Long non-coding RNA AFAP1-AS1 promotes tumor progression and invasion by regulating the miR-2110/Sp1 axis in triple negative breast cancer

Background: LncRNAs have been proved to be involved in the proliferation, apoptosis, invasion, migration and other pathological processes of triple negative breast cancer (TNBC). And the expression level of LncRNA AFAP1-AS1 in TNBC was found to be signicantly higher than that in other subtypes and normal tissue samples, but the specic mechanism of LncRNA AFAP1-AS1 affecting the occurrence and development of TNBC needs to be revealed. Methods: Cell Counting Kit-8 assays, colony formation assays, wound-healing migration, transwell invasion assays and nude mouse xenograft assays were used to conrm the role of LncRNA AFAP1-AS1 in the proliferation, migration of TNBC cells in vitro and in vivo. Bioinformatics analyses, quantitative polymerase chain reaction (qRT-PCR), western blot, and dual-luciferase assays were performed to conrm the interaction between between LncRNA AFAP1-AS1, miR-2110 and Sp1. Results: In the present study, the silencing of AFAP1-AS1 and Sp1 or the upregulation of miR-2110 would result in the suppression of proliferation, migration and invasion of MDA-MB-231 and MDA-MB-468 cells in vitro as well as tumor growth in vivo. Mechanistically, the dual-luciferase reporter assay highlighted that AFAP1-AS1 functioned as a miR-2110 sponge to increase Sp1 expression. AFAP1-AS1 silencing led to a reduction in Sp1 mRNA and protein levels, which could be reverse by the joint transfection of miR-2110 inhibitor. Conclusions: Our ndings demonstrated that AFAP1-AS1 acts as a miR-2110 sponge in TNBC cells, resulting in the regulation of Sp1 expression. And the AFAP1-AS1/miR-2110/Sp1 axis modulated the proliferation, migration and invasion of breast cancer cells and affected the tumorigenesis in mice. Together, our results provide evidence that AFAP1-AS1 could directly bind to miR-2110 in breast cancer cells.


