RETRACTED ARTICLE: MiR-151-3p transferred by cancer-associated fibroblast-derived extracellular vesicles promotes osteosarcoma progression through the CHL1/integrin 1β/TGF-β axis

microRNA-151-3p (miR-151-3p) is a widely reported oncogene with a documented role in tumorigenesis and cancer progression. miRNAs are potent regulators of gene expression that are transferred between cells by extracellular vesicles (EVs). However, there is limited data about the role of EV-derived miR-151-3p in osteosarcoma (OS) progression, or the possible effects of miR-151-3p on the paracrine activity of cancer-associated fibroblasts (CAFs) in OS. To fill this gap of knowledge, we isolated CAF-derived EVs (CAF-EVs) from examined their up-take by human MG63 OS cells and their effects on MG63 biology. We found high expression of miR-151-3p in OS tissues, and miR-151-3p derived from CAF-EVs promoted the process of epithelial-mesenchymal transition (EMT), migration, and invasion of MG63 cells. Notably, bioinformatics analysis, RIP, and dual luciferase report test determined that CHL1 was the direct binding target of miR-151-3p. CAF-EVs mediated the CHL1/integrin 1β axis through miR-151-3p to regulate the TGF-β pathway and then promoted the proliferation, migration, invasion, and EMT of OS cells. Our in vivo results confirmed that EV secretion of miR-151-3p activated the TGF-β pathway through the CHL1/integrin 1β axis to promote the proliferation of OS cells and increase tumor volume and weight. Other effects were to upregulate E-Cadherin and downregulate the expression of N-Cadherin and β-catenin. Hence, our data reveal a potential functional axis mediated by EVs in the tumor microenvironment of OS and suggest potential candidate therapy targets.


Introduction
Osteosarcoma (OS) originates from malignant mesenchymal cells in the bone, which can proceed to develop lifethreatening local or distant metastasis, despite optimal management [1]. OS is regarded as the most frequently occurring primary bone sarcoma among children and young adults, but has a bimodal age distribution, with greater incidence in the elderly than among middle-aged people [2]. Moreover, currently available treatment modalities include surgical resection and systemic chemotherapy, giving 5-year event-free survival of patients with localized OS of about 70%, whereas the overall survival rate of patients suffering from metastatic or recurrent OS is below 20% [3]. Therefore, the better understanding of biology and pathogenesis of OS is urgently required to yield novel targeted therapies and to improve oncologic outcomes [4,5].
In recent years, the tumor microenvironment has attracted research attention regarding its contribution to tumorigenesis, insofar as surrounding fibroblasts are found to be R E T R A C T E D A R T I C L E activated by cancer cells [6]. Of note, cancer-associated fibroblasts (CAFs) can create a favorable environment for invasion and metastasis of cancer cells by interacting with different pathways related to cancer [7]. More importantly, microRNAs (miRNAs) released from extracellular vesicles (EVs) have been recognized as a bridge connecting CAFs, normal fibroblasts, and cancer cells, while dysregulation of miRNAs is closely associated with the formation and function of CAFs [8]. EVs are known to carry diverse molecular cargoes, such as miRNAs, DNA, lipids, and signal peptides [9]. Established roles of EVs include their ability to eliminate unnecessary components for maintaining cellular homeostasis and to mediate intercellular communication [10]. Intriguingly, miRNAs encapsulated in EVs have been reported to possess therapeutic property against malignancy by mediating the metabolism and growth of cells via targeting genes at a post-transcriptional level [11,12]. Moreover, miR-1228 incorporated in EVs (derived from CAFs in OS) has been demonstrated to exhibit pro-migratory and pro-invasive action by targeting the suppressor of cancer cell invasion (SCAI) [13]. Besides, previously-reported microarray-based analysis has identified miR-151-3p as a putative differentially expressed miRNA in CAF-derived EVs for OS, thus arousing our interest in probing its functional role in regulating the progression and development of OS. Though the functional roles of miR-151-3p in malignancies have been validated in breast cancer and cholangiocarcinoma [14,15], their implications in OS remain elusive. Moreover, the role of miR-151-3p has been elucidated to depend on its targeting relation with cell adhesion molecule with homology to L1 cell adhesion molecule (L1CAM) (CHL1) [16], which is consistent with prediction results of the preliminary bioinformatics analysis in our study. Given the above-mentioned evidence, we attempted to unravel the role of CAF-derived EV communication in OS involving miR-151-3p and CHL1, aiming to provide a novel insight into developing therapeutic strategies for the management of OS.

