LAIR-1 overexpression inhibits osteosarcoma epithelial-mesenchymal transition via GLUT1-related energy metabolism CURRENT STATUS: UNDER REVIEW

Background: Leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1) is a collagen receptor belonging to the immunoglobulin superfamily. Although prior studies have evaluated the biological role of LAIR in solid tumors, the precise mechanisms underlying LAIR-1 functions as a regulator of tumor biological functions remains unclear. Methods: LAIR-1 expression was evaluated using an osteosarcoma (OS) tissue microarray by immunohistochemical analysis. Wound healing and Transwell assays were performed to evaluate tumor cell migration. Quantitative PCR and western blotting were conducted to detect the expression of epithelial-mesenchymal transition (EMT)-related molecules. RNA-sequencing (RNA-seq) was conducted to evaluate the mRNA expression profiles after overexpressing LAIR-1 in OS cells. Glucose uptake and glucose transporter (Glut) 1 expression in OS cells in vitro were evaluated by flow cytometry and western blotting. Results: LAIR-1 expression significantly differed between the T1 and T2 stages of OS tumors, and LAIR-1 overexpression inhibited OS cell migration. LAIR-1 expression was inversely correlated with the expression of EMT-associated transcription factors via the Forkhead box O1/Twist1 signal transduction pathway. Furthermore, RNA-seq and quantitative PCR demonstrated that EMT energy metabolism-related molecules were significantly reduced after LAIR-1 overexpression. Conclusions: Notably, overexpression of LAIR-1 in OS cells decreased Glut1 expression. These findings provide insight into the molecular mechanism underlying OS progression.


Background
Osteosarcoma (OS) is the most common malignant solid bone tumor in children and young adults, accounting for 6% of all pediatric cancers and typically originating in the metaphyses of long bones [1,2]. Multidisciplinary approaches have been developed for treating patients with OS [3,4]; however, their overall prognosis remains unsatisfactory, with a 5-year survival rate as low as 20% [5][6][7]. Therefore, new and effective targets for OS diagnosis, therapy, and prognosis are needed.
In this study, we measured LAIR-1 expression in OS tissues to evaluate the roles of LAIR-1 in OS progression. We overexpressed LAIR-1 in OS cell lines by lentiviral transfection, and then measured cell proliferation, epithelial-mesenchymal transition (EMT)-associated transcription factor expression, and cell migration. Furthermore, EMT-related energy metabolism was examined after LAIR-1 overexpression. Our study provides insight into the role of LAIR-1 in OS.

Materials And Methods 2.1. Immunohistochemistry (IHC)
We used a formalin-fixed paraffin-embedded tissue microarray comprising 62 samples from patients with OS and nine samples from adjacent normal rib bone tissues (Alenabio Biological Technology Company; Xi'an, Shaanxi, China). Clinicopathological data were collected from the medical records of surgically treated patients with OS, including age, sex, pathological diagnosis, TNM grading, and stage. No patients had been administered preoperative treatment or had the co-occurrence of other diagnosed tumors. The sample size of patients with the T3 stage was small (n = 3), and thus this group was excluded from analysis. Samples for IHC were prepared using a standard method.

Semi-quantitative analysis of immunohistochemical data and bioinformatics analysis
All tissue samples were evaluated by two independent pathologists blinded to the clinical data. A semi-quantitative score was generated based on the IHC staining intensity as follows: +, weak staining; ++, moderate staining; +++, intense staining. The R2 platform (http://r2.amc.nl) was used to analyze the public OS dataset, which includes 127 OS samples.

Cell culture
Human OS cell lines were purchased from ATCC (Manassas, VA, USA), and the human normal osteoblast cell line hFOB1.19 was obtained from Jennio Biotech (Guangzhou, Guangdong, China). All cells were cultured at 37 °C in a humidified atmosphere containing 5% CO 2 . Foxo1 short interfering RNA (siRNA) and negative control siRNA were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Cells were transfected with 50 nM siRNA or negative control (NC) siRNA using Lipofectamine

Lentivirus infection
Commercially available lentiviral LV-LAIR-1 constructs (Tianyucheng Biotechnology, Xi'an, Shaanxi, China) were modified to overexpress LAIR-1. Human OS cells were infected with LV-negative control (LV-NC) or LV-LAIR-1. The infection efficiency of lentiviral vectors expressing green fluorescent protein (GFP) was evaluated by fluorescence microscopy.

Quantitative PCR (qPCR) assay
Total RNA was extracted from cells using TRIzol Reagent (Invitrogen). SuperScript III Reverse Transcriptase (Invitrogen) was used for reverse transcription, and PCR was performed using SYBR Green Realtime PCR Master Mix (TAKARA, Shiga, Japan). The qPCR primers for human genes were purchased from Tsingke Biotech (Beijing, China). Relative gene expression was quantified using the comparative Ct method (2 −ΔΔCT ) with GAPDH used as an internal control.