Background
Breast cancer is one of the most common cancers among women in the world. Data in 2019 showed that breast cancer accounted for 30% of newly diagnosed cancer cases and was the cause of cancer deaths in 15% of women [1] . Clinically, according to the expression differences of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2), breast cancer is divided into the following four types: Luminal A (ER + and/or PR + , HER2 − ), Luminal B (ER + and/or PR + , HER2 −/+ ), HER2 + (ER − , PR − , HER2 + ) and triple negative breast cancer (TNBC). Among them, TNBC subtype accounts for about 15-20% of all breast cancer cases, which is highly malignant and has the characteristics of high recurrence rate, high metastatic potential, poor treatment response and prognosis. In addition to conventional chemotherapy and radiotherapy, there is no effective targeted therapy. Therefore, there is an urgent need to further understand the molecular mechanism of triple-negative breast cancer in tumor progression, and to develop experimental targets with potential clinical application, and it is of great signi cance for formulating more effective clinical treatment strategies and improving the prognosis of patients [2] . Among them, RNA-based cancer treatment methods have gradually changed from concept to reality. Non-coding RNA (ncRNA) blocked mRNA function by inhibiting its transcription and binding to proteins, which had clinical therapeutic effects on tumors [3,4] .
Long Noncoding RNA (LncRNA), having a length greater than 200 nt, is one type of ncRNA with no protein coding function, and has complicated biological functions. Their abnormal expression or dysfunction has been proved to be closely related to the occurrence and development of human diseases [5] . LncRNAs have been proved to be involved in the proliferation, apoptosis, invasion, migration, EMT and other pathological processes of TNBC. Based on the rapid development of high-throughput sequencing and genome analysis technology, a large amount of information about LncRNA has emerged in the past decade with the help of powerful bioinformatics analysis tools. Reiche et al. identi ed more than 9,500 LncRNA transcripts with signi cant differences in normal breast tissue and cancer tissue [6] . A comprehensive analysis of LncRNAs expression pro les and clinical data from 1097 breast cancer samples from The Cancer Genome Atlas (TCGA) was conducted. 1510 LncRNAs were differentially expressed in TNBC comparing with normal samples, and 672 LncRNAs expression were distinguished between TNBC and non TNBC samples [7] . However, the speci c mechanism of LncRNAs affecting the occurrence and development of TNBC has not been deeply revealed.
Therefore, it is of great signi cance and urgent desire to explore TNBC related LncRNAs, study the molecular mechanism of TNBC, and provide reference data for the understanding, diagnosis, treatment and prognosis of TNBC.
LncRNA actin ber associated protein 1-antisense RNA1 (LncRNA-AFAP1-AS1, abbreviated as AFAP1-AS1) was rst found in the sequencing of esophageal adenocarcinoma (EAC) and normal tissues, which was located on the antisense chain of the protein coding gene AFAP1 on chr4: 7755817-7780655 (+) of human genome (GRCh37/hg19). AFAP1-AS1 has been reported to be closely related to the proliferation and metastasis of different cancers, and was closely related to the poor prognosis of human malignant tumors [8] , such as pancreatic ductal adenocarcinoma [9] , esophageal adenocarcinoma [10] , lung cancer [11] and colorectal cancer [12] .
In breast cancer, the signi cantly up-regulated AFAP1-AS1 indicated a poor prognosis [13,14] , and was identi ed as the most dysregulated LncRNA from analyzed seven pairs of HER2 + subtype breast cancer samples [15] . Limited knockout of AFAP1-AS1 (about 25-50%) was su cient to reduce the proliferation and colony formation in MDA-MB-231 cells and HCC1937 cells [16] , meanwhile repressed BT-549 and MCF-7 cells proliferation, migration by downregulating SEPT2 via sponging miR-497-5p [17] . An in silico analysis from cDNA microarray data con rmed that AFAP1-AS1 was potential prognostic factor for TNBC patients [18] . And it promoted epithelial-mesenchymal transition by Wnt/β-catenin signaling pathway in TNBC [19] . A former study in our lab analyzing AFAP1-AS1 in BC samples of TCGA, and found that the expression level of AFAP1-AS1 in TNBC was signi cantly higher than that in other subtypes and normal tissue samples [20] . These above results suggested that AFAP1-AS1 was involved in the pathogenesis of breast cancer and may become a new biomarker or therapeutic target for TNBC. However, despite a small amount of research, the role of AFAP1-AS1 speci c targets and related signaling pathways in the development and progression of TNBC has still been fragmentary and needs further study.
In this study, bioinformatics analysis, molecular biology, cell biology and tumorigenesis assay in nude mice were used to study the downstream regulatory relationship of AFAP1-AS1. We found that AFAP1-AS1 and Sp1 knockdown or miR-2110 overexpression would suppress MDA-MB-231, MDA-MB-468 cells proliferation, migration and invasion in vitro as well as tumor growth in vivo. AFAP1-AS1 silencing led to a reduction in Sp1 mRNA and protein levels, which could be reverse by the joint transfection of miR-2110 inhibitor.
Mechanistically, the results revealed that AFAP1-AS1 competitively bound to miR-2110, affecting the expression of Sp1, thus regulating the proliferation and migration in TNBC cell and tumor progression in vivo.

Methods
Cell culture MDA-MB-231 (ATCC® HTB-26™, The Chinese Academy of Sciences, China) and MDA-MB-468 (BNCC339862, Bnbio, China) cells were cultured in L15 medium containing 20% fetal bovine serum at 37°C and 100% air constant temperature incubator. The original medium was discarded and the cells were digested by 1ml of 0.25% trypsin when the cells grow to 80% con uence. After the adherent cells become round, stop the digestion with 1ml of medium, centrifuge in a low-speed centrifuge at 1000 r/min for 3 min. The supernatant was aspirated, an appropriate amount of culture medium was added to the cell pellet. Cells were passaged in the ratio of 1:4 to 1:3 every 2 to 3 days. Observe the morphological changes with an inverted microscope.
The shSp1 plasmid of Sp1 was also constructed by the pcDNA3.1-EGFP vector, and the target sequences (GCTAGCGCTGGTGGTGATGGAATACATCTCGAGATGTATTCCATCACCACCAGCTTTTTGAATTC) were inserted between the restriction site of NheI (GCTAGC) and EcoRI (GAATTC).

Cell transfection
The cells in the logarithmic growth phase were used for the subsequent experiments. The pcDNA3.1-NC plasmid and shlncRNA-AFAP1-AS1 plasmid were transiently transfected with Lipofectamine 3000 (L3000015, Thermo Fisher, China) according to the manufacturer's instructions. Taking the 12-well plate transfection as an example. The cells were seeded on the 12-well plate the day before transfection, and cultured overnight.
1μg per well plasmid was transfected when reaching 60-80% density. After transfection, incubate at 37°C for 72 hours for subsequent experiments.

