Clinical samples and cell lines
This study used formalin-fixed and paraffin-embedded OS patient tissues (n = 72) and normal tissues (n = 6), as well as fresh primary OS tissues (n = 20) and normal tissues (n = 5) that were collected from the Sun Yat-sen University Cancer Center, First Affiliated Hospital of Shantou University Medical College and First Affiliated Hospital of Zhejiang University Medical College from 2010 to 2023. None of the patients had received preoperative radiotherapy or chemotherapy. This study was approved by the ethical review committees of the Shantou University Medical College Cancer Center. All participants involved in our study provided written informed consent.
OS cell lines (MG63, U2OS), obtained from Cell Bank of the Chinese Academy of Science (Shanghai, China), were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Gibco, Shanghai, China), at 37oC in a humidified incubator containing 5% CO 2.
Drugs, siRNA, plasmid transfection and stable cell lines
Paclitaxel and cisplatin were purchased from MCE (Shanghai, China). For knockdown, short interfering RNAs against SOX2 (si-SOX2), miR-5047 and control mimics and inhibitors were purchased from the GenePharma Company (Shanghai, China). For gene transfection, WAC-AS1, pcDNA3.1-SOX2 and the negative control vectors (NC) were purchased from the Vigene Company (Jiangsu, China). jetPRIME (Polyplus Transfection, USA) was used to transfect the above-mentioned plasmids or oligonucleotides. Transfection was performed according to the user’s manual. For stable cell lines, WAC-AS1 shRNA and control shRNA were separately inserted into the pGLV3/H1/GFP/Puro vector. Wildtype and mutant WAC-AS1 sequences were cloned into the lentiviral expression vector pCDGEF1/GFP/Puro. OS cells were infected by the lentivirus following the user’s instructions. To isolate stably expressing cells, the infected cells were selected in 1 µg/ml puromycin (Gibco, USA) for two weeks.
RNA sequencing and qRT-PCR
RNA sequencing was performed by Gene Denovo Biotechnology Co. (Guangzhou, China) according to the manufacturer’s protocol. Briefly, total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA,USA) The enriched mRNAs and ncRNAs were fragmented into short fragments and reverse transcribed into cDNA with random primers. After purification and ligation to Illumina sequencing adapters, the second-strand cDNA was digested and selected by agarose gel electrophoresis, PCR amplified, and sequenced using an Illumina HiSeqTM 4000. The RNA-seq data is available online: SRA accession: PRJNA640969.
Cytoplasmic and nuclear RNA extraction was performed using a FastPure Cytoplasmic & Nuclear RNA purification kit (ECOTOP Scientific, Guangzhou). Total RNAs were extracted using an RNA extraction kit purchased from the Vazyme Company (Nanjing, China). The cDNA was reverse-transcribed using a cDNA Synthesis Kit (Vazyme). Then, SYBR Green Master Mix (Vazyme) was used to perform qRT-PCR in a Bio-Rad 7500 Fast RT-PCR System. β-actin and U6 served as controls. The primer sequences are listed in Table S1.
Cell proliferation, colony formation and migration assays
A Cell Counting Kit-8 (CCK-8) (Solarbio, China) was utilized to measure cell proliferation as previously described . For colony formation, 500 cells were seeded in a 6-well plate and stained after 14 days. Colonies with > 30 cells were scored. Transwell and wound healing assays were used to examine cell migration as previously described .
Tumorsphere formation assay
One thousand cells were seeded in ultralow attachment six well plates (Corning) and cultured for 1 week in DMEM/F12 medium (Invitrogen, Shanghai, China) supplemented with B27 (1:50, Gibco), 20 ng/ml bFGF (Sigma, Shanghai, China), and 20 ng/ml EGF (Sigma). Pictures were taken using a bright-field microscope and the number of the spheres was counted.
Flow cytometry analysis
Cell apoptosis was examined using an Annexin V-FITC/PI Double Staining Kit (Beyotime, China) according to the user’s manual. To detect the OS stem cell subpopulations, APC-anti-CD133 antibody was used. A total of 1 × 106 cells were incubated with antibodies in the dark at 4 °C for 30 min. After washing, the cells were re-suspended in 500 µl of PBS and analyzed using a flow cytometer(C6, Japan).