Bioinformatics analysis
The miRNA expression dataset (GSE28423) of OS was retrieved from the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/), which included four normal bone samples and 19 OS cell lines. The differentially expressed genes (DEGs) were screened by R limma package with |logFC|> 1 and p < 0.05 set as the thresholds. EVs miRNAs data were retrieved from the EV-miRNA database (http://bioinfo.life.hust.edu.cn/EVmiRNA#!/). The intersection between the significant differentially expressed miRNAs in GSE28423, and EVs miRNAs from fibroblasts were obtained to predict the OS-related fibroblasts EVs miRNAs with the jvenn tool (http://jvenn.toulouse.inra.fr/app/example. html). To predict further the downstream target genes of miRNAs, we used three bioinformatics websites, which differed from the binding site matching algorithms, including TargetScan (http://www.targetscan.org/vert_72/), miRDB (http://www.mirdb.org/), and mirDIP (http://ophid.utoronto. ca/mirDIP/). We then obtained the intersection to increase the credibility of the prediction results from jvenn tool. The gene expression levels of OS samples in the TCGA database were obtained through the UALCAN website (http://ualcan.path.ua b.edu/) and the predicted target genes were further screened. Finally, the GeneCards database (https://www.genecards.org/) was used to query the downstream regulatory factors of genes, and further predicted the mechanism of action of genes.

The isolation, culture, and identification of CAFs
All fibroblasts were derived from previously operated OS patients. The methods of tissue acquisition and its characteristics have been previously described [13]. CAFs and corresponding para cancer-associated fibroblasts (PAFs) were isolated from OS and the paracancerous tissues digested by collagenase I. Six pairs of matched primary OS adjacent tumor-free tissues (5 cm from the resection margin of the tumor) were obtained. The tissues were immersed in serumfree Dulbecco's modified Eagle's medium (DMEM). Tissues were cut into 1-3 mm fragments and collected into C-tubes supplemented with 5 mL of serum-free DMEM containing 0.5% collagenase I and digested for 1 h. After gentle treatment with MACS dissociator (Miltenyi Biotec, Germany) for 2 min, the single-cell suspension was collected and centrifuged at 1000 rpm/min for 5 min. Cell pellets were suspended with DMEM containing fetal bovine serum (FBS) and seeded into T25 tissue culture flasks. The cells were cultured in high-glucose DMEM (Gibco, Grand Island, NY, USA) supplemented with 10% FBS (Gibco), penicillin (100 IU/ mL), and streptomycin (100 μg/mL), at 37°C with 5% CO 2 . Non-adherent cells and tissues were removed by washing twice with phosphate-buffered saline (PBS) after 48 h and the adherent stromal fibroblasts were then cultured for 4-7 days.
The isolated cells above were grown on overslips at 4 × 10 4 cells/well, and fixed in 4% paraformaldehyde for 30 min. After washing with PBS, the 0.1% Triton X-100 was added to the mixture for 15 min. Then the cells were blocked in 5% BSA for 1 h, and incubated with the primary antibodies against α-SMA, FSP1, and FAP at 4°C for 12 h. After washing with TBST for three times, the cells were incubated with the fluorescent secondary antibody for 1 h at room temperature in the dark, and then stained with DAPI for 5 min. After washing three times with PBS, the surface markers α-smooth muscle actin (αSMA), fibroblast-activated protein (FAP), and P. Wang et al.

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fibroblast-specific protein 1 (FSP1) of CAFs and PAFs were detected by the flow cytometry [17].

Extraction and identification of EVs from CAFs and PAFs
The cancer-associated fibroblast extracellular vesicles (CAF-EVs) and para cancer-associated fibroblast extracellular vesicles (PAF-EVs) were isolated from CAFs and PAFs cells, respectively, in the 3rd-5th generation. Before isolation of EVs, the cell medium was replaced with RPMI 1640 containing 0.5% FBS (Sigma-Aldrich), while CAFs and PAFs were then incubated for further 24 h. The cell culture supernatant was collected and centrifuged at 2000×g and 4°C for 20 min to remove cellular debris. Then the EVs were collected after 1-h ultracentrifugation at 4°C, and 10,000×g. The EVs were suspended in medium containing 25 mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid before ultracentrifugation. Finally, the supernatant was removed and the EVs were resuspended in PBS and stored at −28°C. The method was used to enrich CAF-EVs and PAF-EVs by multiple rounds of ultracentrifugation.
As reported previously [18], extracted EVs were characterized by transmission electron microscope (TEM) observation, nanoparticle tracking analysis (NTA), and Western blot analysis. In brief, extracted EVs were fixed with 1% glutaraldehyde, applied as a drop on a carboncoated copper grid, and stained with 1% phosphotungstic acid. The samples were tested using a JEM-2100 TEM (JEOL, Tokyo, Japan). LSM images were recorded using the PARTICLEMEIRIX system. Nanoparticle tracking analysis (NTA) was performed using a NanoSight NS300 system (Malvern Instruments, Malvern, UK). Brownian motion of extracellular vesicles in PBS was recorded and tracked, while size distribution data were generated by applying the Stokes-Einstein equation. The characteristics of extracellular vesicles were identified by the Western blot analysis. For this purpose, the total protein concentration was determined according to the bicinchoninic acid (BCA) protein assay kit (Beyotime Institute of Biotechnology, Shanghai, China), an 10% SDS gel loading buffer was prepared. The mixture of extracted protein and the loading buffer was boiled for 10 min, cooled on ice, centrifuged, and equivalently added into each lane for electrophoresis by a micropipette, and then the gel protein was transferred to nitrocellulose membrane. The membrane was added with the primary antibodies i.e., CD63 (1:2000, ab216130, rabbit antibody, Abcam, Cambridge, UK,), TSG101 (1:10,000, ab125011, rabbit antibody, Abcam), CD81 (1:10,000, ab109201, rabbit antibody, Abcam), and Calnexin (1:100,000, ab92573, rabbit antibody, Abcam). With an addition of the secondary antibody of goat antirabbit IgG conjugated with horseradish peroxidase (HRP) (1:5000, Beyotime Institute of Biotechnology, Shanghai, China.), the membrane was shaken and incubated at 37°C for 1 h. The membrane was immersed in enhanced chemiluminescence (ECL) reaction solution (Pierce, Waltham, MA, USA) for 1 min, and then exposed after removal of the liquid to the chemiluminescence machine (Shanghai Jing Science and Technology Co., Ltd., Shanghai, China). In this experiment, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out and the antigen-antibody reaction was conducted after the membrane was transferred. The semiquantitative gray analysis was adopted to analyze the bands through the FluorChem FC2 system (Alpha Innotech, San Leandro, CA, USA) and the gray value of each band was calculated.