RT-qPCR
Total RNA was extracted using TRIzol reagent (Invitrogen, USA) and the RNA quantity and density were veri ed by a Nanodrop. PrimeScript TM RT Reagent Kit (RR037A, Takara, China) was used to reverse transcription. RT-qPCR was performed using the SYBR Premix Ex Taq TM (RR820A, Takara, China) according to the manufacturer's instructions. The assays were operated in triplicate and relative gene expression was determined by using the 2 -ΔΔCt method.
For miRNA RT-qPCR, 0.5 μg total RNA and miR-2110 RT Primer (or U6 RT Primer, Table S1) were used for miRNA RT reaction. The primers 5'-TGCGGTTGGGGAAACGGCCGCTG-3' (miR-2110, forward), 5'-CCAGTGCAGGGTCCGAGGT-3' (miR-2110, reverse); 5'-GCTCGCTTCGGCAGCACA-3' (U6, forward) and 5'-AACGCTTCACGAATTTGCGTG-3' (U6, reverse) were used in the miRNA qPCR reaction. For colony formation assay, cells were digested into single-cell suspension and 500 cells seeded in each culture dish (6 cm). The appropriate complete medium was added to each dish with the culture medium being refreshed every three days. The cells were then washed twice with PBS, xed with 4% paraformaldehyde and stained with 1 mL Giemsa stain for 15 min. The number of colonies was counted manually and averaged from the duplicate wells. The clone formation rate (%) = (number of cell clones / total number of cells added)

Wound-healing migration, and transwell invasion assays
For wound-healing migration assay, the cell monolayers were mechanically disrupted using a sterile 10 μL micropipette tip to generate a linear wound, and the cells were washed off by PBS. After that, the culture medium was added. The 24 well plates were taken out at 0h, 24h and 48h for micrograph. The migration distance of cells was observed, the migration rate of cells at different time points was calculated, and the column diagram was drawn. The migration rate (%) = (scratch area of 0h -scratch area of Nh) / (scratch area of 0h) X100%.
For transwell invasion assays, cells were incubated using 24-well transwell plates (8 μm pore size, Corning, NY, USA). 1X10 5 cells suspended in serum-free medium were plated in the upper chambers with Matrigel (BD Biosciences, USA), and 600 μL complete medium was added to the lower chamber. After incubation for a suitable amount of time(24h for MDA-MB-231 and 48h for MDA-MB-468), the cells were xed in 4% iced paraformaldehyde for 30min, stained by Giemsa stain for 15min and counted under a microscope.

Luciferase reporter assay
The vectors of luciferase reporters were synthesized to pmirGLO vector to constructed wildtype and mutation plasmids. Cells were inoculated into a 24-well plate and were co-transfected with miRNA mimics or the negative control and the luciferase reporter vector as well as lipofectamine 3000 reagent. After 48h, the dualluciferase reporter was detected by Dual-Luciferase® Reporter Assay System (E1910, Promega) and relative luciferase activity was normalized to Renilla luciferase activity. The sequence of miRNA-NC mimics was 5'-UCACAACCUCCUAGAAAGAGUAGA-3', and the sequence of hsa-miR-2110 mimics was 5'-UUGGGGAAACGGCCGCUGAGUG-3'.

Western blot assays
For total protein extraction, cell lysates were obtained using RIPA buffer. A total of 10 μL protein was injected into a 10% SDS-PAGE gel and transferred to PVDF membranes at 4°C, 200 mA for 1h. After blocking with blocking solution, the membranes were incubated with primary antibody overnight at 4°C. After PBS washing, membranes were then exposed to the secondary antibody (HRP-Goat anti rabbit / mouse IgG antibody) for 1h.
Bands were incubated with an ECL kit and analyzed with an imaging system. The primary antibodies CLDN4 In vivo assays 72 of 1-5 weeks old BALB/C SPF nude mice were purchased from SPF (Beijing) Biotechnology Co.,Ltd., all of them were females (not pregnant), healthy and mature, with an average weight of 18±2g. They were raised in the barrier system of the Laboratory Animal Center of the Institute of Radiation Medicine, Chinese Academy of Medical Sciences. All feed, water, air, bedding and various supplies that enter the barrier system must be sterilized by high temperature and high pressure; all people and animals entering the laboratory must undergo strict microbial control.
After 1 week of adaptive growth, they were randomly divided into 12 groups with 6 mice in each group. 0.1ml of 10 8 /ml cells diluted with PBS were inoculated subcutaneously into the armpits. Observing the nude mice's mental state, activity, responsiveness, diet and appearance of the subcutaneous vaccination area every two days. The tumor size was measured every week by a Vernier caliper and the tumor volume (V) was calculated as V (cm 3 ) = 1/2AB 2 with A and B representing the largest and smallest diameters, respectively. With the tumor volume as the Y axis and growth weeks as the X axis, the tumor growth curves of each group were drawn. Animals were euthanized 30 days after inoculation, and the tumors were taken out, weighed, and then placed in 4% paraformaldehyde for xation.

