Lncrna LINC00958 Promotes Tumor Progression Through Mir-4306/CEMIP Axis in Osteosarcoma

European Review for Medical and Pharmacological Sciences 2021; 25: 3182-3199 LncRNA LINC00958 promotes tumor progression through miR-4306/CEMIP axis in osteosarcoma

Y. ZHOU1, T. MU2

1Department of Orthopedics, Beijing Chao-Yang Hospital, Capital Medical University, Beijing, China 2Department of Stomatology, Emergency General Hospital, Beijing, China

Abstract. – OBJECTIVE: To investigate the er gene assay verified that LINC00958 competi- mechanism by which LINC00958 affects osteo- tively bound miR-4306 and repressed its expres- sarcoma progression through miR-4306/CEMIP sion. Silencing of LINC00958 inhibited prolifera- axis. tion, cell cycle, metastasis, and invasion of os- PATIENTS AND METHODS: The microarray teosarcoma cells while inducing cellular apopto- data (GSE66673) for gene expression in osteo- sis. The introduction of miR-4306 inhibitors re- sarcoma cells were obtained from the Gene Ex- versed the tumor-suppressing effect of silenc- pression Omnibus (GEO) database, and differ- ing LINC00958. miR-4306 binds to CEMIP and entially expressed genes were analyzed by bio- suppressed its expression. Xenograft tumor ex- informatics tools. Real-time quantitative PCR periments and tumor metastasis assays in nude (RT-qPCR) was performed to detect the expres- mice demonstrated that silencing LINC00958 in- sion levels of LINC00958, miR-4306, and CEMIP hibited osteosarcoma cells’ growth and metas- in osteosarcoma tissues and cell lines. West- tasis while inhibiting miR-4306 reversed this ef- ern blot was performed to detect the expression fect. Kaplan-Meier analysis showed that high ex- levels of CEMIP. Subcellular fractionation analy- pression of LINC00958 was significantly associ- sis and RNA Fluorescence in situ hybridization ated with poor prognosis of osteosarcoma pa- (FISH) assay were performed to analyze the sub- tients. cellular localization of LINC00958. The target re- CONCLUSIONS: LINC0095 promotes tumor- lationship between LINC00958, CEMIP, and miR- igenesis and metastasis in osteosarcoma by 4306 was verified by public bioinformatics da- competitively inhibiting miR-4306 expression, tabase and dual-luciferase reporter assay. RNA leading to elevated expression of CEMIP. immunoprecipitation (RIP) assay was performed to detect LINC00958 and miR-4306 bound to Key Words: AGO2. The biological functions of LINC00958 LncRNA LINC00958, MiR-4306, CEMP, Osteosar- and miR-205 on proliferation, cell cycle, apop- coma. tosis, migration, and invasion of osteosarcoma cells were evaluated by gain-of-function and loss-of-function experiments. Tumorigenic and metastatic abilities of cells in vivo were detect- Introduction ed by xenograft tumor experiments and tumor metastasis assays in nude mice. Correlation be- Osteosarcoma, also known as osteogenic sar- tween miR-4306 and LINC00958 or CEMIP ex- coma, is one of the most common primary ma- pression in osteosarcoma tissues was analyzed lignant tumors of bone, most often occurring using Pearson correlation analysis. Kaplan-Mei- in adolescents under the age of 20 or children1. er analysis was performed to assess the relationship between LINC00958 expression and Nearly 60% of all osteosarcoma lesions occur overall survival of osteosarcoma patients. in the distal femur, proximal tibia, femur, and RESULTS: LINC00958 expression levels sig- pelvis2. Clinically, osteosarcoma is highly ma- nificantly increased in osteosarcoma tissues lignant, rapidly growing, highly aggressive, and while miR-4306 expression levels significant- early metastatic3,4. Osteosarcoma has the highest ly decreased, and the expression of these two mortality and morbidity rate among all types of genes was negatively correlated. Subcellu- 5 lar fractionation analysis and RNA FISH assay bone cancer, especially in adolescents . Due to demonstrated that LINC00958 was mainly local- the lack of specificity of clinical symptoms in ized in the cytoplasm. Dual-luciferase report- the early stage of osteosarcoma, the prognosis of

3182 Corresponding Author: Yu Zhou, MD; e-mail: [email protected] LncRNA LINC00958 promotes tumor progression through miR-4306/CEMIP axis in osteosarcoma patients is poor, with a 5-year survival rate of 52- untranslated region (3’UTR) of target mRNAs, 56% and a high rate of pulmonary metastasis and resulting in degradation or translational repres- recurrence after treatment6,7. Therefore, strength- sion of target mRNAs and negative regulation of ening research on the pathogenesis of osteosarco- gene expression at the post-transcriptional lev- ma can provide an important basis and reference el18,19. Some studies indicate that the interaction for clinical diagnosis and treatment. between lncRNA and miRNA plays a critical Long non-coding RNA (LncRNA) is a class of role in tumor development. Wang et al20 found nucleotide fragments whose transcripts are longer that miR-335 expression was downregulated in than 200 nt without coding capacity, which plays osteosarcoma tissues and cell lines and func- a biological role in embryonic development, cell tioned as a tumor suppressor gene. Upregulation growth and disease development through splic- of miR-335 can inhibit migration and invasion of ing regulation, chromatin remodeling and RNA osteosarcoma cells by targeting Rho-associated degradation8-10. Some studies11-13 have shown that coiled-coil protein kinase 1 (ROCK1). Accumu- lncRNAs are often aberrantly expressed in hu- lating evidence suggests that miRNAs regulate man tumors and are involved in tumor initiation, a variety of osteosarcoma cell functions, includ- progression, and metastasis. With the in-depth ing proliferation, differentiation, metastasis, and study of lncRNAs, a large amount of evidence apoptosis. has revealed that the upregulation or downregula- The ceRNA regulatory network is a recently tion of lncRNAs is closely related to mechanisms discovered novel mechanism by which RNAs such as the development of osteosarcoma14. In os- interact with each other in vivo and can regulate teosarcoma cells, elevated expression of lncRNA the action of coding genes, which also extends tends to promote cell proliferation. Liu et al15 the previous understanding of the large number found that LINC01296 expression was elevated of non-coding RNAs in vivo21,22. Numerous in- in osteosarcoma, while silencing LINC01296 ex- vestigations23,24 have demonstrated that ceRNA pression inhibited osteosarcoma cell proliferation networks are widespread in different tumors, and and induced apoptosis. It was also found that the ceRNAs may have an impact on tumor develop- expression level of cyclin D1 was positively cor- ment and prognosis, which could be an indicator related with LINC01296 expression in osteosar- for early diagnosis, prognostic evaluation, and a coma tissues, which partly explained the pro-tu- target for cancer therapy. In this study, we iden- morigenic effect of LINC01296 in osteosarcoma. tified a long non-coding RNA LINC00958 sig- Recent studies have shown that lncRNAs also nificantly overexpressed in osteosarcoma tissues play an important role in tumor invasion and me- and cells and investigated its biological function tastasis. According to Ma et al16, the expression and mechanism on promoting cell metastasis and of FOXF1 and its antisense transcript FOXF1- invasion in osteosarcoma cells. We found that AS1 (lncRNA) were significantly upregulated in LINC00958 acts as a ceRNA for miR-4306 and osteosarcoma tissues and cell lines, which were regulates metastasis and invasion of osteosarco- associated with poor prognosis in osteosarcoma cells via CEM P. ma patients. Furthermore, silencing FOXF1-AS1 and FOXF1 significantly inhibited osteosarcoma cell migration and invasion in vitro and in vi- Patients and Methods vo by decreasing the expression of MMP2 and MMP9, whereas enhancing the expression of Bioinformatics Analysis FOXF1-AS1 had the opposite effect. In this study, The microarray data (GSE66673) for gene ex- LINC00958 was found to be abnormally overexpression in osteosarcoma cell was obtained from pressed in highly metastatic osteosarcoma cells the GEO database (https://www.ncbi.nlm.nih. in the GSE38666 dataset deposited in the GEO gov/geo/), and the parental osteosarcoma cells database, which prompted us to further define its SAOS-2 and SAOS-2-derived LM5 cells with functional role in the mechanism of osteosarcoma increased metastatic potential were selected from metastasis. this microarray study. The parental osteosarcoma MicroRNAs (miRNAs) are small non-coding cells were used as controls for differential gene RNAs composed of 19-25 nucleotides17. miR- expression analysis. Then, background correction NAs affect cell proliferation, metastasis, dif- and standardized preprocessing were performed ferentiation and apoptosis through complete or using the affy package in the R language, and incomplete complementary binding to the 3’ “limma” package was used for screening dif-

