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

Human normal breast epithelial cell (MCF-10A) and BC cells (MDA-MB-231, T47D, BT-474, SKBR3, MCF7) were bought from Chinese Academy of Sciences (Beijing, China). Cells were grown in RPMI-1640 medium (Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS; Invitrogen) and 1% penicillin/streptomycin (Sigma-Aldrich, Milan, Italy) with 5% CO₂ at 37 °C. LiCL (Taili industrial co. LTD, Shanghai, China), the Wnt/β-catenin pathway activator, was added into culture medium for treating SKBR3 and MCF7 cells.

SKBR3 and MCF7 cells were transfected with specific shRNAs against FLVCR1 (sh-FLVCR1-AS1#1#2), MYC (sh-MYC) and negative control (shCtrl), as well as pcDNA3.1/CTNNB1, pcDNA3.1/MYC and the empty pcDNA3.1 vector (all purchased from GenePharma, Shanghai, China), separately. The miR-381-3p mimics, miR-381-3p inhibitor, NC mimics and NC inhibitor were simultaneously constructed by GenePharma. Transfection was conducted for 48 h in light of the protocol of Lipofectamine2000 (Invitrogen)

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.

Cells (1 × 10⁴) in 96-well plates were cultured with10 µL CCK-8 reagent over specific time points. Absorbance was evaluated at 450 nm using a microplate reader (Bio-Tek Instruments, Hopkinton, MA, USA).

1 × 10³ cells were cultured in 6-well plates for 2 weeks. After fixed in methanol (Solarbio, Beijing, China), colonies were processed with crystal violet (Sigma-Aldrich). Visible colonies were counted manually under microscope (Olympus, Tokyo, Japan).

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).

2 × 10⁴ 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).

Total protein was obtained from cells which were lysed by RIPA lysis buffer. Protein concentration was measured with BCA Kit (Pierce, Appleton, WI, USA). We isolated proteins with SDS-PAGE, which were moved to PVDF membranes. 5% skimmed milk was used for blocking membranes, and then were co-cultured with primary antibodies for p53 (ab32389), Bax (ab32503), Bcl-2 (ab185002), MMP2 (ab215986), MMP7 (ab205525), AKT (ab179463), p-AKT (ab38449), β-catenin (ab32572), p65 (ab16502), p-p65 (ab6503) and GAPDH (ab8245) from Abcam (Cambridge, USA). GAPDH was used as internal control. Secondary antibodies were added to incubate for 1 h. Amount of proteins was examined via chemiluminescence detection system. IMAGEJ software (National Institutes of Health, Bethesda, Maryland, USA) was utilized to quantify protein bands, with GAPDH as normalized control.

ChIP experiment was processed with usage of Magna ChIP Kit (Millipore, Darmstadt, Germany). After cross-linked 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.

For FLVCR1-AS1 promoter analysis, the pGL3-Basis reporter (Promega, Madison, WI, USA) vector containing FLVCR1-AS1 promoter was co-transfected with pcDNA3.1/MYC and empty pcDNA3.1 vector, or shCtrl and sh-MYC into cells. The wild-type (WT) and mutant (Mut) binding sites of miR-381-3p mimics in FLVCR1-AS1 sequence or CTNNB1 3′UTR was sub-cloned into pmirGLO dual-luciferase vector (Promega) to construct FLVCR1-AS1-WT/Mut or CTNNB1-WT/Mut, then co-transfected with miR-381-3p mimics or NC mimics into cells. To conduct TOP/FOP-Flash analysis, TOP/FOP-Flash (Genechem) was co-transfected into SKBR3 and MCF7 cells along with sh-FLVCR1-AS1#1 or shCtrl. The luciferase activity was lastly determined via Dual-Luciferase Reporter Assay System (Promega).

1 × 10³ cells in 96-well plates were mixed with 20 µL MTT reagents over 24, 48,72 and 96 h. Later, dimethyl sulfoxide (DMSO) was added. The optical density at 490 nm was analyzed by micro-plate reader (Bio-Tek Instruments, Hopkinton, MA, USA).

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-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).

The FLVCR1-AS1-WT, FLVCR1-AS1-Mut and NC were biotin labeled into Biotin FLVCR1-AS1 WT, Biotin FLVCR1-AS1 Mut and Biotin Ctrl, severally. Then, cell lysates were cultured with the biotinylated probe and M-280 streptavidin magnetic beads (Sigma-Aldrich). The miR-381-3p levels were analyzed by qRT-PCR.

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.

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).

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.

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.

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://​jaspar.​genereg.​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).

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 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

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 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).

Potential miR-381-3p binding site inCTNNB1 3′UTR was determined via Starbase (Fig. 4m). Luciferase reporter assay exhibited that enhanced expression of miR-381-3p remarkably attenuated the luciferase activity of CTNNB1-WT (Fig. 4n). RIP found significant enrichment of miR-381-3p, CTNNB1 and FLVCR1-AS1 in RISC (Fig. 4o). These findings indicated that FLVCR1-AS1 competitively bind to miR-381-3p against CTNNB1.

Finally, we intended to validate whether CTNNB1 mediated the function of FLVCR1-AS1 in BC. As observed, CTNNB1 possessed a significant up-regulation in BC cell lines relative to normal MCF-10A cells (Fig. 5a). Then, we verified that co-transfection of pcDNA3.1/CTNNB1 could normalize CTNNB1 level in FLVCR1-AS1-inhibited BC cells (Fig. 5b, c). Result of western blot analysis showed the same effects on β-catenin level (protein form of CTNNB1) (Fig. 5d). Functionally, silencing FLVCR1-AS1 inhibited cell vitality was enhanced again by CTNNB1 overexpression (Fig. 5e). TUNEL result manifested that upregulated CTNNB1 abolished the apoptosis-facilitating effects of FLVCR1-AS1 knockdown (Fig. 5f). Transwell results suggested that overexpressing CTNNB1 sharply reversed the hindered motility caused by depleted FLVCR1-AS1 (Fig. 5g, h). Such observation was further confirmed in Fig. 5i.

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.