LncRNA FLVCR1-AS1 promotes proliferation, migration and activates Wnt/β-catenin pathway through miR-381-3p/CTNNB1 axis in breast cancer

Understanding the molecular mechanism of long non-coding RNAs (lncRNAs) in carcinogenesis is conducive for providing potential target for cancers. The role of FLVCR1-AS1 in breast cancer (BC) has not been probed yet. qRT-PCR and western blot assays were used to estimate relevant expressions of mRNAs and proteins. CCK8, MTT and EdU were implemented to assess cell proliferation ability. TUNEL was performed to investigate cell apoptosis, whereas transwell assay was performed to test cell migration and invasion capacities. TOP/FOP Flash assay was conducted to determine the activity of Wnt/β-catenin pathway. Luciferase reporter, RNA pull down and RIP assays were performed to verify interaction between genes. FLVCR1-AS1 was abnormally up-regulated in BC cells. Silencing FLVCR1-AS1 inhibited cell proliferation, migration, invasion, yet accelerating apoptosis. Inhibition of miR-381-3p reversed the tumor restraining impacts of FLVCR1-AS1 depletion on BC progression. Additionally, CTNNB1 was recognized to be targeted by miR-381-3p. FLVCR1-AS1 aggravated BC malignant progression via up-regulation CTNNB1 through sponging miR-381-3p. FLVCR1-AS1 regulates BC malignant behavior via sequestering miR-381-3p and then freeing CTNNB1, implying a promising target for BC therapy.

have emerged as novel focuses of clinical applications, since they play pivotal role in human malignancies [10][11][12].
The prevalent ceRNA mechanism has proofed that lncRNAs can function as competing endogenous RNAs (ceRNAs) for their interaction with sequestered microR-NAs (miRNAs), resulting in elevated expression of downstream target genes [13]. In BC, previous studies have found that some lncRNAs were dysregulated and predicted clinical prognosis. For instance, RHPN1-AS1 was found as abnormally up-regulated in BC and facilitated malignant phenotypes in vitro [14]. In addition, Li et al. [15] discovered that lncRNA HOXC13-AS, which was significantly up-regulated in BC, enhanced cell proliferation ability through up-regulating PTEN expression via suppressing miR-497-5p.
Previous studies have revealed the aberrant elevation of FLVCR1-AS1 in several tumors via being engaged in the lncRNA-miRNA-mRNA ceRNA network. For example, in gastric cancer (GC), enriched FLVCR1-AS1 promoted malignant behaviors of GC cells by its ceRNA role of c-Myc through targeting miR-155. Besides, FLVCR1-AS1 overexpression tended to result in poor prognostic outcomes in GC cases [16]. FLVCR1-AS1 contributed to cellular activities in glioma through targeting miR-4731-5p/ E2F2 signaling [17]. Also, FLVCR1-AS1 facilitated biological behaviors of ovarian cancer cells via regulating miR-513/YAP1 signaling [18]. FLVCR1-AS1 sponged miR-485-5p to modulate biological behaviors in human cholangiocarcinoma [19]. However, the expression profile, specific function and acting mechanism of FLVCR1-AS1 in BC have not been elucidated yet. Hence, present study aimed to investigate whether and how FLVCR1-AS1 functions in BC.

qRT-PCR
TRIzol reagent (Takara, Otsu, Japan) was used for extracting total RNA from SKBR3 or MCF7 cells. Subsequently, total RNAs were reversely transcribed into cDNAs under Reverse Transcription Kit (Takara). The qRT-PCR was performed with utilization of SYBR Green real-time PCR Kit (Takara) on the Bio-Rad CFX96 system (Bio-Rad, Hercules, CA). Fold expression changes were calculated via 2 −ΔΔCt method, with GAPDH/U6 as reference gene.

TUNEL assay
TUNEL staining assay was performed using In Situ Cell Death Detection Kit (Roche, Mannheim, Germany). Following nuclei staining with DAPI (Sigma-Aldrich), relative fluorescence intensity was determined via EVOS FL microscope (Olympus).

