Knockdown of lncRNA FEZF1-AS1 Inhibits metastasis of Osteosarcoma Cells by miR-4456/GALNT10

Background: Osteosarcoma (OS) is among the malignant tumors with high mortality and low survival, especially in children and adolescents. Research shows that LncRNA FEZ family zinc nger 1 antisense RNA 1 (FEZF1-AS1) enhances osteosarcoma progression. Nevertheless, the function and mechanism of FEZF1-AS1 in metastasis of OS remains unclear. Methods: We used qRT-PCR to assay for the expression of FEZF1-AS1, miR-4456, and GALNT10 in OS tissue specimens and cell lines. We also investigated the progression of OS through metastasis using the wound healing and Transwell assays. Moreover, we used the dual-luciferase reporter test, RIP assays, and western blot to validate whether FEZF1-AS1 serves as a competing endogenous RNA (ceRNA), modulating the expression of GALNT10 through sponging miR-4456 in OS. Results: FEZF1-AS1 was up modulated in OS tissues. Silencing FEZF1-AS1 repressed OS cell migration and invasion. microRNA-4456 (miR-4456) was involved in FEZF1-AS1-induced migration and invasion. miR-4456 was down modulated in OS tissue specimens and cell lines. Functionally, the up modulation of miR-4456 reversed the facilitative inuence of FEZF1-AS1 on OS cell inltration and migration. Mechanically, FEZF1-AS1 interacted with miR-4456 in a reciprocal suppressed manner. Moreover, miR-4456 targets GALNT10 via the Luciferase assay. Besides, the up modulation of GALNT10 reversed the migration and invasion inhibited by FEZF1-AS1 knockdown. Silencing of FEZF1-AS1 inhibits OS cell inltration and migration through miR-4456 /GALNT10 sponging. Conclusion: Herein, we demonstrated that FEZF1-AS1 is a prospective bio signature of metastasis in OS patients. Mechanistically, we showed that the FEZF1-AS1/miR-4456/GALNT10 axis is a target for novel therapeutic development for OS.


Introduction
OS is the most frequent bone tumor in children and teenagers with high fatality rates [1]. Despite the much effort spent over the decades, the overall survival of OS patients remains dismal [2]. In most instances, the malignancies metastasize to the lung tissues in OS patients [3]. Standard neoadjuvant chemotherapy successfully raises the likelihood of limb rescue, which considerably enhances the 5-year survival rate and quality of life in OS patients [4]. The results of previous studies posit that lymph-node metastasis and distant metastasis of malignancies is a multiplex and multiphase process entailing the participation of genes, proteins, miRNAs, and signaling cascades [5,6]. Therefore, it is urgent to comprehend the pathogenesis of OS metastasis to enhance the outcomes in patients.
Nevertheless, the role of FEZF1-AS1 in OS metastasis has not been elucidated.
Research evidence shows that miRNAs participate in the pathogenesis of OS by functioning as oncogenes or tumor inhibitors via various cascades [4,15]. The ndings of early studies indicate that the atypical expression of miRNAs is closely linked to OS progress [16]. For instance, miR-451a hinders the growth and metastatic phenotypes of papillary thyroid carcinoma cells through repressing ZEB1 [4] and miR-134/ VEGFA suppresses tumorigenesis and progression by mediated IRF1 in OS [15]. These data imply that miRNAs play a critical role in OS. Nevertheless, the function of miR-4456 in OS remains unclear. miR-4456 plays a suppressive role in AKI [17]. Additionally, miR-4456 is down modulated in Hypersexual disorder [18].
Glycosyltransferase, also referred to as glycogene, is a kind of enzyme that moderates the modi cations of proteins and lipids [19]. It serves an essential function in normal cell development and other physiological processes [20]. Moreover, it is a crucial biomarker in cancer [21]. For instance, it represses the Hnf4α/ miR-122 cascade in hepatitis B virus-correlated hepatocellular carcinoma and facilitates the activity of the prospective oncogenic GALNT10 protein [22]. Nevertheless, the function and mechanism of miR-4455 and GALNT10 in OS metastasis remains unclear.
Here, we elucidated the expression of FEZF1-AS1 in OS tissues and cell lines, as well as the knockdown of FEZF1-AS1suppressed metastasis. Therefore, we inspected the therapeutic in uence of FEZF1-AS1 on OS and the ceRNA modulatory network of FEZF1-AS1/miR-4456/GALNT10.