Western blotting
Total protein was prepared using a routine procedure and blotted with following primary antibodies:

Wound healing and Transwell migration assays
Cells were seeded into six-well plates, and a scratch was produced in the monolayer after 48 h.
Images of the wound area were captured immediately after the scratch and after 6 and 12 h (T0, T6, and T12, respectively) to monitor cell migration into the wounded area. The percentage of the scratch area (% scratch) that had closed was calculated as follows: (width at T0 − width at T6 or T12)/width at T0 × 100.
A Transwell migration assay was performed using a Transwell chamber with 8-µm pores (Millipore, Billerica, MA, USA). Untreated OS cells (blank) and their corresponding transfectants overexpressing LV-NC and LV-LAIR-1 were seeded (2 × 10 4 into each well) into the upper chambers in 500 µL serumfree medium. The lower chambers were filled with complete medium, and all chambers were incubated at 37 °C for 24 h. The cells on the upper surface of the membrane were removed, and those in the lower chamber were fixed and stained with 0.1% crystal violet. Images were obtained using an inverted microscope (CX41, Olympus, Tokyo, Japan).

Immunofluorescent staining
Cells were cultured on glass chamber slides. The cells were fixed and permeabilized, and then incubated with primary antibodies for 1 h at room temperature. After washing, the cells were incubated with Cy3-labeled goat anti-rabbit secondary antibody and stained with DAPI (Roche Diagnostics, Basel, Switzerland). Images were obtained using an Olympus microscope.
Immunofluorescence intensities were quantified using ImageJ software (NIH, Bethesda, MD, USA). Generation of the transcriptome library and RNA-seq were performed on the BGISEQ platform (BGI Technologies, Shenzhen, China). The Dr. TOM2 system was used to analyze the transcriptome data.

Statistical analysis
Data were statistically analyzed using GraphPad Prism version 5.0 software (GraphPad, Inc., La Jolla, CA, USA); all data are presented as the mean ± standard deviation. Data were analyzed using an independent sample t-test for comparisons between two groups. Clinical data were statistically analyzed using SPSS software (version 10.0; SPSS, Inc., Chicago, IL, USA). The Pearson χ 2 test was used to evaluate the statistical significance of the association between LAIR-1 expression and clinical features (n = 59). Statistical significance was defined as P < 0.05.

Increased LAIR-1 expression is associated with advanced T stage in patients with OS
IHC staining was performed to detect LAIR-1 expression in 62 human OS samples and 9 adjacent normal bone tissues. Unlike the membrane expression pattern in lymphocytes, which have a large nucleus and small cytoplasmic volume, we observed higher LAIR-1 expression in the cytoplasm of OS cells than in the cell membrane (Fig. 1A). Comparisons between LAIR-1 expression and the clinicopathological characteristics of patients with OS are shown in Table 1. LAIR-1 expression was significantly higher in patients in the T2 stage than in those in the T1 stage of OS tumors (P = 0.006). Table 1 Relationship between LAIR-1 expression and clinicopathological features in OS patients (n = 59 Moreover, analysis of 127 OS samples using the R2 platform indicated that cases with higher levels of LAIR-1 expression had better survival rates than cases with lower LAIR-1 expression levels (P = 0.015,  Fig. 1C) and western blotting (Fig. 1C), respectively.
Additionally, western blotting revealed no differences in the expression of PCNA, a protein marker related to cell proliferation, among all groups (Fig. 1C). An EdU assay and western blotting for PCNA were performed to analyze whether LAIR-1 overexpression affects the growth and proliferation of human OS cells. The EdU assay revealed no difference in the number of positively stained HOS cells between the LAIR-1 overexpression group and LV-NC or blank groups ( Supplementary Fig. 1D). These findings demonstrate that LAIR-1 overexpression did not affect OS cell growth or proliferation.
Next, we investigated the effect of LAIR-1 expression on OS metastasis in wound-healing and Transwell assays. The scratch assay showed a significantly lower repair efficiency in LAIR-1overexpressing cells than in LV-NC-overexpressing and untreated control cells (Fig. 2A). The results of statistical analysis of the scratch closure ratios at 6 and 12 h are shown in Fig. 2B. We also conducted a cell migration assay to analyze the migration ability of OS cells after ectopic LAIR-1 overexpression.
The results indicated that the migration ability of OS cells infected with LV-LAIR-1 was dramatically lower than that of LV-NC and untreated blank cells (Fig. 2C and D). These results demonstrate that LAIR-1 overexpression inhibits OS cell migration.

LAIR-1 overexpression suppressed EMT in OS cells
We further explored the mechanism underlying the suppression of OS cell migration by LAIR-1 overexpression. By qPCR, E-cadherin mRNA expression was found to be upregulated and N-cadherin mRNA expression was downregulated in LAIR-1-overexpressing cells compared to in blank and LV-NC control group cells ( Fig. 2E and F); however, no change was observed in vimentin mRNA expression in LAIR-1-overexpressing OS cells (Fig. 2G).
Here, LAIR-1 overexpression significantly decreased the mRNA and protein levels of Twist1 in HOS cells compared to in control cells ( Fig. 3A and B). Additionally, immunofluorescence staining revealed that Twist1 expression was significantly downregulated after LAIR-1 overexpression in HOS cells (Fig. 3D).