Transwell assay
2 × 10 4 cells in the top compartment were added with serum-free medium, while medium containing 10% FBS was placed into bottom chamber. After 48 h, migrated cells were fixed with paraformaldehyde (Solarbio) and dyed in crystal violet solution. Invasion assay was performed using the upper chamber was pre-coated with Matrigel (BD Biosciences, Shanghai, China). The number of migrated or invaded cells was captured via a microscope (Olympus).

Chromatin immunoprecipitation (ChIP)
ChIP experiment was processed with usage of Magna ChIP Kit (Millipore, Darmstadt, Germany). After crosslinked chromatin was sonicated to 200-300-bp fragments by ultrasound, lysates were immunoprecipitated with anti-MYC or anti-IgG. Precipitated chromatin DNA was detected by RT-qPCR.

Subcellular fractionation
Subcellular isolation of RNAs in SKBR3 and MCF7 cells was performed by Cytoplasmic and Nuclear RNA Purification Kit (Norgenbiotek Corporation, Thorold, ON, Canada), followed by fraction analysis via qRT-PCR.

Fluorescence in situ hybridization (FISH) Assay
Fluorescence-conjugated FLVCR1-AS1 probes were produced by Bersinbio Company (Guangzhou, China). BC cells in paraformaldehyde were dehydrated with ethanol. Air-dried cells were denatured for incubation with FISH probes utilizing hybridization reaction buffer overnight. After washing by ×2 saline-sodium citrate, cells were dyed in Hoechst and the results were recorded by Zeiss LSM800 confocal laser microscopy (Zeiss, Oberkochen, Germany).

RNA immunoprecipitation (RIP)
RNA-binding protein immunoprecipitation kit (Millipore) was applied for performing the RIP assay. SKBR3 and MCF7 cells were lysed with lysis buffer and then incubated with anti-Ago2 and negative control anti-IgG. RNA enrichment was analyzed by qRT-PCR.

TUNEL assay
The apoptosis of SKBR3 and MCF7 cells were studied via TUNEL Apoptosis Kit (Invitrogen), with employment of DAPI (Koritai Biotechnology, Beijing, China) for dying. Cells were then observed and captured by fluorescence microscopy (Olympus, Tokyo, Japan).

Tumor growth in nude mice
Male nude mice were obtained commercially from Shi Laike Company (Shanghi, China). Cells transfected with sh-FLVCR1-AS1#1 or shCtrl were injected subcutaneously into mice. Tumor volumes were recorded every 4 day. All mice were sacrificed after 4 weeks, and tumors were removed, weighed. Approval of this animal study was obtained from the Animal Research Ethics Committee of Minhang Hospital, Fudan University.

Statistical analysis
GraphPad Prism 7.0 software (La Jolla, CA, USA) was applied for statistical analysis. Results were manifested as mean ± SD. The difference of groups was compared via Student's t test or one way ANOVA analysis. P < 0.05 Pan et al. Cancer Cell Int (2020) 20:214 indicated the statistical significance and all experiments were run in no less than triplicate.

FLVCR1-AS1 is aberrantly overexpressed in BC cells and silencing FLVCR1-AS1 can dampen the malignant behavior of BC
Firstly, qRT-PCR was carried out to explore the expression status of FLVCR1-AS1 in BC cells. As a result, we discovered that FLVCR1-AS1 was significantly up-regulated in BC cell lines (MDA-MB-231, T47D, BT-474, SKBR3 and MCF7) than normal MCF-10A cells (Fig. 1a). Before conducting loss-of-function experiments, we ensured the suppressed expression of FLVCR1-AS1 in SKBR3 and MCF7 cells (Fig. 1b). Thereafter, CCK8 and colony formation assays evaluated that FLVCR1-AS1 inhibition evidently crippled cell vitality (Fig. 1c). After counting colonies, we confirmed that FLVCR1-AS1 depletion could suppress BC cell proliferation (Fig. 1d). Next, TUNEL assay indicated significant increase of TUNEL positive cells after FLVCR1-AS1 knockdown, revealing suppression of FLVCR1-AS1 enhanced apoptosis ability (Fig. 1e). Moreover, we unveiled that compared with control group, knockdown of FLVCR1-AS1 apparently diminished the number of migrated and invaded cells (Fig. 1f, g). These functional experiments revealed that FLVCR1-AS1 overexpression in BC was positively related to BC progression in vitro. Subsequently, western blot manifested that silencing FLVCR1-AS1 notably augmented the expression of p53, Bax, but declined the level of Bcl-2, MMP2 and MMP7 (Fig. 1h). Expression change of these proteins further validated the tumor inhibition effect of FLVCR1-AS1 knockdown.

