Journal: Frontiers in Oncology

Article Title: Tspan9 Induces EMT and Promotes Osteosarcoma Metastasis via Activating FAK-Ras-ERK1/2 Pathway

doi: 10.3389/fonc.2022.774988

Figure Lengend Snippet: The siRNA and shRNA sequences used in this study.

Article Snippet: A FLAG-tagged PGMLV-6946 Tspan9 expression vector was purchased from GenePharma Technology Co., Ltd (Shanghai, China).

Techniques: shRNA, Negative Control

Journal: Frontiers in Oncology

Article Title: Tspan9 Induces EMT and Promotes Osteosarcoma Metastasis via Activating FAK-Ras-ERK1/2 Pathway

doi: 10.3389/fonc.2022.774988

Figure Lengend Snippet: Human OS cells and tumor tissue samples exhibit Tspan9 upregulation. (A–C) Tspan9 mRNA levels were significantly elevated in OS tumor tissues and cell lines in the GSE12865, GSE33383, and GSE42352 datasets relative to levels in normal MSCs and OBs. (D) Relative Tspan9 mRNA levels were markedly increased in HOS cells relative to control hFOB1.19 cells, whereas no changes were evident in U2OS or Mg63 cells as assessed via qRT-PCR. GAPDH served as a normalization control. (E) Western blotting results revealed that Tspan9 protein expression in HOS but not U2OS and Mg63 was significantly higher compared to hFOB1.19 cells. β-actin was used as a loading control and for normalization. Data are means ± SD from two independent experiments. (F) ROC curves and AUC values were determined using the GSE33383 and GSE42352 datasets. * P < 0.05; ** P < 0.01; *** P < 0.001; Student’s t-test. Tspan9, Tetraspanin-9; MSC, mesenchymal stem cell; OB: osteoblast; OS: osteosarcoma; GEO: Gene Expression Omnibus; qRT-PCR, quantitative reverse -transcription polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; SD, standard deviation; ROC, receiver operating characteristic; AUC, area under the curve.

Article Snippet: A FLAG-tagged PGMLV-6946 Tspan9 expression vector was purchased from GenePharma Technology Co., Ltd (Shanghai, China).

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction, Standard Deviation

Journal: Frontiers in Oncology

Article Title: Tspan9 Induces EMT and Promotes Osteosarcoma Metastasis via Activating FAK-Ras-ERK1/2 Pathway

doi: 10.3389/fonc.2022.774988

Figure Lengend Snippet: Tspan9 knockdown suppresses OS cell proliferation. (A) Tspan9 mRNA levels in siTspan9 cells (siTspan9#1, #2, and #3) were significantly lower than those in siNC cells, as measured via qRT-PCR. (B) The viability of HOS cells in the siNC and siTspan9 HOS cells was assessed via CCK-8 assay. (C) The impact of Tspan9 knockdown on HOS cell proliferation was measured via colony formation assay. Statistical results of colony formation numbers normalized to shNC were presented. * P < 0.05; ** P < 0.01; *** P < 0.001; Student’s t-test. NC, negative control; si, small interfering RNA.

Article Snippet: A FLAG-tagged PGMLV-6946 Tspan9 expression vector was purchased from GenePharma Technology Co., Ltd (Shanghai, China).

Techniques: Quantitative RT-PCR, CCK-8 Assay, Colony Assay, Negative Control, Small Interfering RNA

Journal: Frontiers in Oncology

Article Title: Tspan9 Induces EMT and Promotes Osteosarcoma Metastasis via Activating FAK-Ras-ERK1/2 Pathway

doi: 10.3389/fonc.2022.774988

Figure Lengend Snippet: RNA-seq-mediated identification of the biological roles of Tspan9 in OS cells. (A) Heatmaps demonstrating DEGs identified via RNA-seq in HOS cells in which shTspan9 or shNC were stably expressed. (B) DEGs (n=211) are represented in a volcano plot, including 96 upregulated DEGs (red) and 115 downregulated DEGs (green), with DEGs having been identified using the following criteria: adjusted log fold-change ≥ 1 and P ≤ 0.05. (C, D) GO analyses of DEGs identified following Tspan9 knockdown were conducted, with top enriched biological processes, molecular functions, and cellular components being shown in a bubble chart in which darker coloration is indicative of more significant enrichment. (E, F) KEGG pathway enrichment analyses of identified DEGs were conducted, with the results being shown in a bubble chart in which bubble size is proportional to the number of DEGs in a given pathway, and the bubble color is indicative of P-value significance (red = significant, blue = non-significant). DEGs, differentially expressed genes; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Article Snippet: A FLAG-tagged PGMLV-6946 Tspan9 expression vector was purchased from GenePharma Technology Co., Ltd (Shanghai, China).