3183 Y. Zhou, T. Mu ferentially expressed genes with the screening Cell culture: The human normal osteoblast cell threshold of | log2FolfChange | > 2.0 and adj.P.Val line hFOB 1.19, osteosarcoma cell lines HOS, (the corrected p-value) < 0.0525,26. The “pheat- MG-63, U2OS, 143B, and human embryonic map” package was used to construct the differ- kidney cells HEK-293T were purchased from ential gene expression heatmap. Next, the down- the American Type Culture Collection (ATCC; stream miRNAs of LINC00958 were predicted Manassas, VA, USA). The cells were cultured in using the RNA22 database (https://cm.jefferson. Dulbecco’s Modified Eagle’s Medium (DMEM; edu/rna22/) and the starBase 3.0 database (http:// Gibco, Grand Island, NY, USA) supplemented starbase.sysu.edu.cn/). Prediction of miRNA tar- with 10% fetal bovine serum (FBS; Gibco, Grand get genes was performed using the miRDB da- Island, NY, USA), 100 mg/mL penicillin and 100 tabase (http://www.mirdb.org/) and the RNA22 mg/mL streptomycin in an incubator at 37°C with database (https://cm.jefferson.edu/rna22/). 5% CO2. The culture medium was changed every 2 days, and the cells were passaged when they Study Samples reached about 90% confluence. Clinical Sample Collection: 63 osteosarcoma cancer tissues and paired normal tissues were Cell Transfection collected from 63 patients who received tumor re- The sequences of LINC00958, miR-4306, and section in the Department of Oncology of Beijing CEMIP were retrieved from the National Center Chao-Yang Hospital from September 2015 to De- of Biotechnology Information (NCBI). The design cember 2019. The 63 patients included 34 males and construction of plasmids for the experiments and 29 females with a mean age of 20.71± 7.26 were based on the pCMV-Flag-N/C vector and years old. All specimens were immediately fro- entrusted to the Shanghai Sangon Biotechnology zen in liquid nitrogen at -80°C. Patient inclusion Company. The density was adjusted according criteria: 1) patients were pathologically diagnosed to the cell growth rate and inoculated in 6-well with only osteosarcoma as the primary tumor; plates, and the cells were transfected with a den- 2) patients did not undergo radiotherapy, che- sity of 80%-90% according to the manufactur- motherapy or biological therapy before surgery; er’s instructions of lipofectamine 3000 kit (Invi- 3) patients had complete clinicopathological and trogen, Carlsbad, CA, USA). Stably transfected follow-up data. Pathological specimens were con- cells were selected with G418 (1000 μg/mL) and firmed in the Pathology Department, and relevant cultured for 4 weeks after 48 h of transfection. clinical data were collected through retrospective The sequence of sh-LINC00958 were shown as review of patient files. The detailed parameters of follows: sh-LINC00958-1: 5’-GCAGGCCCAGAC- the patients are shown in Table I. CACTTTATA-3, sh-LINC00958-2: 5’-GGC-

Table I. The correlations between lncRNA LINC00958 expression and clinicopathological characteristics in OS patients.

LINC00958 expression

Characteristics Cases (n) High (n = 32) Low (n = 31) p-value

Age (years) 0.502 < 18 38 18 20 ≥ 18 25 14 11 Gender 0.382 Male 34 19 15 Female 29 13 16 Metastasis 0.011 Yes 22 16 6 No 41 16 25 Tumor location 0.348 Tibia/Femur 39 18 21 Other 24 14 10 Enneking stage 0.021 I-IIA 18 5 13 IIB-III 45 27 11