MYC enhances the transcriptional activity of FLVCR1-AS1
We supposed that transcription factor might play a role for the abnormal overexpression of FLVCR1-AS1 in BC. We found potential transcription factor responsible for FLVCR1-AS1 regulation via UCSC and Jaspar database (http://jaspa r.gener eg.net/). We selected MYC among all the transcription factors. Because MYC has been reported to drive signaling pathways and further promotes aggressive BC tumors. Identification of MYC target genes is crucial in signaling pathways that facilitates tumor development [20].
To determine the effects of MYC on FLVCR1-AS1, we overexpressed the expression of MYC (Fig. 2a) and then observed a significant up-regulation of FLVCR1-AS1 in MYC-overexpressed BC cells (Fig. 2b). Conversely, FLVCR1-AS1 expression was significantly decreased after knockdown of MYC (Fig. 2c, d). Above findings suggested the positive influence of MYC in modulating FLVCR1-AS1 expression. Through ChIP assay, we found that FLVCR1-AS1 promoter was largely enriched by antibodies targeting MYC rather than by those against IgG, which suggested that MYC interacted with FLVCR1-AS1 promoter (Fig. 2e). To probe into the impact of MYC on FLVCR1-AS1 transcription, luciferase reporter assay was performed in HEK-293T cell lines. Consequently, we observed that the promoter activity was predominantly attenuated after knockdown of MYC, but enhanced in the presence of MYC overexpression (Fig. 2f ). This demonstrated that MYC expedited the transcription of FLVCR1-AS1.
We then detected specific sequence on FLVCR1-AS1 promoter for the binding with MYC by browsing Jaspar. We found that − 697 to − 708 sites were the putative MYC-binding sites at the fragment P4 in FLVCR1-AS1 promoter region (Fig. 2g). Next, we performed ChIP assay to verify this putative MYC binding site. We found that MYC could bind to the P4 fragment in FLVCR1-AS1 promoter region (Fig. 2h). Then, luciferase report was performed to examine the effects of MYC on the transcription of FLVCR1-AS1 in SKBR3 and MCF7 cells. After co-transfection with WT-P4 or MUT-P4, we noticed that MYC overexpression sharply stimulated FLVCR1-AS1 promoter activity, while MYC knockdown greatly suppressed promoter activity in WT-P4 group. However, no significant promoter activity change was observed in MUT-P4 group (Fig. 2i). These data suggested that MYC stimulates FLVCR1-AS1 transcription in BC via interacting with FLVCR1-AS1 promoter at − 697 to − 708 site upstream transcriptional start site (TSS).