Cell transfection
Human OS MG63 cells and human embryonic kidney (HEK) 293 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The MG63 cells were cultured in DMEM supplemented with 10% FBS (Gibco), 1% penicillin, and 1% streptomycin.

Immunofluorescence
Firstly, cells were fixed with 4% paraformaldehyde for 20 min and permeated with the 0.1% Triton X-100 for 5 min. Then, cells were incubated overnight with the specific primary antibodies (1:100) at 4°C followed by incubation with the fluorescent secondary antibody (Life Technologies, USA). The nuclei were stained with DAPI (100 ng/mL, Roche, USA). All labeled cells were examined with a Leica confocal fluorescence imaging microscope and LAS AF version 2.0 software (Leica Microsystems, Germany). The specific primary antibodies included Patient-derived α-SMA (ab5694, Abcam), vimentin (ab20346, Abcam), FAP (ab28244, Abcam).

Real-time qPCR
Total RNA (500 ng) was extracted using the Takara Prime Script RT master mix kit (Takara, No. RR037B) according to the manufacturer's instructions. The RT-qPCR was performed using the SYBR Premix Ex Taq II kit (Takara, No.2) in a 20 μL reaction mixture containing equal amounts of cDNA. miRNAs were reversely transcribed and relatively quantified using the TaqMan MicroRNA Reverse Transcription Kit (Thermo Fisher Scientific, 4366596). All mRNA primers used in this study were in accessory material (Table 1). TaqMan® Probes were purchased from Thermo Fisher Scientific (miR-151-3p, Thermo Fisher Scientific, No.1). β-actin was used as an internal reference gene for mRNA and U6 for microRNA, because they are stably expressed in cells and have been widely used for determining gene expression. The calculation of each sample is based on their relative horizontal threshold period (Ct) value normalized Ct value internal reference gene using formula 2 −ΔΔCt (ΔCt = Ct target gene-Ct β-actin/U6).

Laser confocal microscopy
After CAF-EVs were extracted, they were stained with PKH67. According to kits instructions (pkh67gl-1kt, Sigma-Aldrich, St. Louis, USA), EVs were mixed with Diluent C, and PKH67-Diluent C dye was then added rapidly, mixed, and incubated for 5 min. Then staining was terminated by adding 2 mL 10% BSA in PBS (D8537). The liquid was transferred to the bottom of the tube slowly and carefully, added with 1.5 mL sucrose solution, and ultracentrifuged at 190,000 r/min for 2 h at 2-8°C. The media and interface layers were carefully aspirated and the extracellular vesicle pellet was resuspended in PBS by gently blowing. Thereafter, it was transferred to the Amicon centrifugal filter and centrifuged at 3000 r/min for 40 min in a high-speed centrifuge to reduce the volume to 0.5-1/mL. MG63 cells were routinely cultured, plated, and replaced with fresh medium after 48 h. Next, the cells were stained with DiL to become red and co-cultured with 0.05 µg PKH67labeled EVs per 10 3 cells [19] or PBS for 24 h [20]. After washing three times with PBS, the cells were fixed with 4% paraformaldehyde at room temperature for 30 min. Then, the cells were washed three times with PBS and stained with DAPI at a density of 100 ng/mL (36308ES11, Yisheng Bio, Shanghai, China) for 5 min. The images were observed and captured with a laser confocal microscope (DMi8, Leica, Wetzlar, Germany). There were two groups: (1) PBS group (MG63 cells were added with PBS culture only), (2) CAF-EVs group (MG63 cells were co-cultured with PKH67 labeled CAF-EVs).