3184 LncRNA LINC00958 promotes tumor progression through miR-4306/CEMIP axis in osteosarcoma

CCAGACCACTTTATAAGC-3, sh-LINC00958-3: anti-Cleaved Caspase-3 antibody (Abcam, Cam- 5’-GGATCTGTCAGGTTATTTCAT-3. bridge, MA, USA), anti-E-Cadherin antibody (Abcam, Cambridge, MA, USA), anti-N-Cadher- Reverse Transcription Quantitative in antibody (Abcam, Cambridge, MA, USA) at PCR (RT-qPCR) 4°C overnight. Then the membrane was probed Total RNA was extracted from tissues and with the horseradish peroxidase (HRP)-labeled cells using TRIzol Reagent (Invitrogen, Carlsbad, goat anti-rabbit immunoglobulin G (IgG) anti- CA, USA). Then The total RNA was reversely body (Proteintech, Chicago, IL, USA) at 37°C for transcribed into complementary DNA (cDNA) in 1 h at room temperature, Subsequently, the bands accordance with the instruction of PrimescriptTM were visualized using enhanced chemilumines- RT reagent Kit (TaKaRa Biotechnology, Dalian, cence (ECL), and these bands were imaged on a China) and One Step PrimeScript miRNA cDNA Bio-Rad Gel imager (Bio-Rad, Richmond, CA, Synthesis Kit (TaKaRa Biotechnology, Dalian, USA). The gray value was measured using The China), respectively. RT-qPCR was performed Image J software and compared with the internal on an ABI7500 Quantitative PCR instrument reference to calculate the relative ratio. The ex- (Applied Biosystems, Foster City, CA, USA) us- periment was repeated three times. ing SYBR® Premix Ex TaqTM kit (TaKaRa Bio- technology, Dalian, China). All primers were Fluorescence in Situ Hybridization (FISH) synthesized by Shanghai Sangon Biotechnology The FISH probe of LINC00958 was designed Company, and the primer sequences are listed and synthesized by Ribobio and labeled with in Table II. With U6 regarded as an internal ref- Cy3 fluorescent dye. The RNA FISH assay was erence of miR-4306 and GAPDH as an internal performed according to the instructions of the reference of LINC00958 and other genes, the RiboTM lncRNA FISH Probe Mix (Red) kit relative expression of products was calculated by (Ribobio, Guangzhou, China). Briefly, U2OS the 2-ΔΔCt method. The experiment was repeated and 143B cells cultured on slides were washed three times. with PBS and fixed with 4% paraformaldehyde. They were incubated in 40°C pre-hybridization Western Blot Analysis buffer for 4 h and then hybridized with the Total cellular protein was extracted by the probe at 40°C overnight. Afterward, cells were RIPA lysis buffer (Solarbio Science & Technol- stained with DAPI and imaged with a laser ogy, Beijing, China) supplemented with PMSF. confocal scanning microscope (Leica, Wetzlar, The total protein concentration was measured by Hesse, Germany). BCA kit (Solarbio Science & Technology, Bei- jing, China) and the sample volume was adjusted Subcellular Fractionation Analysis to 30 μg protein/lane with deionized water. The To determine the cellular localization of samples were subjected to SDS-PAGE gel elec- LINC00958, nuclear grade fractions were isolat- trophoresis and transferred onto a polyvinylidene ed from the cytoplasm according to the instruc- fluoride (PVDF) membrane (Solarbio Science tions of NUCLEI EZ PREP NUCLEI Isolation & Technology, Beijing, China). The membranes Kit from the manufacturer (Sigma-Aldrich, St. were blocked with 5% skim milk for 1 h at room Louis, MO, USA). The extracted RNA was then temperature and incubated with the anti-CE- subjected to reverse transcription and RT-qPCR. MIP antibody (Proteintech, Chicago, IL, USA), The primer sequences are shown in Table II.

Table II. Primers used in this study. Sequence

Primers Forward (5’-3’) Reverse (5’-3’)

LINC00958 CCATTGAAGATACCACGCTGC GGTTGTTGCCCAGGGTAGTG miR-4306 AAAGCGCCGCTGGAGAGA TATGGTTGTTCACGACTCCTTCAC CEMIP GCTCTTGAGTTGCATGGACA ACCGCGTTCAAATACTGGAC GAPDH TGGTGAAGACGCCAGTGGA GCACCGTCAAGGCTGAGAAC U6 CGCTTCGGCAGCACATATACTA TCACGAATTTGCGTGTCATCCT

3185 Y. Zhou, T. Mu

Northern Blot taining magnetic beads. RNA was extracted after Total RNA was extracted from U2OS and 143B proteinase K digestion and used for subsequent cells using TRIzol Reagent (Invitrogen, Carlsbad, RT-qPCR assays. CA, USA). Northern blot probes for LINC00958 and 18S were prepared by Biotin RNA Labeling Cell Proliferation Assay Kit (Roche, Basel, Switzerland). After thermal According to the manufacturer’s instructions, denaturation of total cellular RNA, the RNA cell proliferation capacity was assayed using samples were separated by formaldehyde de- the Cell Counting Kit-8 (CCK-8, Dojindo, Ku- naturing gel electrophoresis and transferred to mamoto, Japan). Briefly, transfected cells were NC membranes (Solarbio Science & Technology, seeded into 96-well plates at 1×104/well and in- Beijing, China), which were then incubated with cubated for 24, 48, 72, and 96 h. Subsequently, hydration buffer containing the probes. Finally, the absorbance at 490 nm was measured using the RNA signal was detected using a chemilumi- an enzyme labeling instrument (Bio-Rad, Rich- nescent nucleic acid detection kit (Thermo Fisher mond, CA, USA). The experiment was repeated Scientific, Waltham, MA, USA). three times.

Dual-Luciferase Reporter Assay Cell Cycle Assay Dual-luciferase reporter assays were used Flow cytometry analysis was conducted to to verify the target relationship between miR- evaluate the cell cycle. Transfected cells were 4306 and LINC00958 as well as CEMIP and collected and fixed with 70% ethanol overnight. miR-4306. Luciferase reporter plasmids con- Subsequently, cells were stained with propidium taining wild-type LINC00958 (wt-LINC00958), iodide (PI, Beyotime, Shanghai, China) in the mutant LINC00958 (mut-LINC00958), wild- dark for 30 min. Then, the percentage of the cells type CEMIP (wt-CEMIP) and mutant CEMIP in G0/G1, S and G2/M phases was detected by (mut-CEMIP) were designed and constructed flow cytometry (BD Biosciences, Franklin Lakes, by GenePharma Co., Ltd., (Shanghai, China). NJ, USA). The experiment was repeated three The correctly sequenced wild or mutant lu- times. ciferase reporter plasmids were co-transfected into HEK293T cells with miR-430 mimics or Apoptosis Assay negative control mimics (NC mimic) for 48 h, Cell apoptosis was assessed using Annexin respectively, and the cells were collected and V-FITC/PI Apoptosis Detection Kit (Beyotime, lysed. According to the manufacturer’s instruc- Shanghai, China) according to the manufacturer’s tions, the luciferase activity was determined by instructions. Briefly, transfected cells were har- the Dual-Luciferase reporter assay kit (Prome- vested and resuspended in binding buffer. Then, ga, Madison, WI, USA). Fluorescence intensity Annexin V-FITC (5 μl) and PI (10 μl) were added was measured with a GLomax 20/20 Luminom- and incubated for 20 min at room temperature in eter fluorescence detector from Promega. The the dark. Subsequently, the cells were analyzed experiment was repeated three times. by flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA). The experiment was repeated RNA Immunoprecipitation (RIP) Assay three times. The binding of LINC00958 and miR-4306 to AGO2 protein was detected by RIP kit (Milli- Well Assay pore, Billerica, MA, USA). Osteosarcoma cells Migration and invasion assays were performed were washed with pre-cooled PBS, and the su- using transwell chambers as described previous- pernatant was discarded. RIP Lysis Buffer was ly. Briefly, U2OS and 143B cells were inoculated added, and the products were lysed on ice and in uncoated and Matrigel-coated upper chambers collected. Then, the beads were washed with RIP (BD Biosciences, Franklin Lakes, NJ, USA) for Wash Buffer. After removal of the supernatant, migration and invasion assays, respectively. 1 × the beads were incubated with 5 μg Argonaute2 105 cells were suspended in serum-free DMEM (AgO2) antibody (Abcam, Cambridge, MA, USA) and inoculated in the upper chamber, and the low- for 30 min at room temperature, and normal rab- er chamber was supplemented with a complete bit immunoglobulin G (Abcam, Cambridge, MA, medium containing 10% FBS. After incubation USA) was used as the control. Subsequently, the for 24 h, cells were stained with 0.5% crystal cell lysate was incubated with RIP buffer con- violet and photographed under an inverted micro-