FLVCR1-AS1 contributes to Wnt/β-catenin pathway activation in BC
For the sake of understanding the potential mechanism whereby FLVCR1-AS1 affected BC development, we performed western blot to evaluate expression level of proteins related to major pathways associated with BC, such as PI3K/AKT, Wnt and NF-κB pathways. Interestingly, only the level of β-catenin was greatly decreased after FLVCR1-AS1 knockdown instead of p-AKT (PI3K/AKT pathway) or p-p65 (NF-κB) pathway (Fig. 3a). Hence, we preliminarily judged that Wnt/β-catenin signaling pathway might be implicated in FLVCR1-AS1 regulated BC. TOP/FOP Flash assay was performed to evaluate the activity of Wnt/β-catenin after knockdown of FLVCR1-AS1. It manifested that FLVCR1-AS1 knockdown significantly dampened the activity of this pathway (Fig. 3b).
To further illustrate whether FLVCR1-AS1 impacted Wnt/β-catenin pathway, we used LiCl, the agonist of Wnt/β-catenin pathway, and evaluated the changes of FLVCR1-AS1 silencing impacted cellular functions. We found that cell proliferation ability was impaired after Pan et al. Cancer Cell Int (2020) 20:214 Fig. 1 FLVCR1-AS1 is aberrantly up-regulated in BC. a qRT-PCR assay was used to detect FLVCR1-AS1 expression in BC cell lines and normal cell line. b The relative expression of FLVCR1-AS1 after knocked down was determined by qRT-PCR. c, d CCK8 and colony formation were performed to assess cell proliferation after silencing FLVCR1-AS1. e TUNEL was performed to investigate apoptosis after knockdown of FLVCR1-AS1. f, g Transwell assays were conducted to determine cell migration and invasion abilities respectively after silencing FLVCR1-AS1. h Western blot was performed to examine the expression of proteins associated with cell apoptosis and migration. ** P < 0.01 FLVCR1-AS1inhibition, but recovered in response to LiCl treatment (Fig. 3c). On the contrary, the apoptosis rate was increased after silencing FLVCR1-AS1, but decreased again after adding LiCl (Fig. 3d). Transwell assays found that migrated and invaded cells were decreased after silencing FLVCR1-AS1, but increased sharply again after LiCl treatment (Fig. 3e, f ). We then performed qRT-PCR to validate the effects of FLVCR1-AS1 on CTNNB1. Expectedly, FLVCR1-AS1 knockdown led to obvious down-regulation on CTNNB1 expression (Fig. 3g). These findings indicated that FLVCR1-AS1 affected BC malignant behaviors via Wnt/β-catenin pathway. Thereafter, we wanted to know by which manner FLVCR1-AS1 could regulate this pathway. Thus, we detected the subcellular location of FLVCR1-AS1 and determined FLVCR1-AS1 was mainly a cytoplasmic lncRNA with subcellular fractionation and FISH (Fig. 3h), which indicating the possible ceRNA role of FLVCR1-AS1. Hence, we presumed that FLVCR1-AS1 might function as ceRNA to modulate the expression of CTNNB1

MiR-381-3p is the target of FLVCR1-AS1
We found two shared combinable miRNAs with FLVCR1-AS1 and CTNNB1 by browsing Starbase (Fig. 4a). Of note, the expression of miR-381-3p was enhanced much more dramatically under FLVCR1-AS1 depletion (Fig. 4b). Besides, miR-300 has been reported to be an oncogene in BC [21]. On the contrary, miR-381-3p has been uncovered as tumor inhibitor in BC [22]. Hence, we chose miR-381-3p as the focus of this study. It was disclosed that miR-381-3p was obviously down-regulated in BC cell lines compared with normal one (Fig. 4c). Moreover, putative miR-381-3p binding site to FLVCR1-AS1 was revealed by starBase (Fig. 4d). More importantly, after enhancing miR-381-3p expression, we observed an evident weakness on the luciferase activity of FLVCR1-AS1-WT group, but no distinct luciferase activity change in FLVCR1-AS1-MUT group (Fig. 4e). RNA pull down showed that miR-381-3p expression was more Fig. 3 FLVCR1-AS1 activates Wnt/β-catenin pathway. a Western blot was performed to measure expression of relevant proteins engaging in common tumor signaling pathway after knockdown of FLVCR1-AS1. b FOP/TOP assay was used to verify the activity of Wnt/β-catenin Pathway. c MTT was performed to assess cell proliferation after adding agonist of Wnt/β-catenin pathway. d TUNEL was performed to investigate apoptosis after adding agonist of Wnt/β-catenin pathway. e, f Transwell assays were conducted to determine cell migration and invasion abilities respectively after adding agonist of Wnt/β-catenin pathway. g qRT-PCR was used to determine expression of CTNNB1 after transfecting with sh-FLVCR1-AS1#1. h Subcellular fractionation and FISH were performed to detect the location of FLVCR1-AS1. ** P < 0. enriched by biotinylated FLVCR1-AS1-WT than negative control groups (Fig. 4f ). From above results, we identified that FLVCR1-AS1 served as a miR-381-3p sponge in BC. In subsequence, rescue assays were implemented to determine the role of FLVCR1-AS1/miR-381-3p axis in BC. Prior to that, we confirmed the inhibition effect of miR-381-3p inhibitor on miR-381-3p expression (Fig. 4g). As anticipated, miR-381-3p inhibition reversed the restrained proliferation in FLVCR1-AS1-silenced BC cells (Fig. 4h). TUNEL manifested that inhibiting miR-381-3p abrogated the pro-apoptosis ability of suppressed FLVCR1-AS1 (Fig. 4i). We also observed that the notably lowered number of migrated and invaded cells upon FLVCR1-AS1 suppression was normalized by miR-381-3p inhibitor (Fig. 4j, k). Then, the results of western blot further proved that inhibition of miR-381-3p offset the anti-tumor effects of FLVCR1-AS1 silence (Fig. 4l).
Xenografts model was used to further validate the effects of FLVCR1-AS1 on tumor growth in vivo. As indicated, suppression of FLVCR1-AS1 conspicuously slowed down the rate of tumor growth compared to control group (Fig. 5j). We also found apparently lessened volume and weight in tumors with FLVCR1-AS1 inhibition in comparison to those without (Fig. 5k). These experiments in vivo demonstrated that FLVCR1-AS1 promoted BC tumor growth in vivo. Together, FLVCR1-AS1 exerts its oncogenic effects on the malignant behaviors of BC by elevating CTNNB1 level through sponging miR-381-3p.