Techniques: RNA Sequencing Assay, Stable Transfection

Journal: Frontiers in Oncology

Article Title: Tspan9 Induces EMT and Promotes Osteosarcoma Metastasis via Activating FAK-Ras-ERK1/2 Pathway

doi: 10.3389/fonc.2022.774988

Figure Lengend Snippet: Tspan9 promotes in vitro OS cell migration and invasion. (A) GFP expression was indicative of stable lentiviral transduction of HOS cells with shTspan9 (shTspan9#1 or shTspan9#2) or shNC constructs at near 100% efficiency (fluorescent microscopy). Tspan9 knockdown efficiency was also confirmed via qRT-PCR (lower left panel) and Western blotting (lower right panel). (B) OS cell migration was assessed in a wound-healing assay using cells stably expressing shTspan9 or shNC. (C) The impact of Tspan9 knockdown on OS cell migration and invasion was assessed via a Transwell approach. (D) GFP expression was indicative of successful Tspan9 overexpression, as confirmed via qRT-PCR and Western blotting relative to Mock control. Wound-healing (E) and Transwell assays (F, G) were conducted to assess the impact of Tspan9 on the migratory and invasive activity of OS cells. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; Student’s t-test. GFP, green fluorescent protein; sh, short hairpin RNA.

Article Snippet: A FLAG-tagged PGMLV-6946 Tspan9 expression vector was purchased from GenePharma Technology Co., Ltd (Shanghai, China).

Techniques: In Vitro, Migration, Expressing, Transduction, Construct, Microscopy, Quantitative RT-PCR, Western Blot, Wound Healing Assay, Stable Transfection, Over Expression, Activity Assay, shRNA

Journal: Frontiers in Oncology

Article Title: Tspan9 Induces EMT and Promotes Osteosarcoma Metastasis via Activating FAK-Ras-ERK1/2 Pathway

doi: 10.3389/fonc.2022.774988

Figure Lengend Snippet: Tspan9 regulates EMT, and knocking down of it impairs in vivo OS cell metastasis. (A) EMT marker protein levels and associated transcription factor expression were assessed in HOS and U2OS cells stably expressing shTspan9/OE-Tspan9 or control constructs. (B) Quantification of the Western blotting results presented in (A) . (C, D) HOS cells stably expressing shTspan9 or control constructs were injected via the lateral tail vein into nude mice to establish a model of pulmonary metastasis (n=5/group). Left: representative lung images; Right: representative H&E staining results (1x and 5x). Pulmonary nodules are indicated by red arrows, while normal alveolar tissue is indicated by blue arrows. (E) Numbers of metastatic pulmonary nodules in the indicated groups. (F) Lung weights on day 28 post-HOS tumor cell injection. (G) Murine body weight was assessed every 4 days. * P < 0.05; ** P < 0.01.

Article Snippet: A FLAG-tagged PGMLV-6946 Tspan9 expression vector was purchased from GenePharma Technology Co., Ltd (Shanghai, China).

Techniques: In Vivo, Marker, Expressing, Stable Transfection, Construct, Western Blot, Injection, Staining

Journal: Frontiers in Oncology

Article Title: Tspan9 Induces EMT and Promotes Osteosarcoma Metastasis via Activating FAK-Ras-ERK1/2 Pathway

doi: 10.3389/fonc.2022.774988

Figure Lengend Snippet: Tspan9-β1 interactions promote FAK-Src-Ras-ERK1/2 pathway signaling and OS metastasis. (A) Interactions between Tspan9 and integrin β1 were detected in a co-IP assay. (B) FAK-Ras-ERK1/2 pathway proteins (FAK Y397 , total-FAK, Ras, pERK1/2, and total-ERK1/2) were analyzed via western blotting, with β-actin as a loading control. (C) Quantification of the Western blotting results presented in (B) . (D) Western blotting was used to assess Ras downstream signaling in OE-Tspan9 U2OS cells following Salirasib treatment (50μM). (E) Following treatment with Salirasib (50μM), OE-Tspan9 cells were analyzed in migration and invasion assays, with representative cells being shown. (F) Schematic presentation of mechanism underlying Tspan9-mediated OS metastasisis. All analyzes were repeated two or three times. Data are means ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: A FLAG-tagged PGMLV-6946 Tspan9 expression vector was purchased from GenePharma Technology Co., Ltd (Shanghai, China).

Techniques: Co-Immunoprecipitation Assay, Western Blot, Migration