Fluorescence in situ hybridization (FISH), ISH and immunohistochemistry (IHC)
FISH staining was performed according to the user’s manual. MG63 and U2OS cells, grown on slides, were fixed with 4% (v/v) formaldehyde, and then dehydrated for overnight hybridization with a WAC-AS1 probe. After counterstaining with DAPI, slides were examined under a fluorescence microscope. For ISH, WAC-AS1 probes were synthesized by Boster (Wuhan, China). WAC-AS1 expression was also examined in formalin-fixed, paraffin-embedded (FFPE) samples using procedures outlined in the user’s manual. IHC was performed as described previously . The scores for ISH and IHC in OS samples were quantified by two pathologists, and the average score of each tissue was obtained for statistical analysis.
Western blot analysis
Total protein from cells and tissues was extracted by RIPA lysis buffer (Beyotime) containing 1% protease inhibitors (Invitrogen). Western blotting was performed as previously described . The primary antibodies were anti-SOX2, Nanog, OCT4, PARP, CD133, Bax, Bcl-2, β-actin and GAPDH (all CST, 1:1000).
RNA immunoprecipitation (RIP) assay
A RIP Kit (Bersinbio, Guangzhou, China) was utilized to determine the binding between WAC-AS1, miR-5047 and AGO2 protein according to the manufacturer’s protocol. In brief, OS cells were washed with PBS and resuspended using RIP lysis buffer containing a proteasome inhibitor. Antibody against AGO2 or IgG was incubated with cell lysates overnight. Then RNA-protein complexes were incubated with protein A/G magnetic beads. After proteinase K digestion, the immunoprecipitated RNAs were purified and subjected to qRT-PCR.
Chromatin immunoprecipitation (ChIP) assay
A ChIP Kit (Bersinbio) was used, to determine the binding between SOX2 protein and the predicted WAC-AS1 promoter region, according to the manufacturer’s instructions. In brief, cells were crosslinked in 1% formaldehyde and quenched with glycine. Then 200–600 bp fragments of cross-linked chromatin were obtained by sonication, and then incubated with SOX2 or IgG antibody overnight. Finally, DNA was isolated and quantitative PCR was used to examine the relative enrichment of the WAC-AS1 promoter region.
Dual luciferase reporter assay
WAC-AS1 cDNA, either wild-type (WAC-AS1 WT) or containing a mutant miR-5047 binding site (WAC-AS1 Mut), were cloned into the pmirGLO vector (Vigene). Then, the indicated plasmids were co-transfected with miR-5047 mimics or control into OS cells. After co-transfection for 48 h, a dual Luciferase Reporter Assay System (Promega, MD, USA) was used to detect relative luciferase activity according to the manufacturer’s instructions.
RNA pulldown assay
To capture proteins capable of binding to WAC-AS1, a desthiobiotin-labeled WAC-AS1 was designed. An RNA Pull-Down Kit (Bersinbio) was used according to the manufacturer’s instructions. The biotin–labeled WAC-AS1 was incubated with OS cell protein extracts, then pulled down with magnetic beads and eluted. Anti-AGO2 was detected by western blotting, and miR-5074 was detected by qRT-PCR.
MiRNA target prediction
MiRNAs that potentially bound to the 3’ untranslated region (3’UTR) of an mRNA or lncRNA were predicted using websites, including ENCORI (https://starbase.sysu.edu.cn/), miRDB (http://www.mirdb.org/) and Targetscan (http://www.targetscan.org/vert_72/). MiRNAs potentially sponged by WAC-AS1 were predicted via ENCORI and miRDB. Overlapping miRNAs were chosen as miRNA candidates sponged by WAC-AS1.
U2OS cells transduced with WAC-AS1 or NC lentivirus were subcutaneously injected into the backs of BABL/c nude mice (n = 6). The tumor volume of each mouse was calculated every 7 days according to the formula: length× width2 × 0.5. Four weeks after cell injection, mice were euthanized and the xenografted tumors were collected and weighed. Then, the growth curves of tumors of the two groups were drawn according to the tumor volume. For metastasis, stable WAC-AS1 expression or control OS cells were injected into mice through tail vein (n = 5). After 5 weeks, euthanasia was performed, and the numbers of lung metastatic nodules were counted under the microscope.
Data analysis was conducted with SPSS 19.0 or GraphPad prism 8.0 software. The results are presented as mean ± SD after three independent trials. Student’s t test or one-way analysis of variance (ANOVA) was implemented to compare statistical differences between two groups or among multiple groups. Associations between different target expressions in OS patients were analyzed using Pearson’s correlation analysis. A P < 0.05 was considered to indicate statistical significance.