3186 LncRNA LINC00958 promotes tumor progression through miR-4306/CEMIP axis in osteosarcoma scope. Five fields of view were selected at random Hematoxylin and Eosin (HE) Staining to count the number of permeating cells under a Lung tissues of nude mice were taken and fixed light microscope and were further analyzed sta- in 10% buffered formalin. Paraffin-embedded tistically. tissues were sectioned at 5 mm and stained with HE. Sections were histologically observed and Scratch Wound Healing Assay evaluated under light microscopy. U2OS and 143B cells were inoculated into 6-well plates at a density of 2×105 cells/well, and then replaced with serum-free medium af- Statistical Analysis ter 100% confluence and scratched vertically All experimental data were processed and an- with a 200 μl pipette tip. Scratch healing was alyzed using SPSS 22.0 statistical software (IBM measured, and the scratch healing rate was cal- Corp., Armonk, NY, USA). The measurement culated. data were expressed as mean ± standard devia- tion. Paired t-test was used for the comparison of Animal Model cancer tissues and adjacent normal tissues, and Female BALB/c nude mice (age: 4-6 weeks; one-sample t-test was used for the comparison weight: 16-22 g) were provided by the Animal between the other two groups. One-way ANO- Experimental Center of Beijing Chao-Yang Hos- VA was used for comparison between multiple pital. The mice were fed with regular food with groups, after which pairwise comparisons were free access to drinking water in a well-ventilated made using post hoc tests. Pearson correlation environment of 50-60% humidity and alternative analysis was performed to correlate the expres- light/darkness cycle at room temperature. Animal sion of LINC00958 and miR-4306 or CEMIP in experiments were strictly in line with the Guide osteosarcoma tissues. Kaplan-Meier analysis was for the Care and Use of Laboratory Animal by In- performed to correlate the high and low expres- ternational Committees. The nude mice were ran- sion of LINC00958 in osteosarcoma patients with domly assigned to the following 3 groups (5 mice overall survival (log-rank test). p-value < 0.05 per group): sh-NC + NC inhibitor (co-delivered was considered statistically significant. with sh-NC and NC inhibitor), sh-LINC00958 + NC inhibitor (co-delivered with sh-LINC00958 and NC inhibitor), and sh-LINC00958 + miR- Results 4306 inhibitor (co-delivered with sh-LINC00958 and miR-4306 inhibitor). LINC00958 is Upregulated and For xenograft tumor experiments, 143B cells Associated with Poor Prognosis in with strong growth viability were trypsin digest- Osteosarcoma Patients ed, resuspended in serum-free DMEM medium Initially, we retrieved the osteosarcoma and counted. For each group, 1 × 106 cells were cell-based gene expression microarray data suspended in 100 μL serum-free DMEM, and (GSE45168) from the GEO database, and the uniformly mixed with 100 μL extracellular ma- bioinformatics tools were used to perform dif- trix (ECM) gel, which was then subcutaneously ferential gene expression analysis for osteo- injected into the back of the nude mice (5 mice sarcoma cell. The expression heatmap of the per group). After 3 weeks of subcutaneous inocu- top 25 differentially expressed genes in the lation, the nude mice were euthanized by carbon datasets is shown in Figure 1A, indicating that dioxide asphyxia, and the tumors were excised long non-coding RNA LINC00958 exhibited and weighed. significantly higher expression in cells with For the tumor metastasis assay, 143B cells increased metastatic potential. Therefore, we with strong growth viability were trypsin digest- selected LINC00958 as an important mole- ed, resuspended in serum-free DMEM medium cule for subsequent analysis. To verify the and counted, and the cell concentration was results from differential expression analysis, adjusted to 2 × 107 cells/mL. 100 μL cell sus- we detected the expression of LINC00958 in pension was injected into the nude mice through osteosarcoma tissues and cells by RT-qPCR. the tail vein. After 30 days of culture, the nude The results showed that the expression level of mice were executed and the metastatic foci in LINC00958 was significantly higher in osteo- the lungs were directly visualized in randomly sarcoma tissues compared with corresponding selected fields. adjacent normal tissues (Figure 1B). The ex-

3187 Y. Zhou, T. Mu

Figure 1. LINC00958 is overexpressed in osteosarcoma and correlates with prognosis. A, The expression heat map of the top 25 differential genes in dataset GSE14407. B, The expression levels of LINC00958 in osteosarcoma tissues and corresponding adjacent normal tissues were determined by qRT-PCR (n = 63). C, The expression levels of LINC00958 in hFOB 1.19, HOS, MG-63, U2OS, and 143B cells was determined by qRT-PCR. D, Kaplan-Meier survival curves were used to analyze the correlation between LINC958 expression and overall survival in 63 osteosarcoma patients. Data are represented as mean ± SD. **p < 0.01, ***p < 0.001 vs. normal tissues/hFOB 1.19 cell. pression level of LINC00958 was significantly Biological Characteristics of LINC00958 increased in four osteosarcoma cell lines (HOS, LINC00958 is annotated as a lncRNA gene in MG-63, U2OS, and 143B) compared with the Homo sapiens (NR_038904), located at the p-ter- human normal osteoblast cell line hFOB 1.19, minus of chromosome 11 in the genome, and the and the expression level of LINC00958 was sequence of LINC00958 is conserved among the higher in U2OS and 143B cell lines (Figure mammalian species. First, we detected the pres- 1C). We selected U2OS and 143B cell lines with ence of LINC00958 in the total RNA of U2OS higher LINC00958 expression for subsequent and 143B cells by Northern blotting experiments cell experiments based on the results. To further (Supplementary Figure 1A). To verify the sub- explore the relationship between the expres- cellular localization of LINC00958, we isolated sion level of LINC00958 and the prognosis of the nuclear and cytoplasmic fractions and ex- osteosarcoma patients, Kaplan-Meier analysis amined the transcript levels of LINC00958 by showed that higher expression of LINC00958 RT-qPCR. The results showed that LINC00958 was significantly associated with poor prog- was strongly enriched in the cytoplasmic fraction nosis of osteosarcoma patients (HR = 3.241, of both U2OS and 143B cells (Supplementary p = 0.004) (Figure 1D). Taken together, the Figure 1B). Besides, we further examined it by above findings suggested that LINC00958 was RNA fluorescence in situ hybridization assay expressed at a higher level in osteosarcoma, and obtained consistent results (Supplementary which was associated with a poor prognosis of Figure 1C). These results strongly suggest that osteosarcoma patients. LINC00958 is mainly located in the cytoplasm.

