2.1. Data download and preprocessing
Download GSE33382 expression spectrum data in GEO database, a total of 87 samples. There were 84 tumor samples and 3 control samples. After screening, all zero-valued genes and samples were removed, and the expression of redundant genes was averaged. Finally, 21591 genes and 83 disease samples were left.
Died in Fe database FerrDb database (http://www.zhounan.org/ferrdb) to download Fe death related factors, and the Fe on the reference death factor set[13], with the A total of 291 related factors were obtained for subsequent analysis, of which 193 were expressed in GSE33382.
2.2. Unsupervised cluster analysis
The "ConsensusClusterPlus" package of "R" was used for unsupervised consistency clustering.
2.3. Analysis of differential gene expression
The "limma" package of "R" was used for differential gene expression analysis. P < 0.05 was selected, and the absolute value of LOG2 (FC) was 1.2 as the threshold.
2.4. Functional enrichment analysis
Metascape (https://metascape.org/gp/index.html#/main/step1) online tools was Used to enrichment analysis of differentially expressed genes between the classes.
2.5. Build a co-expression network
"WGCNA software" package of "R" was used to construct weighted co-expression network for differentially expressed gene sets
2.6. Identification of key modules and hub genes
First, we extracted 549 genes from Turquoise module (the key module for identification), and constructed PPI network in String database. There were 279 nodes in the network (excluding solitary points). Key genes in the network were extracted. Here, the genes with a degree in the top 25% were considered as hub genes, and there were 69 genes in total. Secondly, the key Module hub Gene (module-membership and gene-significance) is identified. The threshold value of Gene Significance is selected as required. The default value of 0.2 given on the official website is used here, and the MM is 0.8. After screening, 42 hub genes (GS_MM_hub) were obtained. Finally, the key module hub genes and PPI key genes were taken to intersect to obtain the phenotypically related 4 hub genes.
2.7. Construct hub Gene Multi-factor Regulatory network (ncRNA\TF)
Respectively from mirwalk database (http://mirwalk.umm.uni-heidelberg.de/), lncrna2target database (http://123.59.132.21/lncrna2target/index.jsp), And TRRUS (https://www.grnpedia.org/trrust/Network_search_form.php) in the database, The hub genes and regulatory relationship was download from ncRNA/TF, and visualization with the Cytoscape software.
2.8. Main materials and instruments
Rabbit anti-human YRDC, ARPC5, EIF2S1, CAPZA1 polyclonal antibody (Santa Cruz Company, USA), Universal goat anti-rabbit HRP(K4001, DAKO Company), Ordinary optical microscope (Olympus Company, Japan); Color pathological image analysis system (microvision 98 lines); Q500MC image analyzer (Leica1EICA Company); Primer synthesis (Wuhan Jikai Biological Company); DMEM/F12, DMEM culture medium, FBS, Neomycin G418 (Thermofisher).
2.9. Tissue chip and general information
Osteosarcoma tissue microarrays and control bone and bone marrow tissue microarrays were acquired from Xi'an Zhongke Guanghua Biotechnology Co., Ltd., comprising 70 osteosarcoma specimens and 11 healthy bone and bone marrow samples. The osteosarcoma group included 45 males and 25 females, with ages ranging from 10 to 69 years and an average age of 29.5 years. The tumor locations were 36 cases in the femur, 14 in the tibiofibula, 9 in the humerus, and 9 in other locations. Of the 70 osteosarcoma cases, 47 had detailed tumor diameter records, with 15 cases having a tumor diameter ≤ 5cm and 32 cases having a tumor diameter > 5cm. Based on the Enneking staging system, there were 2 cases at stage I, 31 at stage IIA, 31 at stage IIB, 6 at stage IV, 6 with lymph node metastasis, and 64 without lymph node metastasis. The control group consisted of 8 male and 3 female patients, with ages ranging from 22 to 87 years and an average age of 46.3 years. The study received approval from the Guizhou People's Hospital Ethics Committee.
2.10. Immunohistochemistry
Tissue slides were deparaffinized and heated in an oven at 60°C for 120 minutes. Subsequently, they were immersed in xylene I and II for 5 minutes each, followed by sequential rinses with anhydrous alcohol, 95% alcohol, 80% alcohol, and finally distilled water. Antigen retrieval was performed using preheated EDTA (pH 8.0) solution in a thermal repair instrument at 120°C for 2 minutes, held for 10 minutes, cooled to room temperature for 15 minutes, and washed with PBS thrice for 2 minutes each. The samples were then incubated with 3% hydrogen peroxide for 10 minutes at room temperature, followed by three 2-minute PBS washes. After drying, primary antibodies were added and incubated overnight at 4°C. The slides were rinsed with PBS three times for 2 minutes each, dried, and treated with a universal secondary antibody for 30 minutes at 37°C. After three 3-minute PBS washes and two distilled water rinses, DAB color development was performed for 5 minutes, followed by a running water rinse. The samples were counterstained with hematoxylin for approximately 10 seconds, differentiated with 0.5% hydrochloric acid alcohol, washed with water, and treated with saturated lithium carbonate for bluing. The slides were dehydrated with 95% alcohol and anhydrous alcohol for 1 minute each, cleared with xylene I and II for 1 minute each, and mounted using neutral gum. Two blinded pathologists independently evaluated immunohistochemical staining. In cases of disagreement, a consensus was reached after joint review and discussion. Staining results were analyzed using a two-way scoring system [14, 15], assessing staining intensity (scored 0–3) and the percentage of positive cells (scored 0–3). The final staining score was determined by combining these scores: 0 (negative, -), 1–2 (weak, +), 3–4 (moderate, ++), and 5–6 (strong, +++).