Dual-luciferase reporter assay
The target of miR-151-3p was verified to be CHL1 by luciferase reporter assay while the 3′UTR of miR-151-3p and CHL1 were artificially synthesized. The gene promoter fragments were introduced into the ARE-Luc reporter gene using the endonuclease sites Nhe I and Bgl II (Shanghai Yisheng Biotechnology Co., Ltd.), whereas the complementary sequence mutation site of the seed sequence was designed on miR-151-3p and wild-type (WT) CHL1. After restriction enzyme digestion, the target fragment was inserted into the pGL3 reporter plasmid using T4 DNA ligase. The correctly sequenced luciferase reporter plasmids (pGL3-miR-151-3p WT) (pGL3-CHL1 WT) and (pGL3-miR-151-3p MUT) (pGL3-CHL1 MUT) and (pGL3-Control) were cotransfected into HEK293T cells (purchased from ATCC) with miR-151-3p-mimic and NC-mimic, respectively. After 12 h of transfection, the cells in different groups were treated with DMSO for 24 h. Before being lysed by the buffer of double-luciferase reporter assay kit (E1910, Promega), the cells were washed with 1× PBS. Afterward, the luciferase activity was measured with the Glomax 20/20 luminometer fluorescence detector (Promega). Each group of experiments was repeated three times.

CCK8 assay
Cell viability was analyzed using Cell Counting Kit 8 (CCK-8, Dojindo, Kyushu Island, Japan). After being treated with EVs, MG63 cells were seeded into 96-well Wound healing assay MG63 cells (2 × 10 5 cells/well) were seeded in a 6-well plate for 24 h. Then 100 μg/mL EVs were added into fresh medium (the number of EVs is the same as Transwell analysis). After 48 h of incubation with EVs, the cell monolayer was scraped with a 1 mL pipette tip. Then, the cells were cultured in a serum-free medium, and the migration ability was evaluated under a bright-field microscope at 0 and 48 h.

Transwell assay
Before the experiment, 50 μL Matrigel Matrix (Sigma, USA) was added to the chamber to coat the filter membrane. Next, 200 μL cell suspension (2 × 10 5 MG63 cells/well) was seeded in the upper chamber. The remaining steps were the same as above. The number of stained cells was counted under an inverted microscope (XDS-800D, Shanghai Caikang Optical Instruments Co., Ltd., China) and was expressed as the mean.

RNA-binding protein immunoprecipitation (RIP) assay
The binding of miR-151-3p to Argonaute2 (AGO2) protein was detected using the Magna RIP RNA-Binding Protein Immunoprecipitation kit (Millipore Billerica, MA, USA). Cells were lysed using an equal volume of RIPA lysis buffer (P0013B, Beyotime Biotechnology Co., Shanghai, China) on an ice bath for 5 min and centrifuged at 14,000 rpm at 4°C for 10 min for isolation of the supernatant. A part of the cell extract was used as input, while the other part was incubated with the antibody for coprecipitation. The steps were as follows: In each coprecipitation reaction system, 50 µL of magnetic beads were washed and re-suspended in 100 µL of RIP Wash Buffer (EHJ-BVIS08102 Xiamen Jiahui Biotechnology Co., Ltd., China). Each group was added with 5 µg of antibodies for binding. The magnetic bead-antibody complexes were washed and resuspended in 900 µL of RIP wash buffer, followed by addition of 100 µL of cell extract to incubate at 4°C overnight. The sample was placed on a magnetic pedestal to collect the magnetic bead-protein complexes. The samples and input were digested by protease K, respectively. Then, RNA was extracted for subsequent PCR detection. RNA was extracted from exosomes using the Total Exosome RNA Isolation Kit (Invitrogen, Carlsbad, CA, USA). The extracted RNA was subsequently used for the RT-qPCR assay. Total RNA was extracted using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Besides, the antibody used for RIP was AGO2 (ab32381, 1:50, Abcam) mixed at room temperature for 30 min, and IgG (1:100, ab109489, Abcam, UK) as a negative control.

Western blot analysis
Total cellular proteins were extracted using RIPA lysis buffer according to the instructions (R0010, Solarbio). The cells or tissues were lysed at 4°C for 15 min followed by centrifugation at 15,000 rpm.

Establishment of subcutaneous tumor xenografts model in mice
Male Balb/c nude mice (n = 40, 4-6 weeks, 15-20 g, J004, Nanjing Junke Biological Engineering Co., Ltd., China) were randomly divided into four groups. The clean laminar airflow frame and room in the feed barrier system were regularly exposed to UV rays while cages, bedding, drinking water, and feed were autoclaved and sterilized at room temperature of 24-26°C and relative humidity was maintained at 40-60% in the animal quarters. The study was approved and reviewed by the Clinical Ethics Committee of The People's Hospital of Yizheng City, The Affiliated Hospital of Yangzhou University. CAFs were digested, re-suspended, and then centrifuged multiple times to remove FBS as possible from suspension. Two hundred MiR-151-3p transferred by cancer-associated fibroblast-derived extracellular vesicles promotes. . .