3188 LncRNA LINC00958 promotes tumor progression through miR-4306/CEMIP axis in osteosarcoma

Silencing LINC00958 Inhibits LINC0095 Functioned as a Sponge for Proliferation, Metastasis, and MiR-195-5p in Osteosarcoma Invasion of Osteosarcoma Cells Recent studies have indicated that many ln- While Inducing Cellular Apoptosis cRNA play important regulatory roles in a va- The newly identified lncRNA LINC00958 was riety of cancers through the competing endog- upregulated in osteosarcoma tissues and cells. enous RNA (ceRNA) mechanism27,28. To deter- To investigate the effect of LINC00958 on the mine whether lncRNA LINC00958 regulates biological function of osteosarcoma, we exam- gene expression through the ceRNA mechanism, ined the effect of LINC00958 on osteosarco- the online bioinformatics software, RegRNA ma cells by knocking down or overexpressing 2.0 and starBase 3.0 were used to predict the LINC00958 in U2OS and 143B cells. The expres- downstream miRNAs regulated by LINC00958. sion level of LINC00958 was verified for both The online prediction showed that miR-4306 knockdown and overexpression of LINC00958 targets the 3’ UTR of LINC00958 (Figure 3A). in osteosarcoma cells compared with control To determine whether miR-4306 interacts di- cells (Figure 2A). Sh-LINC00958 target sequenc- rectly with LINC00958, we performed a du- es included sh-LINC00958-1, sh-LINC00958-2, al-luciferase reporter assay. The Dual-Lucifer- and sh-LINC00958-3, among which the first had ase assay results showed that miR-4306 mimic the highest inhibitory efficiency. Therefore, we significantly decreased the luciferase activity of chose sh-LINC00958-1 for the subsequent func- LINC00958-Wt; however, the luciferase activity tional experiments (sh-LINC00958 refers to sh- of LINC00958-Mut was not affected by miR- LINC00958-1 in this study). Subsequently, we 4306 mimic transfection (Figure 3B). Also, RIP further investigated the effect of LINC00958 assay confirmed that LINC00958 and miR-4306 on proliferation, cell cycle, apoptosis, migration were both immunoprecipitated by the anti-Ago2 and invasion of osteosarcoma cells by gain- and antibody but not by the IgG antibody (Figure 3C), loss-of-function experiments. After LINC00958 indicating that LINC00958 as a sponge for miR- knockdown, the proliferation, migration and in- 4306. Then, we further explored the correlation vasion ability of osteosarcoma cells significantly between LINC00958 and miR-4306. LINC00958 decreased, while the apoptosis rate and the per- downregulation significantly increased the ex- centage of G0/G1 phase of the cell cycle signifi- pression level of miR-4306, and conversely, over- cantly increased (Figure 2B-2D, 2F-2H). On the expression of LINC00958 evidently decreased the contrary, when LINC00958 was overexpressed expression level of miR-4306 in U2OS and 143B by transfection with a plasmid containing the cells, as revealed by RT-qPCR analysis (Figure indicated sequence, the proliferation, migration 3D). Notably, we also found that miR-4306 ex- and invasion ability of osteosarcoma cells sig- pression was down-regulated in osteosarcoma nificantly increased while the apoptosis rate and tissues, and the expression of LINC00958 and the percentage of G0/G1 phase of the cell cycle miR-4306 in osteosarcoma tissues was negatively significantly decreased (Figure 2B-2D, 2F-2H). correlated (Figure 3E and 3D). Taken together, Meanwhile, the expression levels of apoptosis-re- our data demonstrate that LINC00958 acts as a lated proteins (cleaved caspase-3) and epitheli- sponge for miR-4306 and negatively regulates al-mesenchymal transition (EMT) related pro- miR-4306 expression in osteosarcoma. teins (E-cadherin and N-cadherin) were measured by Western blot assay. The results showed MiR-4306 Rescues the Function of that knockdown of LINC00958 in U2OS and LINC00958 in Osteosarcoma Cells 143B cells increased the abundance of cleaved Next, we explored whether the effect of caspase-3 and E-cadherin and reduced the con- LINC00958 on the biological function of osteo- tents of N-cadherin (Figure 2E, 2I). On the sarcoma cells was dependent on the sponging contrary, overexpression of LINC00958 in U2OS of miR-4306. First, the efficiency of miR-4306 and 143B cells reduced the contents of cleaved inhibitor was confirmed by RT-qPCR analysis caspase-3 and E-cadherin and increased the con- (Figure 4A). Then, rescue experiments were contents of N-cadherin (Figure 2E, 2I). Based on the ducted to investigate whether LINC00958 exerted above results, a conclusion could be drawn that its function through miR-4306 in osteosarcoma LINC00958 can promote the proliferation, me- cells. After performing the RT-qPCR analysis, tastasis and invasion of osteosarcoma cells while the results showed that sh-LINC00958 and NC reducing cellular apoptosis in vitro. inhibitor cotransfection significantly increased

3189 Y. Zhou, T. Mu

Figure 2. LINC00958 affected the migration and invasion of osteosarcoma cells. A, The efficiency of knockdown or overexpression of LINC00958 in U2OS and 143B cells was determined by qRT-PCR. B, The proliferation of U2OS and 143B cells was detected by CCK-8 assay. C, The cell cycle progression of U2OS and 143B cells was detected by flow cytometry analysis. D, Apoptosis rates of U2OS and 143B cells were measured by Annexin V-FITC/PI Apoptosis Detection Kit and analyzed by flow cytometry.E, The protein levels of cleaved caspase-3 in U2OS and 143B were assessed by Western blot assay. (F) and (G) The migration and invasion ability of U2OS and 143B cells evaluated by transwell assay, scale bar = 50 μm. H, Scratch wound healing assays were performed to examine the migration capacity of two osteosarcoma cell lines, scale bar = 50 μm. I, The protein levels of E-cadherin and N-cadherin in U2OS and 143B cells were assessed by Western blot assay. Data are represented as mean ± SD. *p < 0.05, **p < 0.01 vs. the sh-NC group, #p < 0.05, ##p < 0.01 vs. the oe- NC group.