Human tissue samples
We collected 31 pairs of specimens consisting of osteosarcoma and adjacent normal tissues from osteosarcoma patients (from January 2013 to November 2019) at The Tumor Hospital of Harbin Medical University. The Ethics Committee of The A liated Hospital of Harbin Medical University approved this study, and we obtained written informed permission from all the patients. Clinicopathological parameters of OS patients were showed in Table1. All tissues were harvested and then kept at -80℃ or snap-frozen in liquid nitrogen immediately pending use.

Cell transfection
The FEZF1-AS1-siRNA, miR-4456 mimics, miR-4456 inhibitor, GALNT10 plasmids, and their corresponding negative controls (shNC, NC mimics, NC inhibitor, pcDNA3.1) were constructed and provided by RIBIOBIO (Guangzhou, China). We grew the OS cells in the medium until a con uence of approximately 60-70%. After that, we transfected all the plasmids mentioned above into 143B or U2OS using the riboFECT CP Transfection Kit (C10511-1, RIBIOBIO, Guangzhou, China) as per instructions of the manufacturer. After 48h or 72 h following transfection, we attained the OS cells for downstream experiments.

RT-qPCR
To assess the expression of FEZF1-AS1, GLANT10, and miR-4456 in OS tissue or cells, we performed RT-qPCR. The trizol reagent (Cat: 15596026, Invitrogen, Carlsbad, USA) was utilized to extract total RNA as per the protocol of the manufacturer. The TransScript Green One-Step qRT-PCR SuperMix kit ( Takara, Kyoto, Japan) was used to assay for the relative gene expression via qPCR. GAPDH or U6 was used as an internal control. The 2−ΔΔ-Ct approach was used to examine the relative level of expression of genes in OS. The sequences of the primers used in this study are shown in Table S1.

Western blot assay
We harvested the treated cells and lysed the using RIPA (Radio Immunoprecipitation Assay, Beyotime, Shanghai, China) buffer. We separated the cell lysates on 12.5% SDS/PAGE (sodium dodecyl sulfatepolyacrylamide), then transferred onto PVDF (polyvinylidene uoride membrane, Millipore, Billerica, USA) membranes, followed by blocking with 5% non-fat dried milk for 1h at room temperature (RT). After that, we incubated the membranes with primary antibodies at 4℃ against GALNT10, E-cadherin, Vimentin, and GAPDH. Subsequently, we incubated the membranes with the second antibodies separately at RT for 2h, and the proteins detected using an EasyBlot ECL kit (ECL kit, Santa Cruz Biotechnology, Santa Cruz, USA). The antibodies targeting GALNT10, E-cadherin, Vimentin, and GAPDH were all purchased from Abcam Co., Ltd. (Pudong, Shanghai, China). The density of the bands was imaged via enhanced chemiluminescence. GAPDH was employed as an endogenous control.

Bioinformatics Analysis and Luciferase Reporter Assay
We predicted the putative binding sites of miR-4456 and FEZF1-AS1 or GALNT10 via bioinformatics analysis using starbase 2.0 (starbase.sysu.edu.cn). We generated the wild-type vectors (FEZF1-AS1-WT and GALNT10-WT) and mutant-type vectors (FEZF1-AS1-Mut and GALNT10-Mut) using the pGL3 vector (Promega, Madison, WI, USA). In the luciferase reporter test, we used the riboFECT CP Transfection Kit (Catalogue number, manufacturer, country) to co-transfect the U2OS cells with WT or Mut luciferase reporter vectors and miR-4456 mimic or miR-NC mimic. Following 48h, post-transfection, we conducted the luciferase enzyme activity test in each group using the luciferase reporter assay kit (Catalogue number, Promega, country). The luciferase activity of Renilla was employed as the standard.