Data analysis
The data obtained were expressed as mean ± standard deviation (SD) or standard error (SE). ANOVA and t test were performed to analyze the intergroup differences. P < 0.05 indicated signi cant differences.

Results
AFAP-AS1 promotes the proliferation, migration and invasion of breast cancer cells To explore the biological function of AFAP1-AS1, rst we designed a shlncRNA-AFAP1-AS1 shRNA. Compared to the negative control group, the expression of AFAP1-AS1 was signi cantly decreased in MDA-MB-231 and MDA-MB-468 cells con rming by RT-qPCR (Fig.1A). CCK-8 showed that the cell viability was inhibited after the downregulation of AFAP1-AS1 (Fig.1B). The results of colony formation assay indicated that cell proliferation was decreased after the treatment of shlncRNA-AFAP1-AS1 (Fig.1C). Wound-healing migration and transwell invasion assays revealed that AFAP1-AS1 knockdown decreased cell migration and invasion, suggesting that AFAP1-AS1 was positively correlated with cell migration and invasion ( Fig.1D and 1E).

AFAP1-AS1 targets miR-2110 in breast cancer cells
Increasing studies have shown that LncRNAs might act as sponges of miRNA, thereby interfering with tumor progression. Therefore, we speculated that AFAP1-AS1 may in uence the function of some miRNAs that may play certain roles in breast cancer.
Using bioinformatics prediction assay, 13 miRNAs were predicted by LncBase database (Table.1 Meanwhile, after silencing AFAP1-AS1, the level of miR-2110 was up-regulated compared with the NC (negative control) group (Fig.2C). Together, our results provide evidence that AFAP1-AS1 could directly bind to miR-2110 in breast cancer cells.