3190 LncRNA LINC00958 promotes tumor progression through miR-4306/CEMIP axis in osteosarcoma

Figure 3. LINC00958 targeted the miR-4306 in osteosarcoma cells. A, Schematic representation of the wild-type and mutant binding sites of miR-4306 for LINC00958. B, Dual-Luciferase reporter assay was performed to validate the directly targeted role of miR-4306 on LINC00958 in HEK-293T cell. C, The interaction of LINC00958, miR-4306 and Ago2 in U2OS and 143B cells were evaluated by RIP assays. D, The expression levels of miR-4306 in U2OS and 143B cells were assessed by qRT-PCR after knockdown or overexpression of LINC00958. E, The expression levels of miR-4306 in osteosarcoma tissues and corresponding adjacent normal tissues were determined by qRT-PCR (n = 63). F, The correlation between miR-4306 and LINC00958 expression in osteosarcoma tissues was measured by Spearman’s correlation analysis. Data are represented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 vs. the NC mimic group/the IgG group/the sh-NC group/normal tissues, #p < 0.05 vs. the oe-NC group. the expression level of miR-4306 compared with and invasion but promoted apoptosis and G0/G1 the control group, while these outcomes were phase arrest of the cell cycle compared with the abrogated following cotransfection with miR- negative control; however, miR-4306 inhibitor 4306 inhibitor (Figure 4B). Then, functional restored the inhibitory effect of knocking down experiments showed that cells transfected with LINC00958 in 143B and U2OS cells (Figure sh-LINC00958 and NC inhibitor had dramat- 4C-4E, 4G-4I). Meanwhile, Western blot assay ically suppressed cell proliferation, metastasis showed that the abundance of cleaved caspase-3

3191 Y. Zhou, T. Mu

Figure 4. LINC00958 promoted metastasis and invasion of osteosarcoma cells by targeting miR-4306. A, MiR-4306 expression in osteosarcoma cells transfected with NC inhibitor or miR-4306 inhibitor was determined by qRT-PCR. B, U2OS and 143B cells were cotransfected with sh-LINC00958 and NC inhibitor or miR-4306 inhibitor, and the expression of miR- 4306 in the cells was detected by qRT-PCR. C, The proliferation of U2OS and 143B cells was detected by CCK-8 assay. D, The cell cycle progression of U2OS and 143B cells was detected by flow cytometry analysis. E, Apoptosis rates of U2OS and 143B cells were measured by Annexin V-FITC/PI Apoptosis Detection Kit and analyzed by flow cytometry. F, The protein levels of cleaved caspase-3 in U2OS and 143B were assessed by Western blot assay. (G) and (H) The migration and invasion ability of U2OS and 143B cells evaluated by transwell assay, scale bar = 50 μm. I, Scratch wound healing assays were performed to examine the migration capacity of two osteosarcoma cell lines, scale bar = 50 μm. J, The protein levels of E-cadherin and N-cadherin in U2OS and 143B cells were assessed by Western blot assay. Data are represented as mean ± SD. *p < 0.05, **p < 0.01 vs. the sh-NC + NC inhibitor group, #p < 0.05, ##p < 0.01 vs. the sh-LINC00958 + NC inhibitor group.

3192 LncRNA LINC00958 promotes tumor progression through miR-4306/CEMIP axis in osteosarcoma and E-cadherin increased, and the contents of ure 5G). To determine whether miR-4306 plays N-cadherin decreased in the cells transfected a role in the relationship between LINC00958 with sh-LINC00958 and NC inhibitor; however, and CEMIP, we investigated the expression lev- miR-4306 inhibitor restored the inhibitory ef- els of CEMIP in cells co-transfected with sh- fect of knocking down LINC00958 in 143B and LINC00958 and miR-4306 inhibitors. Indeed, the U2OS cells (Figure 4F, 4J). Our data strongly inhibitory effect of knocking down LINC00958 suggest that LINC00958 exerts an oncogenic ef- on CEMIP was partially blocked by the miR- fect on osteosarcoma progression by acting as a 4306 inhibitor (Figure 5H). Therefore, the results ceRNA for miR-4306. suggest that LINC00958 positively regulates CE- MIP expression in osteosarcoma cells through CEMIP is a Target of miR-4306 in sponge miR-4306. Osteosarcoma Cells and Positively Regulated by LINC00958 Silencing LINC00958 Inhibits To determine whether the signaling pathways Osteosarcoma Cell Tumorigenesis are involved in LINC00958/miR-4306 regulation and Metastasis In Vivo of osteosarcoma metastasis, we used miRDB and In the above study, we found that LINC00958 RNA22 to predict potential target genes of miR- targeting miR-4306 /CEMIP axis regulates the 4306. Bioinformatics analysis revealed that the progression of osteosarcoma in vitro. To further 3’ UTR sequence of CEMIP is complementary investigate the effects of LINC00958 and miR- to the seed sequence of miR-4306 and may serve 4306 on osteosarcoma growth and metastasis as the functional protein of miR-4306 (Figure in vivo, xenograft models and lung metastasis 5A). To seek further proof, we used a Dual-Lu- models were established by subcutaneous or tail ciferase reporter assay to verify the above pre- vein injection of 143B cells with different genetic diction. The results showed that the expression of modifications in nude mice, respectively. Xeno- CEMIP-Wt-driven luciferase cotransfected with graft model showed that the weight of tumors miR-4306 mimics was significantly reduced com- was significantly reduced in the sh-LINC00958 pared with the control, but this inhibition was + NC inhibitor group compared with the control abolished by mutation of the putative miR-4306 group, while co-transfection with miR-4306 in- binding site in the 3’ UTR of CEMIP (Figure hibitor partially rescued the inhibitory effect on 5B). Besides, to confirm whether CEMIP is- di tumor growth (Figure 6A). Lung metastasis mod- rectly regulated by miR-4306, we measured the els showed that the number of macroscopically mRNA and protein levels of CEMIP upon miR- visible pulmonary metastatic nodules in the lungs 4306 overexpression in U2OS and 143B cells. of the sh-LINC00958 + NC inhibitor group was We found that both mRNA and protein levels of significantly reduced compared with the control CEMIP were significantly reduced after overex- group. Similarly, co-transfection with miR-4306 pression of miR-4306 (Figure 5C and 5D). Sub- inhibitor partially restored the inhibitory effect sequently, the expression of CEMIP in osteosar- on tumor metastasis (Figure 6B). Subsequently, coma tissue samples and corresponding adjacent lung tissue sections were prepared and examined normal tissues was examined by RT-qPCR. The after hematoxylin and eosin staining, and the dif- results showed that the expression of CEMIP at ferences in the number of intrapulmonary micro- the mRNA level was significantly higher in os- scopically visible pulmonary metastatic nodules teosarcoma tissues compared with corresponding between groups were consistent with the above adjacent normal tissues (Figure 5E). Interesting- conclusion (Figure 6C). These data strongly sug- ly, Spearman correlation analysis further showed gest that LINC00958 has a key role in promoting a negative correlation between miR-4306 and osteosarcoma genesis and metastasis in vivo via CEMIP mRNA levels in osteosarcoma tissues the miR-4306 /CEMIP axis. (Figure 5F). Collectively, these experiments identified CEMIP as a direct target of miR-4306 in osteosarcoma cells. Discussion Since LINC00958 can sponge miR-4306, we next determined whether LINC00958 can reg- Osteosarcoma is the most common malignancy ulate CEMIP expression. We found that knock- that occurs in children and adolescents2. Although down of LINC00958 significantly reduced CE- the development of new clinical adjuvant chemo- MIP protein levels in U2OS and 143B cells (Fig- therapeutic agents has greatly alleviated the pro-