2.11. Cell Lines and Cell Culture
U-2OS cell line and hFOB 1.19 cell line were purchased from Wuhan Punosai Life Science And Technology Co., LTD., while 143B cell line was purchased by Wuhan Hualingke Biological Co., LTD., and cell culture: U-2OS and 143B cells were routinely cultured in DMEM medium containing 10% fetal bovine serum and placed in a 37℃ incubator with 5% CO2 saturation and humidity. hFOB 1.19 cells were routinely cultured in DMEM/F12 medium containing 10% fetal bovine serum and placed in an incubator at 34℃ with 5% CO2 saturation and humidity. The cells were changed once every 2–3 days. When the cells grew and fused to about 80–90%, the cells were subcultured in bottles at 1:3, and cells at logarithmic growth stage were taken for study. The cells were observed under the microscope. When the cells were full of cells, the old medium was discarded and the cells were washed with sterile PBS. Appropriate trypsin was added to the cell culture dish for digestion for about 2min, and complete medium was added to terminate digestion. After adding an appropriate amount of medium and blowing evenly, the cell suspension was proportionally divided into petri dishes or six-well plates for subsequent treatment such as subculture or plate laying.
2.12. Real-Time Reverse Transcription-PCR
Each cell line RNA was extracted, and reverse transcriptase was performed using the TRIzol (TaKaRa Bio, Dalian, China) and a Prime ScriptTM RT Reagent Kit (TaKaRa Bio, Dalian, China). The most efficient YRDC primer sequence was screened for 5'-CAGAATTCATGAGGCCACTGTGCGTGAC-3'(sense strand);5'-GAAAGCTTTTAGTGGAATGTTGGGGT-3'(antisense strand); ARPC5 primer sequence (sense strand: 5'-TGCCACTGGAGTAACACTGC-3'; antisense strand:5'-CTCCCGTCAACACCAAGAAT-3'); EIF2S1 primer sequence(sense strand:5'-TGGGACGCCTAACCTACAAC-3';antisense strand:5'-TCATCTGACCAGGAAGGACA');CAPZA1 primer sequence(sense strand:5'-GGAAAGAAGCAAGTGACCCC-3';antisense strand:5'-TTAGGCTGAAACTGGTGGCT-3');GAPDH primer sequence(sense strand:5'-CTTTAGCATGATCGACTG-3';antisense strand:5'-GTAGAAGCGATAATTGCT-3'). After a pre-denaturation step at 95°C for 5 min, 40 cycles of PCR were performed as follows: 10 s denaturation at 95°C and 30 s annealing at 60°C. The fold amplification for each gene was calculated using the 2-ΔΔCt method.The experiment was repeated three times.
2.13. Western Blot Analysis
The cells underwent lysis on ice for half an hour, with the resulting suspension gathered in a 1.5 mL tube at 14,000 rpm and spun at 4°C for 20 minutes. Protein quantification was achieved via the BCA method, and samples were prepared with 1x loading buffer. Proteins were separated through 10% SDS-PAGE and transferred using the transwet method. Membranes were blocked with 5% skim milk powder at room temperature for two hours, followed by primary antibody incubation overnight at 4°C. All antibodies were sourced from Abcam (Cambridge, MA, USA). After washing the PVDF membrane thrice for 10 minutes each, it was incubated with secondary antibodies for an hour at room temperature for ECL. A gel imager assessed protein levels in cell groups. The experiment was conducted thrice.
2.14. MTT assay
Cells transfected for 24 h were collected, and seeded into a 96-well plate at 5 × 103 cells/well, followed by the addition of 20 µL of MTT solution (5 µg/mL) at 24, 48, and 72 h after being incubated at 37°C. Then the cells were cultured at 37°C for 4 h. A total of 200 µL dimethyl sulfoxide was added to each well, and then the optical density of each group of cells under 570 nm was measured using a spectrophotometer.
2.15. Plate clone formation experiment
The cells in each group at logarithmic growth stage were digested with 0.25% trypsin and beaten into single cells, respectively. The cells were suspended in 10% FETAL bovine serum DMEM medium for reserve. The cell suspension was gradient multiple diluted, and cells in each group were inoculated into a dish containing 10mL 37℃ preheating medium at a density of 200 cells per dish, and gently rotated to make the cells evenly dispersed. The cells were incubated at 37℃ with 5% CO2 and saturated humidity for 72 hours. Discard the supernatant and carefully soak it twice with PBS. The cells were fixed with 5mL of 4% paraformaldehyde for 15 minutes. Then the fixation solution was removed, GIMSA was added with appropriate amount and dyed with the staining solution for 30 minutes. The plate was inverted and a transparent film with grid was superimposed, and the number of clones of 10 cells was counted under the microscope. Finally, the clone formation rate was calculated. Clone formation rate =(number of clones/number of inoculated cells)×100%。
2.16. Statistical Product and Service Solution
SPSS 23.0 software (IBM, Armonk, NY, USA). Data analysis was performed with the Students, t-test and ANOVA. Bonferroni test was used to validate ANOVA for pairwise comparisons. The comparison of count data was performed by the χ2-test. The overall survival rate was calculated by Kaplan-Meier method. P < 0.05 was considered statistically significant.