AFAP1-AS1 upregulates Sp1 level via sponging miR-2110
According to previous studies, miR-2110 was found to have differentiation-inducing and oncosuppressive function in neuroblastoma, and was signi cantly related to the survival rate of patients [21] . Thus, we hypothesized that miR-2110 may functioned as suppressor in TNBC cells and AFAP1-AS1 promoted tumor progression by protecting downstream oncogenes from downregulation by miR-2110.
In order to verify our hypothesis, rstly, 509 miR-2110 potential target genes were found after the prediction and overlapping of three miRNA interaction database: TargetScan, miRDB and miRTarBase. Subsequently, 462 of them were analyzed by R cluster pro ler package. Combined with the subsequent KEGG analysis and the reported pro-oncogenic and up-regulated genes, ve downstream genes with the highest correlation with miR-2110 were screened (Table 2, Fig.3A and 3B) and miR-2110 possessed binding sites in 3 UTR of predicted gene (Fig.3C). Firstly, miR-2110 mimics were transfected into MDA-MB-231 and MDA-MB-468 cells to observe the changes of protein expression levels of candidate target genes (Fig.3D). The results showed that compared with CLDN4, RALYL, RHBDD1 and ZNF703, the Sp1 protein expression decreased signi cantly after transfection with miR-2110 mimics, while the expression of Sp1 increased after transfection with miR-2110 inhibitor ( Fig.3E and Fig.S1). It is speculated that miR-2110 has the strongest targeting ability to Sp1.
Subsequently, dual-luciferase assay was conducted to verify the target relationship between miR-2110 and Sp1. The results in MDA-MB-231 and MDA-MB-468 cells showed that the relative luciferase reporter intensity was 50% lower after the transfection of miR-2110 mimics comparing with the wild type plasmid alone (pmirGLO/Sp1-WT), while miR-2110 mimics had no effect on the mutation plasmid (pmirGLO/Sp1-MUT), which demonstrated that Sp1 was a critical target of miR-2110 (Fig.3F).
To further demonstrate the link among AFAP1-AS1, miR-2110, and Sp1 expressions, cells were subjected to different transfections. First, RT-qPCR assay showed that Sp1 mRNA level was signi cantly decreased after transfected with miR-2110 mimics in MDA-MB-231 and MDA-MB-468 cells, meanwhile the miR-2110 inhibitor caused an up-regulated mRNA level of Sp1 (Fig.3G). Moreover, cells were then transfected with NC or shlncRNA-AFAP1-AS1, and AFAP1-AS1 silencing led to a reduction in Sp1 mRNA and protein levels, which could be rescued by the co-transfection of miR-2110 inhibitor (Fig.3H and 3I). Together, our results show that miR-2110 levels were negatively correlated, whereas AFAP1-AS1 levels were positively correlated to the Sp1 expression, suggesting that AFAP1-AS1 upregulated Sp1 expression via sponging miR-2110.
We then evaluated the roles of Sp1 in breast cancer cells by Sp1 knockdown. After the transfection, the expression of Sp1 was veri ed by western blot, which was signi cantly decreased in shSp1 group than that in NC group (Fig. 4A). CCK-8 and colony formation assays showed that the cell viability was inhibited after Sp1 knockdown, and wound healing, transwell invasion assays revealed that Sp1 knockdown decreased cell migration, invasion of breast cancer cells (Fig. 4B-4E, group 1 vs group 5 and Fig.S2). In a similar way, all these effects are offset by the joint addition of miR-2110 inhibitor ( Fig. 4B-4E, group 5 vs group 6 and Fig.S2).
These results indicated that downregulated Sp1 inhibited tumor progression, and miR-2110 downregulation reversed the Sp1 silencing-induced phenotype of breast cancer cells.
The ndings showed that tumors in the AFAP1-AS1-silencing group had a slower growth rate and particularly less average volume and weight (Fig. 5, group 1 vs group 2). And the tumor volume and weight of the miR-2110-silencing group increased signi cantly (Fig. 5, group 1 vs group 3), while these phenotype were all balanced by AFAP1-AS1 silencing in the shlncRNA-AFAP1-AS1+ miR-2110-inhibitor group (Fig. 5, group 3 vs group 4), in which the tumor size and growth rate were not signi cantly different from the negative control group (Fig. 5, group 1 vs group 4). At the same time, the silence of Sp1 also caused a signi cant decrease in tumor volume and growth rate (Fig. 5, group 1 vs group 5). After the joint addition of miR-2110 inhibitor (shSp1+miR-2110-inhibitor group), related parameters such as tumor volume and growth rate increased (Fig.5, group 5 vs group 6). The results showed that the AFAP1-AS1/miR-2110/Sp1 axis affected the growth rate, average size and weight of tumor.