3193 Y. Zhou, T. Mu

Figure 5. LINC00958 promoted CEMIP expression in osteosarcoma cells by inhibiting miR-4306. A, Schematic representation of the wild-type and mutant binding sites of miR-4306 for CEMIP. B, The binding of miR-4306 and CEMIP mRNA verified by dual-luciferase reporter assays in HEK-293T cell. C, The expression of CEMIP mRNA in osteosarcoma cells transfected with NC mimic or miR-4306 mimic was determined by qRT-PCR. D, The protein levels of CEMIP in U2OS and 143B cells were assessed by Western blot assay. E, The expression levels of CEMIP mRNA in osteosarcoma tissues and corresponding adjacent normal tissues was determined by qRT-PCR (n = 63). F, The correlation between miR-4306 and CEMIP mRNA expression in osteosarcoma tissues was measured by Spearman’s correlation analysis. G, The expression of CEMIP protein in osteosarcoma cells transfected with sh-NC or sh-LINC00958 was determined by Western blot assay. H, U2OS and 143B cells were cotransfected with sh-LINC00958 and NC inhibitor or miR-4306 inhibitor, and the expression of CEMIP in the cells determined by Western blot assay. Data are represented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 vs. the NC mimic group/normal tissues/the sh-NC group/the sh-NC + NC inhibitor group, ##p < 0.01 vs. the sh-LINC00958 + NC inhibitor group. gression of osteosarcoma patients29,30, malignant play important regulatory roles in a variety of metastasis remains a major cause of treatment biological processes, and abnormally expressed failure in osteosarcoma patients31. Therefore, it is lncRNAs may be involved in the development of necessary to probe into the molecular mechanism multiple human diseases, especially in malignant of osteosarcoma development and explore new tumors like osteosarcoma14,32-34. We found that therapeutic strategies and methods to break the lncRNA LINC00958 was markedly upregulated bottleneck of osteosarcoma treatment. With the and significantly associated with poor survival in continuous development of molecular biotech- osteosarcoma patients. Mechanistic studies fur- nology, researchers have found that lncRNAs ther showed that LINC00958 functions as ceRNA

3194 LncRNA LINC00958 promotes tumor progression through miR-4306/CEMIP axis in osteosarcoma

Figure 6. LINC00958 promoted osteosarcoma growth and metastasis by regulating miR-4306 in vivo. A, Representative images of subcutaneous xenograft tumors in vivo and the final tumor weight of each group. B, Representative images of metastatic lung nodules and quantitation of metastatic lung nodules after intravenous injection of 143B cells in the whole lung. C, Representative HE analysis of sections of lung metastases from mice injected with 143B cells, scale bar = 1000 μm. Data are represented as mean ± SD. **p < 0.01, ***p < 0.001 vs. the sh-NC + NC inhibitor group, ###p < 0.001 vs. the sh-LINC00958 + NC inhibitor group. to sponge miR-4306, leading to increased CEMIP as development, metastasis, and invasion35,36. expression and thus promoting osteosarcoma cell We found that LINC00958 promotes the mi- tumorigenesis and metastasis in vitro and in vivo. gration and invasion of osteosarcoma cells in It is suggested that LINC00958 plays an oncogen- vitro and distant lung metastasis in vivo. To our ic role in the development of osteosarcoma and knowledge, the present study is the first to report can be used as a prognostic indicator for osteo- that LINC00958 regulates osteosarcoma cell sarcoma patients. metastasis. Notably, in lymph node metastatic Currently, an increasing number of studies bladder cancer, the expression of LINC00958 have identified functional lncRNAs, previously (also named bladder cancer-associated transcript referred to as “junk RNAs”, that regulate a vari- 2, BLACAT2) was significantly upregulated and ety of osteosarcoma pathological processes such associated with lymph node metastasis. Overex-

3195 Y. Zhou, T. Mu pression of LINC00958 promoted bladder can- In general, lncRNAs function as ceRNAs in cer-associated lymphatic vessel generation and different ways to influence intracellular signal- lymphatic metastasis37. Besides, the research- ing and cell fate, which are largely dependent on ers observed abnormally elevated expression of miRNA targets44. Then, we predicted CEMIP LINC00958 in lung adenocarcinoma (LUAD). as a potential target of miR-4306 using an on- The tumorigenic properties of LINC00958 in line database and further verified the prediction LUAD were revealed by reversing the inhibitory by a dual-luciferase reporter gene assay. Fur- effect of LINC00958 on LUAD cell prolifera- thermore, cellular experiments demonstrated that tion, migration, and invasion38. These findings overexpression of miR-4306 or knockdown of further support the potential pro-oncogenic role LINC00958 significantly inhibited the expres- of LINC00958 in tumorigenesis development. sion of CEMIP protein. CEMIP, also known as However, the molecular mechanisms underlying KIAA1199, is localized at 15q25.1 and encodes the pro-oncogenic role of LINC00958 in human a protein of approximately 150 kDa, which was tumors, including osteosarcoma, have not been identified as a novel protein involved in hyal- fully elucidated. uronan degradation45,46. Recent studies47,48 have Since 2007, the first ceRNA hypothesis was shown that CEMIP is expressed in a variety of proposed that the non-protein-coding transcript tissues and that its expression is abnormally high of IPS1 increases PHO2 mRNA by complemen- in tumor tissues and is strongly associated with tarily binding to miR-399, which has attracted tumor metastasis and invasion, progression, and significant global interest in deciphering the un- prognosis. For example, Zhao et al49 revealed mi- derlying mechanism39. Some scholars40,41 support crotubule destabilization regulated by KIAA1199 the existence of a novel and extensive interaction through the PP2A/stathmin pathway, which in network involving ceRNAs, in which lncRNAs turn promotes metastasis in colorectal cancer regulate the level of protein-coding mRNA mol- cells, while Shen et al50 found that CEMIP may ecules by competitively binding miRNAs. For promote ovarian carcinogenesis and progression example, MT1JP acts as a ceRNA that compet- through activation of the PI3K/AKT signaling itively binds to miR-92a-3p and upregulates the pathway. In our study, we demonstrated that expression of FBXW7 to regulate gastric cancer LINC00958 upregulated CEMIP expression by progression42. In addition, LINC00958 acts as competitively sponging miR-4306, which pro- a sponge for miR-330-5p in pancreatic cancer moted osteosarcoma tumorigenesis and metasta- and promotes PAX8-mediated tumor cell growth, sis accordingly. invasion and metastasis43. Interestingly, in our In summary, we comprehensively investigated study, FISH and subcellular fractionation analy- the functional role and molecular mechanisms of sis revealed that LINC00958 was located in the LINC00958 in osteosarcoma. Our results showed cytoplasm of osteosarcoma cells, which made it that LINC00958 expression was upregulated likely to function as a ceRNA. Next, we further in osteosarcoma cell lines and tissues. Higher explored the molecular mechanisms by which expression of LINC00958 was significantly as- LINC00958 regulates the metastasis and invasion sociated with poor prognosis in osteosarcoma of osteosarcoma cells. Bioinformatics analysis patients. Functional and mechanistic analyses in- and in vitro experimental studies revealed that dicated that LINC00958 increases the expression LINC00958 negatively regulates miR-4306 ex- level of CEMIP by acting as a ceRNA for miR- pression through complementary binding sites. 4306, which in turn promotes the tumorigenesis Moreover, cellular experimental studies revealed and metastasis of osteosarcoma cells in vitro and that miR-4306 inhibitor antagonized the antican- in vivo. Therefore, we suggest that LINC00958 cer effect induced by LINC00958 knockdown. could be both a significant marker for the prog- These data demonstrate that LINC00958 exerts nosis of osteosarcoma patients and an important oncogenic effects by downregulating miR-4306 target for osteosarcoma gene therapy. expression, suggesting that miR-4306 is an important tumor suppressor gene in the process of metastasis and invasion in osteosarcoma. In Conclusions conclusion, LINC00958 may act as a ceRNA to regulate the proliferation, cell cycle, apoptosis, We observed that the expression of LINC00958 metastasis, and invasion of osteosarcoma cells by is markedly elevated in osteosarcoma and is targeting miR-4306. significantly associated with metastasis and

