interact in breast cancer cells by constructing wild-type and mutated pmiGLO/ ATF6-3′-UTR and pmiGLO/ATF6-3′-UTR plasmids ( Figure S5) and performed luciferase reporter assays. Our www.nature.com/scientificreports www.nature.com/scientificreports/ results showed that hsa-miR-145-5p also was able to directly bind to ATF6-3′-UTR, but this targeting effect disappeared when the seed sequences were mutated (Fig. 6A). miR-145 expression or knockdown also changed the levels of AFT6 mRNA and protein in MDA-MB-231 cells (Fig. 6B,C). Moreover, knockdown of ATF6 expression effectively reduced the levels of ATF6 mRNA and protein (Fig. 6D,E) as well as the level of AFAP1-AS1 in breast cancer cells (Fig. 6F). In addition, ATF6 could directly bind to the promoter fragment of AFAP1-AS1 (Figures S7  and S8). After ATF6 was knocked down, the promoter activity of AFAP1-AS1 was reduced (Fig. 6G).

Discussion
TNBC is characterized by a high recurrence rate, high potential metastasis, and poor treatment response and prognosis [35][36][37] . Research to better understand the molecular mechanisms and to support the development of novel molecular targeting therapeutic strategies for TNBC is a significant and hot topic in the field. In this study, we first analyzed TCGA dataset and found that AFAP1-AS1 expression was significantly higher in TNBC vs. other subtypes of breast cancer, while the expression levels of miR-145 and MTH1 were obviously lower and higher in TNBC than those in luminal breast cancer, respectively. Our in vitro and in vivo experiments further showed that AFAP1-AS1 expression was up-regulated in breast cancer cells and promoted TNBC cell proliferation and invasion in vitro as well as tumor formation and growth in nude mice. These data are consistent with previous studies showing that AFAP1-AS1 expression is elevated in breast cancer and promotes tumor proliferation 14,38 .
These results indicate that AFAP1-AS1-miR145-MTH1 is an important CeRNA network in TNBC. Furthermore, AFAP1-AS1 has been demonstrated to be associated with poor prognosis in some cancer patients 39,40 . Based on this, we analyzed the relationships between AFAP1-AS1, miR-145, MTH1 and disease-free survival (DFS) and overall survival (OS) in TNBC patients from TCGA dataset and found no significant relationship ( Figure S9). The possible reason is that the number of cases in TCGA is small and more cases are needed for verification. On the other hand, the prognosis is related to multiple factors, and the corresponding regulatory mechanisms require further research.
Altered expression of different miRNAs occurs and has been reported in breast cancer, but which miRNA interacts with AFAP1-AS1 is unclear. We performed RNAhybrid bioinformatics analysis and found that miR-145 could be a target gene of AFAP1-AS1. In the present study, dual luciferase reporter assays showed that AFAP1-AS1 could directly target miR-145, which confirmed the results of the bioinformatics analysis. For the effect on cell proliferation, we observed that knockdown of AFAP1-AS1 alone could reduce cell proliferation and invasion, but co-transfection of miR-145 rescued tumor cell viability and colony formation ability. These results are consistent with the previous report that miR-145 is one of nine miRNAs in a miRNA signature that may serve as a potential diagnostic marker for breast cancer 24 . A previous genetic association study showed that miR-145 single nucleotide polymorphisms (SNPs) are associated with breast cancer susceptibility 25 , while downregulation of miR-145 can be used to predict the risk of postmenopausal breast cancer 26 . Furthermore, upregulated miR-145 expression through demethylation of the miR145 promoter inhibits breast cancer cell migration and invasion 23 .
Furthermore, we also observed a positive association between AFAP1-AS1 and MTH1 and found that AFAP1-AS1 can increase MTH1 expression through downregulation of miR-145 in breast cancer cells in vitro. These results were consistent with a previous report that miR-145 expression can reduce MTH1 expression to suppress cancer cell proliferation 31 . Because cancer cells grow fast, they produce a large amount of ROS and are in a state of high oxidative stress. MTH1 can convert oxidized nucleoside triphosphates to nucleoside monophosphates, thereby preventing these oxidized nucleoside triphosphates from being incorporated into DNA to reduce cell death. Therefore, cancer cells have increased MTH1 expression to avoid ROS-induced cell damage, while normal cells have low expression levels of MTH1 because intracellular ROS levels are low [27][28][29][30] . Consistently, we found that the expression levels of MTH1 were higher in breast cancer cells compared with those in MCF-10A cells in this study.
In addition, our current study also revealed that ATF6 is a target gene of miR-145, or in other words, miR-145 inhibits ATF6 expression in TNBC cells, while ATF6 can directly bind to the AFAP1-AS promoter. Thus, knockdown of ATF6 expression led to reduced AFAP1-AS1 promoter activity and expression in TNBC cells. Taken together, these findings reveal a positive feedback among these three genes; i.e., ATF6 increases AFAP1-AS1 promoter activity and expression, which leads to a decrease in the miR-145 level and an increase in ATF6 expression in TNBC cells. However, the importance of ATF6 in breast cancer requires further study, because to date, there has been no study reporting the role of ATF6 in breast cancer.

Conclusions
In summary, our current study established a ceRNA-based regulatory network in TNBC cell proliferation and invasion. We also demonstrated the important role of the lncRNA AFAP1-AS1 in the promotion of TNBC proliferation via regulation of MTH1 expression through targeting of miR-145 in breast cancer cells. The identified ATF6/AFAP1-AS1/miR-145 feedback mechanism could play an important role in the regulation of TNBC proliferation and invasion, and the interaction of these factors represents a novel therapeutic target in the treatment of TNBC patients that warrants further investigation.