Discussion
To date, a large number of LncRNAs have been recently discovered through functional genomics studies.
Although many studies have shown that LncRNAs can participate in the pathological process of breast cancer, there are relatively few studies on TNBC-related LncRNAs, especially the speci c mechanism that affects the occurrence and development of TNBC has not yet been deeply revealed.
AFAP1-AS1 was found on the antisense chain of the protein coding gene AFAP1, and involved in the development of a variety of cancers, such as, relating to the poor prognosis of tongue squamous cell carcinoma (TSCC), played a role of oncogene in TSCC by activating Wnt / β-catenin signaling pathway and inhibiting the expression of EMT related genes [22] . In gastric cancer tissues and cells, AFAP1-AS1 was signi cantly up-regulated and regulated the proliferation and apoptosis of gastric cancer cells through PTEN / p-Akt pathway [23] . The expression of AFAP1-AS1 was also up-regulated in esophageal squamous cell carcinoma, and was signi cantly correlated with TNM stage and tumor size [24] . Hypo-methylation and high expression of AFAP1-AS1 was found in Barrett's esophagus and EAC. Its expression interference could also inhibit the proliferation and colony forming ability of EAC cells [25] . In breast cancer, AFAP1-AS1 also showed a signi cantly up-regulated expression compared with adjacent tissues [13] . Here, after analyzing the expression of AFAP1-AS1 in the BC samples of the TCGA database, our previous study found that the expression of AFAP1-AS1 in TNBC was signi cantly higher than other subtypes and normal tissue sample [20] , suggesting that AFAP1-AS1 may be involved in the pathogenesis of breast cancer and may become a new biomarker or therapeutic target for TNBC. After discovering the potential importance of AFAP1-AS1, we deeply studied its function on breast cancer cells, and found that AFAP1-AS1 could promote cell proliferation, clone formation and was positively correlated with cell migration and invasion. Our research will undoubtedly enrich the research eld of LncRNA in TNBC.
Studies have shown that LncRNAs participate in multiple regulatory mechanisms, such as ceRNAs, regulation of transcription, translation, protein modi cation, and the formation of RNA-protein or protein-protein complexes. The ceRNA networks link the function of mRNAs with non-coding RNAs such as miRNAs and LncRNAs, which assumes that LncRNAs serve as miRNA sponge to eliminate miRNA inhibition on its target genes. In this study, dual-luciferase reporter assay con rmed that AFAP1-AS1 acted as a sponge for miR-2110.
There are few studies on miR-2110, but it was considered to be a tumor suppressor in neuroblastoma [21] , which may be functioned by directly targeting Tsukushi [26] . Meanwhile, our research also proved that decreased miR-2110 promoted cell proliferation, migration, and invasion in TNBC cells. MiRNAs are capable of regulating physiological processes by inhibiting target mRNA translation or promoting mRNA degradation.
The bioinformatics results of miRNA target prediction showed ve potential miR-2110 target genes with the highest correlation, including CLDN4, RALYL, RHBDD1, ZNF703 and Sp1. Compared with CLDN4, RALYL, RHBDD1 and ZNF703, the Sp1 expression had more signi cantly distinction after miR-2110 mimics and inhibitor transfection. And it was a well-known transcription factor with pro-oncogenic function in multiple tumors, therefor Sp1 was selected for the following research.
Sp1 is a transcription factor that binds to GC-rich motifs of many promoters and is involved in many cellular processes, including cell differentiation, cell growth, apoptosis, immune responses, response to DNA damage, and chromatin remodeling. It is overexpressed in multiple tumors and is a negative prognostic factor for patient survival. For example, Kim et al. studied the expression pro le of 203 TNBC patients during adjuvant chemotherapy and found that the increase in Sp1 expression was associated with poor prognosis. The Sp1 expression in the multivariate Cox regression model was an effective indicator for predicting the long-term prognosis of TNBC patients treated with doxorubicin [27] . In addition, a large number of studies have shown that Sp1-regulated genes were related to the pro-oncogenic activity [28] . Such as, ERK/Sp1 signaling pathways mediated the TGF-β-induced EGFR upregulation, resulting in the promotion of migration and invasion in breast cancer cells [29] . STAT3 and Sp1 cooperated to induce high expression of the small GTPase Ras Homolog Family Member U RhoU and enhanced breast cancer cells migration [30] . In the present study, the downregulated Sp1 suppressed the proliferation, migration and invasion of MDA-MB-231 and MDA-MB-468 cells as well as the tumor growth in vivo. And LncRNA-AFAP1-AS1 was shown to competitively bind to miR-2110 to reduce the inhibition effect of Sp1 by miR-2110, resulting in the promoted breast cancer progression. All these above ndings indicated that AFAP1-AS1 served as a ceRNA to contribute to breast cancer progression through the miR-2110/Sp1 axis.
On the other hand, some studies have shown that Sp1, as a sequence-speci c DNA binding protein, could initiate the transcription of many cellular genes (including LncRNAs) and participate in various biological processes such as cell proliferation, differentiation, and tumor formation. For example, in retinal malformation cells, Sp1 directly bound to the LncRNA PADAR promoter region and promoted its transcription to regulate apoptosis caused by the Bcl-2/caspase-3 pathway [31] . Sp1 could bind LncRNA SPRY4-IT1 promoter and activate its transcription, playing a carcinogenic role in cholangiocarcinoma [32] . It was noteworthy that, JASPAR platform predicted that there were 18 Sp1 binding sites in the AFAP1-AS1 promoter region, indicating that Sp1 may bind to the AFAP1-AS1 promoter region and regulate the expression of AFAP1-AS1. So, we presumes that AFAP1-AS1 acts as miR-2110 sponge reducing its inhibition effect on Sp1, and in turn, the elevated Sp1 binds to the AFAP1-AS1 promoter region and activates its transcription, forming a positive feedback system. Certainly, these hypothesis also need further exploration and discovery in follow-up research.

Conclusion
In summary, we detected an LncRNA (AFAP1-AS1) that was overexpressed in TNBC tissues, upregulating Sp1 level via sponging miR-2110. Meanwhile the AFAP1-AS1/miR-2110/Sp1 axis modulated the proliferation, migration and invasion of breast cancer cells and affected the tumorigenesis in mice. Our results not only elucidated the potential mechanism by which LncRNAs regulate the TNBC progression, but also suggested that the AFAP1-AS1/miR-2110/Sp1 axis could be a potential target for triple negative breast cancer.

RALY
Increasing expression in BC cells and tumors, and correlated with decreased patient survival [35] .