RNA Immunoprecipitation (RIP)
We harvested 1×10 7 U2OS and 143B cells with or without miR-4456 mimic and then lysed them in RIP buffer. We performed the RIP test using a Magna RNA immunoprecipitation kit (Millipore, Billerica, MA, USA), as well as magnetic beads, pre-coated with Ago2 or IgG antibody. The levels of FEZF1-AS1 in the complex were assayed via qRT-PCR after the isolation of RNA using the Trizol reagent.

Trans-well assay
Trans-well assays were used to detected OS cell invasion. We used the Trans-well chambers (Corning, NY, USA) to observe OS cell in ltration. We seeded 200µl of OS cell suspension (1 × 10 5 cells) in the serumfree medium into the upper chamber, which was coated with Matrigel (BD Biosciences, San Diego, CA, USA). After that, we added 800µl of DMEM added with 10% FBS to the lower chamber. Following 48h of incubation, we removed the cells on the top side of the membrane via a swab. We xed the in ltrative cells on the bottom and stained with 0.1% crystal violet for 15min. The number of invading cells was estimated using a phase-contrast microscope (Olympus, Tokyo, Japan).

Wound healing assays
We seeded the OS cells into 6-well plates and then utilized a sterile 10μl plastic micropipette tip to scratch through the monolayer. After that, we washed the cells thrice using physiological saline, then replaced it with DMEM medium added 0.2% FBS. The cells were scratches for 24h and 48h, then observed the migration of the cells using a microscope (indicate which type of microscope and the manufacturer).

Immunohistochemistry
We xed the tissue samples in 10% formalin and then sliced them into 4μm sections. Subsequently, we incubated the sections at 60ºC for 1h, followed by conventional xylene dewaxing and gradient alcohol dehydration. Then, we blocked the sections blocked using normal goat serum solution at 37°C for 10min, followed by incubation with the primary antibody, anti-GALNT10 for 12h at 4°C. After that, we incubated the sections with the secondary antibody, biotinylated anti-mouse for 10min at RT. The image analysis software (Nikon, Tokyo, Japan) was utilized to count the number of positive cells. The percentage proportion of the positive cells in each eld was computed; the percentage of <10% was regarded as negative, and > 10% was considered as positive.

Statistical analysis
All statistical analyses were conducted independently three to six times in the SPSS 20.0 software (Chicago, IL, USA). Student's t-test and ANOVA were applied in determining the signi cance of differences. A P < .05 signi ed a marked difference.

FEZF1-AS1 is up modulated in OS
We performed qRT-PCR assays to determine the expression level of FEZF1-AS1 in OS, and the results are shown in Figure 1A. FEZF1-AS1 was up modulated in OS tissue samples. At the same time, the up modulation of FEZF1-AS1 was more frequent in OS tissue samples with lymph node metastasis and distant metastasis ( Figure 1B). We additionally examined FEZF1-AS1 levels in normal human osteoblast cell line (hFOB) and osteosarcoma cell lines (143B, U2OS, MG63, and HOS). The levels of FEZF1-AS1 in the OS cell lines were 1.5-3.6-fold higher than in the hFOB cells (Fig. 1C). These data indicate that the upregulation of FEZF1-AS1 in OS causes a dismal prognosis.

FEZF1-AS1-si represses OS cell migration and invasion
We conducted the qRT-PCR test to assess the expression of FEZF1-AS1 in 143B and U2OS cells inserted with FEZF1-AS1-siRNA via transfection (Figure 2A). The wound-healing assay indicated that the metastasis potential was distinctly elevated in the 143B and U2OS cells with the transfection of FEZF1-AS1-siRNA ( Figure 2B-C). The transwell assays revealed that FEZF1-AS1-siRNA silencing suppressed remarkably the migration of 143B and U2OS compared with the control (Figure 2D-E). These data show that FEZF1-AS1-siRNA inhibits osteosarcoma cell in ltration and migration. We evaluated the protein levels of EMT biosignatures to gather more evidence. Consequently, E-cadherin was remarkably elevated, whereas Vimentin markedly decreased in the FEZF1-AS1 silenced group. These data indicated that FEZF1-AS1 reduces cell metastasis in OS.