Evaluation of the Foxo1 levels in OS cells showed that LAIR-1 overexpression decreased Foxo1
phosphorylation; immunofluorescence staining further demonstrated increased nuclear retention of Foxo1 ( Fig. 3B and E). Phosphorylation of Akt, the direct upstream regulator of Foxo1, was markedly decreased in LAIR-1-overexpressing OS cells, indicating that decreased Foxo1 phosphorylation and increased Foxo1 retention in the nucleus are regulated by decreased Akt activation.
The role of Foxo1 in reducing Twist1 was further confirmed in OS cells. Twist1 expression was decreased in HOS cells transfected with Foxo1 siRNA compared to in control siRNA-transfected cells (Fig. 3C). These results suggest that LAIR-1 overexpression significantly decreased the EMT in OS cells via Twist1, which functions downstream of phosphorylated Foxo1.

Characterization of EMT-related genes in LAIR-1-overexpressing OS cells
To further clarify the mechanism involved in the LAIR-1-related EMT process, RNA-seq was performed to detect the mRNA expression profiles in OS cells (reference genome: GCF_000001405.38_GRCh38.p12). We identified 974 mRNAs expressed specifically in LAIR-1-

LAIR-1 inhibits EMT via Glut1-related energy metabolism
The three RNAs differently expressed between the two groups were validated by qPCR, and the results agreed with the RNA-seq data (Fig. 4D). The EMT is an energy-demanding process fueled by glucose metabolism-derived ATP. Notably, ANGPTL4, STC1, and PARP2 are involved in energy metabolism in the EMT. These data suggest that overexpression of LAIR-1 inhibits the EMT via metabolic-related processes. The EMT is accompanied by up-regulated glucose consumption, as evidenced by up-regulated Glut1 expression [15]. Here, we observed significantly decreased Glut1 in LAIR-1-overexpressing OS cells compared to in controls (Fig. 4E). As shown in Fig. 4F, immunofluorescence staining assay also demonstrated reduced Glut1 expression in LAIR-1 highly expressed OS cells. These results indicate that LAIR-1 is involved in the tumor EMT via a Glut1-related energy metabolism process.

Discussion
The inhibitory receptor LAIR-1 is a member of the immunoglobulin superfamily and binds extracellular matrix collagens as high-affinity ligands [16]. Functionally, LAIR-1 tyrosine-based inhibition motifs utilize tyrosine phosphorylation to recruit phosphatases and negatively regulate the immune response and cell differentiation [17]. Additionally, the interaction between LAIR-1 and collagen can facilitate the binding of tumor cells to inhibitory molecules on immune cells to inhibit antitumor immune responses, suggesting their role in tumor immune evasion [18]. Previous studies demonstrated the biological role of LAIR-1 in solid tumors including ovarian cancer, cervical cancer, and hepatocellular carcinoma.
Although OS is a mesenchymal-derived tumor type, studies have demonstrated an association between the EMT and OS migration [19]. Interestingly, the collagen matrix can induce the EMT Up-regulated of ANGPTL4 predicts poor prognosis in cancers and promotes tumor cell proliferation and migration, including in OS cells [24,25]. STC1, together with STC-2, was originally identified as a calcium/phosphate-regulating hormone. Recent studies indicated that the STC-1 gene is closely related to glucose and lipid metabolism, as well as to mitochondrial function [26,27]. Additionally, PARP or PAR alterations have been described in tumors, specifically those influencing the EMT [28][29][30]. Our RNA-seq and qPCR results strongly suggest that LAIR-1 inhibits the EMT through metabolic pathways.
Glucose metabolism in cancer cells contributes to their proliferation, metastasis, and therapy resistance [31,32]. Glut1 is abundantly expressed in cancer cells and plays a pivotal role in the glucose metabolism of tumors [33]. Overexpression of LAIR-1 in OS cells inhibits Glut1 expression and may further regulates the glucose metabolism.

Conclusions
Together, our findings demonstrate that LAIR-1 is overexpressed in human OS tissues and its expression is significantly higher in the T2 stage than in the T1 stage of OS tumors. LAIR-1 overexpression remarkably reduces OS cell migration and the EMT via Twist1 regulation. Evaluation of the underlying molecular mechanism revealed a regulatory role for LAIR-1 in Glut1-related glucose metabolism. Thus, LAIR1 is a potential diagnostic and prognostic marker and therapeutic target for OS.

Consent for publication
Not applicable.

Availability of data and material
All data generated or analysed during this study are included in this published article.

Competing interests
The authors declare that they have no competing interests.

Funding
This investigation was supported by the Natural Science Foundation of China (No. 81671575 and No.

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