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microliter of cell suspension at 5 × 10 6 cells/mL were injected into the right shoulder blades of each nude mouse slowly. The next day, miR-151-3p mimic or miR-151-3p inhibitor, and their negative controls (100 nM) were injected into the tail vein. The mice were observed and injected every two days. After 4 weeks, mice were sacrificed by overdose with 9% pentobarbital sodium (P3761, SIGMA, St. Louis, USA) and the tumors were isolated. During treatment, the shortest diameter (a) and longest diameter (b) of the tumors were measured weekly with Vernier calipers; the tumor volume was calculated according to the formula π (a 2 b)/6, and the tumor mass was weighed with a balance.
Peripheral blood was collected when the mice were sacrificed and centrifuged to prepare serum. EVs in the serum were extracted using the above method and then their miR-151-3p expression was detected by RT-qPCR analysis. The tumor tissues were fixed in 10% formaldehyde and the routinely dehydrated and embedded for later examination.

Statistical analysis
GraphPad Prism 8.0 (GraphPad Software, La Jolla, CA, USA) was used for statistical analysis and all experiments were repeated at least three times. All data conform to normal EVmiRNA DEGs_up distribution and homogeneous variance test. Measurement data were presented as the mean ± standard deviation (SD). Comparisons between the two groups were conducted with an independent sample t-test, while comparisons among multiple groups were performed with one-way ANOVA analysis of variance. Tukey's test was used for posthoc testing. p < 0.05 was considered statistically significant.

High expression of miR-151-3p in OS
Through analysis of the OS miRNA expression dataset GSE28423, we screened out 258 differential genes, among which 136 genes were upregulated and 122 genes were downregulated (Fig. 1A). Then top 105 genes were selected to draw the expression heatmaps (Fig. 1B). The 680 miRNAs of fibroblast EVs were obtained from the EVmiRNA database. Then, 25 miRNAs (the intersection of the highly expressed differential miRNAs of dataset GSE28423 and the fibroblast EVs miRNAs) were obtained by jvenn (Fig. 1C). Moreover, the EVmiRNA database showed that miR-151-3p had the highest mean expression in fibroblast EVs (Fig. 1D), while the heatmap of GSE28423 differential gene expression showed that miR-151-3p was highly expressed in OS. Therefore, miR-151-3p was selected as the objective gene for the subsequent in vivo and in vitro studies.

Release and identification of CAF-EVs and PAF-EVs
The main CAFs and corresponding PAFs from OS tumor tissues were isolated by treatment with 0.5% type I collagenase and the cell's morphology and their growth were observed under an inverted microscope. Our results exhibited the slow growth of PAFs, which formed a flat fusiform shape, had a flat oval sac-like nucleus, significantly visible nuclear cuts, and abundant cytoplasm. On the other hand, CAFs grew rapidly, with a long fusiform or polygonal shape, a slightly wider medial aspect mainly protruding at both ends, blurred outline, and an irregular arrangement ( Fig. 2A). For further analysis of CAFs and PAFs, the surface markers FAP, α-SMA, and FSP1 were tested by Western blot analysis. We found that the expressions of CAFs-specific markers FAP, FSP1, and α-SMA were higher than that of PAFs (Fig. 2B).
After sequential centrifugation to obtain the supernatant of CAFs, CAF-EVs, and PAF-EVs were extracted by ultracentrifugation. TEM results showed that CAF-EVs exhibited typical morphological characteristics of EVs like cup-shaped or spherical (Fig. 2C). NTA analysis found that the size of CAF-EVs ranged from 40 to 200 nm and the mean size was 108 nm (Fig. 2D). Western blot analysis demonstrated positive expression for EVspecific surface markers CD63, TSG101, Alix, and negative expression for Calnexin. In contrast, in cell lysates there was poor expression for CD63, TSG101, MiR-151-3p transferred by cancer-associated fibroblast-derived extracellular vesicles promotes. . .

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Alix, but abundant expression for Calnexin (Fig. 2E). According to their characterization, size, and expression of biomarkers it was confirmed that EVs were successfully isolated from the conditioned medium [21]. Furthermore, the EVs extracted from PAF-EVs had the above typical characteristics.