Discussion
Despite rapid progression in the diagnosis and treatment and of BC, the mortality rate still remains a challenge that needs the determination of sensitive targets. To improve early diagnosis and therapeutic methods, identifying novel molecular targets for BC is becoming increasingly paramount. Accumulating studies have uncovered the crucial ceRNA roles of lncRNAs in the occurrence and development of a wide array of human diseases [23][24][25][26]. Increasing reports have demonstrated that lncRNAs can play important regulatory role in many biological activities and are correlated with the carcinogenesis of cancers [25]. Determining the relationship between lncRNAs and their downstream targets would contribute to the diagnosis and treatment of patients with BC.
In present study, we found that FLVCR1-AS1 was significantly up-regulated in BC cell lines. Knockdown of FLVCR1-AS1 sharply suppressed BC cell proliferation, migration and invasion, while stimulating cell apoptosis in vitro. Besides, the expression of FLVCR1-AS1 was found to be positively correlated with tumor growth, size and volume in vivo, which supported that FLVCR1-AS1 played an oncogenic role in BC. Besides, MYC transcriptionally activated FLVCR1-AS1 in BC.
MiR-381-3p was identified as a potential target gene of FLVCR1-AS1. MiR-381-3p has been discovered to be a tumor suppressor gene and reported to be downregulated in various human malignancies, including cervical cancer, bladder cancer, oral squamous cell carcinoma as well as BC [27][28][29]. We verified the interaction between miR-381-3p and cytoplasmic FLVCR1-AS1. Furthermore, miR-381-3p inhibition reversed the suppressing effects of sh-FLVCR1-AS1 on malignant behaviors of BC cells in vitro, which indicated that FLVCR1-AS1 exerted its oncogenic role in BC via sponging miR-381-3p. We observed that only β-catenin associated with Wnt/β-catenin was significantly down-regulated after FLVCR1-AS1 knockdown. We observed that LiCl, as agonist of Wnt/β-catenin pathway, could abolish the anti-tumor effects of FLVCR1-AS1 knockdown. Subsequently, we identified that CTNNB1 was the target gene of miR-381-3p. Additionally, CTNNB1 has been extensively reported to play an oncogenic role and predicted poor prognosis in multiple cancers. In present study, we found that overexpression of CTNNB1 could abrogate the tumor-inhibiting ability of sh-FLVCR1-AS1. In other words, CTNNB1 up-regulation could rescue the antioncogenic function of FLVCR1-AS1 depletion. Together, our finding initially suggested that lncRNA FLVCR1-AS1 could function as miR-381-3p sponge and up-regulate the expression of CTNNB1 and activate Wnt/β-catenin pathway, consequently aggravating BC malignant progresses.

Conclusion
This study found the MYC/FLVCR1-AS1/miR-381-3p/ CTNNB1 axis in BC initially. We identified the oncogenic role of FLVCR1-AS1 in BC progression. It could hopefully be applied as a potential diagnostic and therapeutic biomarker for patients with BC.