FEZF1-AS1 is a ceRNA of miR-4456
CeRNAs serves a crucial role in the mechanisms of lncRNAs, miRNA, and mRNA in OS [23]. Therefore, we analyzed the miRNA-binding site in FEZF1-AS1 using bioinformatics tools. We found a potential miR-4456 binding site in FEZF1-AS1 (Fig. 3A) and, then, investigated the level of miR-4456 in U2OS cells. The expression of miR-4456 was distinctly reduced in U2OS cells compared with the hFOB cells (Fig. 3B), which was negatively associated with FEZF1-AS1 levels. Depletion of FEZF1-AS1 led to increased levels of miR-4456 (Fig. 3C), whereas over-expression of miR-4456 suppressed FEZF1-AS1 levels (Fig. 3D). We additionally found that miR-4456 mimics remarkably reduced luciferase activity in FEZF1-AS1-WT compared with the FEZF1-AS1-Mut group (Fig. 3E). Therefore, FEZF1-AS1 is a ceRNA of miR-4456. FEZF1-AS1 serves as a ceRNA for miR-4456 to enhance GALNT10 expression miRNAs generally suppress gene functions by targeting the 3′-UTR of their target genes [24]. Therefore, we used several bioinformatics tools to identify miR-4456 targets in OS. Subsequently, the potent oncogene GALNT10 was revealed as a possible target (Fig. 4A). In the tissues obtained from the OS patients, GALNT10 expression was remarkably higher (2.3-fold) than that in the normal tissues (Fig. 4B-C). Furthermore, we found that the expressions of GALNT10 and FEZF1-AS1 were positively associated (Fig.4D). The transfection of miR-4456 mimics decreased GALNT10 mRNA and protein levels (Fig. E, F). A dual-luciferase reporter test was conducted to examine if GALNT10 is a functional target of miR-4456. Our data indicated that only the co-transfection with miR-4456 mimics and the wild-type binding site of GALNT10 (GALNT10-WT) reduced luciferase activity (Fig. 4G). These data reveal that GALNT10 is a direct target of miR-4456.
Since we identi ed FEZF1-AS1 as a ceRNA of miR-4456, we next examined if FEZF1-AS1 controlled GALNT10 expression. GALNT10 protein levels were repressed by FEZF1-AS1 siRNA transfection (Fig.4H). A dual-luciferase reporter test demonstrated that the depletion of FEZF1-AS1 markedly reduced the luciferase activity in GALNT10-WT (Fig. 4I). Next, we examined the presence of FEZF1-AS1 and miR-4456. U2OS cell extracts were immunoprecipitated against Ago2, and mRNA levels in immunoprecipitants were examined by real-time PCR. Both FEZF1-AS1 and miR-4456 were augmented in Ago2 pellets relative to the IgG control (Fig. 4J). These data demonstrate that FEZF1-AS1 is a ceRNA for miR-4456 in promoting GALNT10 expression.
Over-expression GALNT10 expression reverses the repression of OS metastasis stimulated by FEZF1-AS1 silencing A GALNT10 expression vector was prepared and co-transfected with FEZF1-AS1 siRNA into U2OS cells to con rm the role of FEZF1-AS1 via GALNT10 in OS cells. Consequently, FEZF1-AS1 siRNA decreased FEZF1-AS1 levels and GALNT10 expression. Co-transfection with a GALNT10 expression vector reestablished GALNT10 expression in U2OS cells (Fig. 5A). The metastasis assay showed that restored GALNT10 expression augmented metastasis development (Fig. 5B). Therefore, FEZF1-AS1 regulated OS cell growth via GALNT10.