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was significantly upregulated (Fig. 3A). The phagocytosis of CAF-EVs by MG63 cells was detected after the extracted CAF-EVs were co-incubated with MG63 cells. In CAF-EVs-MG63 co-incubation group, the EVs labeled with PKH67 showed green fluorescence and main labeling in the cytoplasm while there was no green fluorescence in PBS-MG63 group, which indicated that CAF-EVs were successfully endocytosed into cells (Fig. 3B). Besides, the  Tumor   AGO2  FXR1  CLK1  PITPNA  TRA2B  UPP2  PFN2  NIPAL2  YTHDF3  CASD1  AKT3  CRK  ZFAND5  FAM104A  CHL1  CLASP2  ATP2A2  HIF1A  18  19  20  21  22  into HEK-293T cells, respectively. The dual-luciferase report detected the binding of miR-151-3p to CHL1. G RIP experiment proved that CHL1 was the target of miR-151-3p. H After transfected with miR-151-3p-inhibitor and NC-inhibitor, EVs were extracted to treat with MG63 cells, and then Western blot analysis was used to detect the expression of CHL1. (U6 is the internal reference for miR-151-3p expression and GAPDH is the internal reference for CHL1 expression. *p < 0.05. The data result is measurement data, mean ± SD indicates that the comparison between the two groups is analyzed by independent sample t-test. The experiment was repeated in triplicate.

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qPCR analysis showed that, compared to the PBS-MG63 group, the expression of miR-151-3p in CAF-EVs-treated MG63 cells was also significantly increased (Fig. 3C).
To confirm the effect of miR-151-3p derived from CAF-EVs on the migration, invasion, and EMT of OS cells, the CAFs cells were transfected with NC-inhibitor and miR-151-3p-inhibitor. The qPCR analysis showed that, compared to the NC-inhibitor, the expression of miR-151-3p was effectively suppressed in the miR-151-3p-inhibitor group (Fig. 3D). Afterward, EVs were extracted from CAFs transfected with NC-inhibitor and miR-151-3p-inhibitor were used to treat with MG63 cells, while CCK-8 was used to detect the cell viability of MG63 cells. Being distinct from the PBS group, the cell viability of the CAF-EVs group was significantly increased, whereas, the cell viability of CAF-EVs + miR-151-3p-inhibitor group was inhibited (Fig. 3E) compared to the CAF-EVs + NC-inhibitor group. In wound healing and Transwell experiments, the migration and invasion of MG63 cells in the CAF-EVs group was higher than that of PBS, while the migration and invasion in CAF-EVs + miR-151-3p-inhibitor group were inhibited (Fig. 3F, G), compared to CAF-EVs + NC-inhibitor group. Next, the expression of EMT-related biomarkers was detected by Western blot analysis. Compared to PBS group, the expression of N-Cadherin and β-catenin was upregulated while that of E-Cadherin was suppressed in the CAF-EVs group (Fig. 3H). Besides, compared to the CAF-EVs + NC-inhibitor group, the expression of N-Cadherin and β-catenin was downregulated and E-Cadherin expression was upregulated in the CAF-EVs + miR-151-3p-inhibitor group. In addition, as shown in Fig. S1, there was no similar result trend in PAF-EVs, suggesting that PAF-EVs had no obvious effect on the migration, invasion and EMT of OS cells. These results suggested that the miR-151-3p derived from CAF-EVs promoted the EMT, migration, and invasion of OS cells.

MiR-151-3p bound to CHL1 3′UTR
To investigate further the role of miR-151-3p in the molecular etiology of OS, we searched for its targets. Previous research had indicated that CHL1 could bind with miR-151-3p to promote the development of OS [22]. Bioinformatics prediction revealed 112, 95, and 88 genes targeted by miR-151-3p from TargetScan, miRDB, and mirDIP, respectively, among which18 common target genes stood out (Fig. 4A). Further analysis of expressions of genes in the OS samples of the TCGA database through the UALCAN website (Fig. 4B) exhibited low expressions of UPP2, NIPAL2, and CHL1, in contrast to the higher expression trend of miR-151-3p. Previously reported studies have indicated CHL1 to be an important tumor suppressor possessing anti-proliferative and anti-metastatic properties [23].
Moreover, the TCGA database showed a low expression of CHL1 in OS (Fig. 4C). Furthermore, the TargetScan website was employed to obtain the 504-511 position on the 3′ UTR of CHL1 and the targeted binding site of miR-151-3p (Fig. 4D). The predicting binding site was detected by the dual-luciferase reporter test. After transfection of miR-151-3p-mimic and NC-mimic into HEK-293T cells, qPCR assay was used to detect the transfer efficiency of miR-151-3pmimic. The expression of miR-151-3p was significantly upregulated in miR-151-3p-mimic group (Fig. 4E). Compared with the NC-mimic + CHL1WT co-transfection group, the fluorescence intensity of miR-151-3p-mimic + CHL1WT co-transfection group was markedly reduced. However, there was no corresponding change observed in the miR-151-3p + CHL1MUT co-transfection group (Fig. 4F). The abundance of AGO2 binding miRNAs is negatively associated with the inhibitory potential of their target gene. Accordingly, our results from the RIP assay demonstrated that more miR-151-3p was immunoprecipitated upon applying AGO2-specific antibodies relative to the IgG control in miR-151-3p mimic-transfected MG63 cells, suggesting that miR-151-3p could indeed bind with CHL1 (Fig. 4G). After transfection with miR-151-3p-inhibitor and NC-inhibitor, MG63 cells were treated with extracted EVs. Western blot analysis showed, that compared to the PBS group, the expression of CHL1 in EVs group was significantly downregulated, while compared to the EVs + NC-inhibitor group, the expression of CHL1 in EVs + miR-151-3p-inhibitor group was upregulated (Fig. 4H). Collectively, the above-described results of the bioinformatics analysis, RIP, and dual luciferase report test suggest that CHL1 was a direct binding target of miR-151-3p.
MiR-151-3p delivered by CAF-EVs promoted activation of the TGF-β pathway via CHL1/ integrin1β axis in vitro to promote OS occurrence Further analysis by the GeneCards database revealed that integrin 1β is a key interaction factor of CHL1 (Fig. 5A). Existing literature showed that the interaction between CHL1 and integer 1β inhibited OS tumor growth and metastasis [24], while overexpression of integer 1β promoted proliferation, invasion, and migration of tumor cells [22]. Intriguingly, it has been demonstrated that integrin 1β promoted the activation of the TGF-β signaling pathway [25], which could promote the growth and metastasis of OS cells [26]. Thus, we speculated that miR-151-3p carried by CAF-EVs may activate the TGF-β signaling pathway by mediating the CHL1/integrin 1β axis, and then promote the growth and metastasis of OS.
To verify further this putative mechanism of oncogenesis, the MG63 cells were subjected to different treatments to elucidate the effect of miR-151-3p delivered P. Wang et al.