3196 LncRNA LINC00958 promotes tumor progression through miR-4306/CEMIP axis in osteosarcoma poor prognosis of osteosarcoma. In addition, Brown KLB, Butterfass-Bahloul T, Calaminus G, LINC00958 may promote malignant progression Capra M, Dhooge C, Eriksson M, Flanagan AM, of osteosarcoma by regulating the miR-4306/ Friedel G, Gebhardt MC, Gelderblom H, Goldsby R, Grier HE, Grimer R, Hawkins DS, Hecker-Nolt- CEMIP axis. ing S, Sundby Hall K, Isakoff MS, Jovic G, Kühne T, Kager L, von Kalle T, Kabickova E, Lang S, Lau CC, Leavey PJ, Lessnick SL, Mascarenhas Conflict of Interest L, Mayer-Steinacker R, Meyers PA, Nagarajan The Authors declare that they have no conflict of interests. R, Randall RL, Reichardt P, Renard M, Rech- nitzer C, Schwartz CL, Strauss S, Teot L, Tim- mermann B, Sydes MR, Marina N. Survival and prognosis with osteosarcoma: outcomes in more Funding than 2000 patients in the EURAMOS-1 (European The work was funded by the Teaching Research and Re- and American Osteosarcoma Study) cohort. Eur form Foundation of Gansu University of Chinese Medicine J Cancer 2019; 109: 36-50. (YBXM-16). 7) Daw NC, Chou AJ, Jaffe N, Rao BN, Billups CA, Rodriguez-Galindo C, Meyers PA, Huh WW. Re- current osteosarcoma with a single pulmonary Availability of Data and Materials metastasis: a multi-institutional review. Br J Can- The datasets and supporting materials generated during cer 2015; 112: 278-282. and/or analyzed during the current study are available from 8) Chandra Gupta S, Nandan Tripathi Y. Potential of the corresponding author on reasonable request. long non-coding RNAs in cancer patients: from biomarkers to therapeutic targets. Int J Cancer 2017; 140: 1955-1967. Ethics Approval and Consent to Participate 9) Li W, Zhang L, Guo B, Deng J, Wu S, Li F, Wang The whole study was approved by the Ethics Committee of Y, Lu J, Zhou Y. Exosomal FMR1-AS1 facilitates Beijing Chao-Yang Hospital. All animal research was car- maintaining cancer stem-like cell dynamic equi- ried out following the Declaration of Helsinki. Written in- librium via TLR7/NFkappaB/c-Myc signaling in fe- formed consent was obtained from all patients. male esophageal carcinoma. Mol Cancer 2019; 18: 22. 10) Peng WX, Koirala P, Mo YY. LncRNA-mediated Authors’ Contribution regulation of cell signaling in cancer. Oncogene Y Zhou designed the study, conducted the experiments and 2017; 36: 5661-5667. drafted the manuscript. T Mu collected and analyzed the 11) Fu X, Zhu X, Qin F, Zhang Y, Lin J, Ding Y, Yang data. All authors read and approved the final manuscript. Z, Shang Y, Wang L, Zhang Q, Gao Q. Linc00210 drives Wnt/beta-catenin signaling activation and liver tumor progression through CTNNBIP1-dependent manner. Mol Cancer 2018; 17: 73. References 12) Li Z, Hou P, Fan D, Dong M, Ma M, Li H, Yao R, Li Y, Wang G, Geng P, Mihretab A, Liu D, Zhang 1) Taran SJ, Taran R, Malipatil NB. Pediatric osteo- Y, Huang B, Lu J. The degradation of EZH2 me- sarcoma: an updated review. Indian J Med Pae- diated by lncRNA ANCR attenuated the invasion diatr Oncol 2017; 38: 33-43. and metastasis of breast cancer. Cell Death Dif- 2) Ferguson JL, Turner SP. Bone cancer: diagno- fer 2017; 24: 59-71. sis and treatment principles. Am Fam Physician 13) Raveh E, Matouk IJ, Gilon M, Hochberg A. The 2018; 98: 205-213. H19 Long non-coding RNA in cancer initiation, 3 Hadjimichael AC, Foukas AF, Savvidou OD, Mav- progression and metastasis – a proposed unify- rogenis AF, Psyrri AK, Papagelopoulos PJ. The ing theory. Mol Cancer 2015; 14: 184. anti-neoplastic effect of doxycycline in osteosar- 14) Jiang N, Wang X, Xie X, Liao Y, Liu N, Liu J, Miao coma as a metalloproteinase (MMP) inhibitor: a N, Shen J, Peng T. lncRNA DANCR promotes tu- systematic review. Clin Sarcoma Res 2020; 10: 7. mor progression and cancer stemness features in 4) Shweikeh F, Bukavina L, Saeed K, Sarkis R, osteosarcoma by upregulating AXL via miR-33a- Suneja A, Sweiss F, Drazin D. Brain metastasis 5p inhibition. Cancer Lett 2017; 405: 46-55. in bone and soft tissue cancers: a review of in- 15) Yu X, Pang L, Yang T, Liu P. lncRNA LINC01296 cidence, interventions, and outcomes. Sarcoma regulates the proliferation, metastasis and cell 2014; 2014: 475175. cycle of osteosarcoma through cyclin D1. Oncol Rep 2018; 40: 2507-2514. 5) Ottaviani G, Jaffe N. The epidemiology of osteosarcoma. Cancer Treat Res 2009; 152: 3-13. 16) Kun-Peng Z, Chun-Lin Z, Xiao-Long M. Antisense lncRNA FOXF1-AS1 promotes migration and in- 6) Smeland S, Bielack SS, Whelan J, Bernstein vasion of osteosarcoma cells through the FOXF1/ M, Hogendoorn P, Krailo MD, Gorlick R, Jane- MMP-2/-9 pathway. Int J Biol Sci 2017; 13: 1180- way KA, Ingleby FC, Anninga J, Antal I, Arndt C, 1191.

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