Discussion
Herein, we elucidated the therapeutic function of FEZF1-AS1 silencing in the progression of OS. We established that the expression of FEZF1-AS1 was augmented in OS tissues as well as cell lines. Knockdown on FEZF1-AS1 was correlated with reduced OS cell in ltration and migration. Besides, we established that the mechanism through which FEZF1-AS1 participates in OS metastasis is mediated by miR-4456 and GALNT10. Additionally, we identi ed a novel prospective mechanism of FEZF1-AS1, which could be essential in OS drug design.
Research evidence shows that the ceRNA modulatory network of lncRNA/miRNA/mRNA is signi cant in the progression, reoccurrence, and therapy of OS [25]. FEZF1-AS1 enhances oncogenesis and malignancy in OS [13,14]. Nevertheless, the function and mechanism of FEZF1-AS1 in OS metastasis remains unclear. Here, we demonstrated that FEZF1-AS1 is up modulated in OS tissues and cell lines and is associated with metastasis, implying that FEZF1-AS1 is an oncogene in OS consistent with the ndings of previous studies (insert citations). We used the loss-of-function assays in this study and demonstrated that silencing FEZF1-AS1 represses OS cell metastasis vial the wound-healing and trans-well tests. Hence, the FEZF1-AS1 knockdown has a therapeutic effect on OS.
In the present study, we focused on unraveling the novel molecular cascade of FEZF1-AS1 in OS progression. The ceRNA modulatory network constitutes the primary mechanism of lncRNA in human tumors [26]. The ndings of several studies indicate that FEZF1-AS1 promotes NSCLC and invasion through the up-regulation of NOTCH1 by serving as a sponge of miR-34a [10]. FEZF1-AS1 serves as a ceRNA sponge of miR-144/CXCR4 to facilitate cell proliferation, Warburg Effect, and represses cell death in OS [13]. The prospective interaction of FEZF1-AS1 with miRNA was explored using bioinformatics tools to investigate the potential mechanism of FEZF1-AS1 in OS. Here, we rst veri ed FEZF1-AS1 as a mimic of miR-4456 in OS cells using the luciferase reporter test. Our data showed that miR-4456 is down modulated in OS, revealing that miR-4456 has a tumor-inhibitory function. miR-4456 was inversely correlated with FEZF1-AS1 mRNA expression in OS tissues. The knockdown of FEZF1-AS1 up modulated the expression of miR-4456, while over-expression of miR-4456 inhibited the expression of FEZF1-AS1.
Luciferase assay data showed that miR-4456 mimics the interface the 3'UTR of FEZF1-AS1-WT, whereas it cannot regulate FEZF1-AS1-Mut. These data suggest that FEZF1-AS1 moderated OS progression by sponging miR-4456. To explore further the ceRNA modulatory network, we validated GALNT10 as a functional target of miR-4456 via bioinformatics analyses and the luciferase reporter test. We established that the expression of GALNT10 is up modulated in OS. GALNT10 is an oncogene in many cancers [21].
GALNT10 participates in viral invasion or replication, immune cell migration, and viral clearance from tissues [27]. Our data showed GALNT10 was down modulated by miR-4456 but positively associated with FEZF1-AS1 in OS. This indicates that FEZF1-AS1 acts as a ceRNA for miR-4456 to repress GALNT10 levels in OS cells. Additionally, we demonstrated that the over-expression of GALNT10 reverses the metastasis inhibition of OS cells induced by FEZF1-AS1 knockdown, indicating that FEZF1-AS1 promotes OS cell metastasis, at least partially through GALNT10.
In conclusion, here, we elucidated the in uence of silencing FEZF1-AS1 on OS metastasis by targeting miR-4456/GALNT10. We rst provide the ceRNA modulatory network of FEZF1-AS1/miR-4456/GALNT10 in OS metastasis, revealing FEZF1-AS1 as a novel target of OS metastasis therapy.

Declarations
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