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by CAF-EVs on growth and metastasis of OS. Then, qPCR was used to detect CHL1 mRNA expression with the results showing that compared to the NC-mimic + oe-NC group, CHL1 mRNA expression was increased in the NC-mimic + oe-CHL1 group while it was decreased in the miR-151-3pmimic + oe-NC group. An inverted trend was evident in the miR-151-3p-mimic + oe-CHL1 group regarding the CHL1 mRNA expression as compared to the miR-151-3p-mimic + oe-NC group (Fig. 5B). In addition, Western blot analysis yielded similar results in the protein expression of CHL1 (Fig. 5C). Meanwhile, protein expression of integrin 1β was inhibited in the NC-mimic + oe-CHL1 group while an increase was noted in the miR-151-3p-mimic + oe-NC group compared to the NC-mimic + oe-NC group. Simultaneous overexpression of miR-151-3p and CHL1 decreased the integrin 1β protein expression.
For further validation, MG63 cells were treated with TGF-β pathway pharmacological inhibitor SB-431542 before performing the Western blot analysis. Compared to the NC-mimic + DMSO group, the protein expression of TGF-β was downregulated and that of CHL1 and integrin 1β showed no changes in the NC-mimic + SB-431542 group. However, when compared to the NC-mimic + DMSO group, in the miR-151-3p-mimic + DMSO group, CHL1 protein expression was reduced while TGF-β and integrin 1β protein expression was upregulated, all of which were reverted in the miR-151-3p-mimic + SB-431542 group (Fig. 5D).

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NC-mimic + SB-431542 group while a contrary trend was evident in the miR-151-3p-mimic + DMSO group (Fig. 5E-G). On the other hand, Western blot analysis showed that, compared to NC-mimic + DMSO group, the expression of E-Cadherin was elevated while that of N-Cadherin and β-catenin was decreased in the NC-mimic + SB-431542 group. However, opposite results were observed in the miR-151-3p-mimic + DMSO group when compared with the NC-mimic + SB-431542 group (Fig. 5H). Collectively, these results suggested that the miR-151-3p delivered by CAF-EVs upregulated integrin 1β expression by targeting CHL1 and then activated the TGF-β pathway, thus promoting the proliferation, migration, invasion, and EMT of OS cells.

MiR-151-3p delivered by CAF-EVs promoted OS tumor growth in vivo
To verify further the pathological mechanism of CAF-EVs delivering miR-151-3p on OS in vivo, we established a mouse tumor model. The miR-151-3p-inhibitor and NCinhibitor were transfected into CAF-EVs and then the transfected CAFs were injected intravenously into the mice, with PBS used as a control). Then, the OS tissue from the mice was isolated and the protein expression of CHL1, integer 1β, and TGFβ was detected by Western blot analysis. Contrary to findings in the PBS group, the expression of CHL1 in the CAF-EVs and CAF-EVs + NCinhibitor groups was downregulated, while that of integ-rin1β and TGFβ was upregulated. Compared to the CAF-EVs + NC-inhibitor group, the expression of CHL1 was significantly up-regulated in the CAF-EVs + miR-151-3pinhibitor group, while that of integrin 1β and TGFβ was downregulated (Fig. 6A). Afterwards, the effects of miR-151-3p on mouse tumor formation were also detected, with determination of the tumor volume and weight ( Fig. 6B-C). We found that, compared to the PBS group, the volume and weight of tumors were significantly increased in the CAF-EVs and CAF-EVs + NC-inhibitor groups. However, compared to the CAF-EVs-NC-inhibitor group, the volume and weight of tumors were significantly decreased in the EVs-miR-151-3p-inhibitor group. Hence, the abovementioned results consistently showed that CAF-EVs delivering miR-151-3p facilitated the tumor growth of OS in vivo.

Discussion
Despite great research efforts to obtain better prognosis, the overall survival rate of patients with OS has remained dismal for decades [27]. Therefore, it is imperative to identify a   [28]. Correspondingly, in the present study, a series of gain-of-function and loss-offunction assays were performed to explore the CAF-derived EV communication in the tumor microenvironment of OS. Summarizing the present experimental data, we conclude that CAF-derived EVs promoted the acquisition of metastatic potential of cancer cells in OS by activating the TGFβ signaling pathway via the miR-151-3p-mediated CHL1/ integrin 1β axis. A fundamental observation made in our study was that miR-151-3p had high expression in OS and functioned as an oncogene. Intriguingly, in the context of breast cancer, miR-151 expression has been reported to be elevated while ectopically expressed miR-151 exhibited an inhibitory effect on breast cancer cell migration and invasion [14]. Moreover, high expression of miR-151-3p has been demonstrated in resected cholangiocarcinoma samples [15]. Furthermore, we now report that found miR-151-3p is highly expressed in EVs derived from CAFs, and contributes to the EMT process, migration, and invasion of OS cells. Nonetheless, CAFs have been recognized to be a driving force for colorectal cancer progression by releasing miRNA-loaded EVs [29]. Likewise, aggressive phenotypes of breast cancer cells have indicated to be significantly enhanced by treatment with EVs derived from CAFs containing multiple miRNAs [30,31]. Largely in agreement with our present finding, a previous report has unraveled the tumor-promoting action of miR-1228 shuttled by EVs derived from CAFs in OS, and showed a mechanism of action via effects on the target gene, SCAI [13].
Correspondingly, our findings from the mechanistic investigation revealed CHL1 to be target gene of miR-151-3p, due to the presence of a specific binding site in CHL1 WT for miR-151-3p. Moreover, our data retrieved from the TCGA database on the UALCAN website indicated that CHL1 was expressed at a low level in OS samples. Concordantly, the downregulation of CHL1 has also been detected in colon adenocarcinoma, where it functioned as a direct target gene of miR-21-5p [32]. Intriguingly, a previously reported study has documented the tumorsuppressive property of CHL1 in esophageal squamous cell carcinoma by curbing proliferation and metastasis [23], suggesting an anti-tumor potential of CHL1 in OS. However, we have not yet validated the expression profile of CHL1 in clinically collected OS samples. We did subsequently explore the downstream signaling pathways possibly involved in the interaction between miR-151-3p and CHL1 in EVs released from CAFs, finding that CAFsreleased EVs activated the TGF-β signaling pathway via the miR-151-3p-mediated CHL1/integrin 1β axis. Besides, CHL1 has been elaborated to suppress the growth and metastasis of nasopharyngeal carcinoma by interacting with integrin 1β [24]. Another study has indicated that the overexpression of integrin 1β promotes colorectal cancer cellular capabilities of proliferation, migration, and  Fig. 7 The mechanism diagram. The transfer of miR-151-3p via CAF-EVs downregulates CHL1 and activates the TGF-β pathway via CHL1/ integrin 1β axis to promote OS progression.

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invasion [22]. Moreover, in the context of non-small cell lung cancer, upregulated integrin 1β is considered as the hallmark of poor overall survival, while integrin 1β knockdown leads to the suppression of proliferative potential of cancer cells in vitro [33]. Accordingly, our study examined that integrin 1β expression was elevated in response to the delivery of miR-151-3p inhibitor, which gave rise to potentiated aggressive phenotypes of OS cells, suggesting that integrin 1β is worthy of future research in relation to OS. The TGF-β signaling pathway, which is conducive to the growth and metastasis of OS is reportedly activated by the integrin 1β [26,34], providing further validation of our present results. Taken together, we reported functional axis involving EV communication that is involved in the tumor microenvironment of OS. Herein, miR-151-3p loaded in EVs derived from CAFs possessed a pro-tumorigenic property through the target-inhibition of CHL1. Collectively, our study shows that CAFs can transfer miR-151-3p via EVs to OS cells, facilitating the EMT, migration, and invasion of OS cells both in vitro and in vivo (Fig. 7). Thus, we present novel insights into the pathogenesis of OS with identification of potential therapeutic targets to be tested in future studies.

Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of interest.
Ethical statement All clinical studies were approved by the Ethics Committee of The People's Hospital of Yizheng City, The Affiliated Hospital of Yangzhou University, and conducted according to the guidelines of the Declaration of Helsinki. All OS and paracancerous tissue specimens were obtained from the oncology department of The People's Hospital of Yizheng City, The Affiliated Hospital of Yangzhou University. Patients signed informed consent for tissue donation in advance of their surgery.
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