Table1_Construction of prognostic signature of patients with oral squamous cell carcinoma based on pyroptosis-related long non-coding RNAs.xlsx

Background and objectiveOral squamous cell carcinoma (OSCC) is the most common malignant tumor in the head and neck, and its morbidity and mortality are increasing year by year. Changes in key genes are thought to be closely related to the occurrence and development of OSCC. Pyroptosis is an inflammatory form of programmed cell death that has been implicated in malignancies and inflammatory diseases. Changes in the expression of long noncoding RNAs may also affect tumorigenesis and progression. In this study, our main objective was to evaluate the association between pyroptosis-related lncRNAs and prognosis in patients with OSCC.MethodsThe RNA-seq data and clinicopathological data of OSCC patients are from The Cancer Genome Atlas database. The pyroptosis gene set is obtained from Gene Set Enrichment Analysis database. Univariate COX, Lasso and multivariate COX regression analyses were used for the construction of risk prognostic models of OSCC, eight lncRNAs were incorporated into prognostic models. The Kaplan-Meier method and log-rank test were used to evaluate the differences of overall survival between patients in high-risk and low-risk groups. The reliability of predictions across the dataset was analyzed by receiver operating characteristic (ROC) curves. The immune signature score was calculated using the single-sample gene set enrichment analysis.ResultsEight pyroptosis-related lncRNAs were used to construct prognostic signature of OSCC, including AC136475.2, AC024075.2, JPX, ZFAS1, TNFRSF10A-AS1, LINC00847, AC099850.3 and IER3-AS1. According to this prognostic signature, patients with OSCC were divided into high-risk and low-risk groups. Kaplan-Meier survival analysis showed that the survival rate of the high-risk group was significantly lower than the low-risk group. ROC area for risk score was 0.716, and ROC area of the 8 lncRNAs are all between 0.5 and 1, implied that these lncRNAs had high accuracy in predicting the prognosis of OSCC patients. Immune Infiltration findings suggested that these lncRNAs affected immune responses in the microenvironment of OSCC.ConclusionThe prognostic signature based on pyroptosis-related lncRNAs potentially serves as an independent prognostic indicator for OSCC patients. And this signature facilitates research on targeted diagnosis and treatment of patients diagnosed with OSCC.</p

Immunohistochemical staining with MAB1281 at different group after 12 weeks.

<p>USCs were identified by arrows. Scale bar = 50 μm.</p

Mineral apposition rate.

<p>(a) Alizarin red and calcein reveal mineralization. (b) Quantitative analysis shows a significant difference in the appositional rate of empty control, β-TCP and USCs/β-TCP (*P<0.05). Scale bar = 200 μm.</p

Proliferation of USCs on a β-TCP was determined using the CCK-8 assay on days 1, 2, 3, 4, 5, 6, and 7 (a).

<p>(b) Comparison of ALP activity after culturing USCs in osteogenesis media. (c) Calcium content quantification of USCs cultured on β-TCP for 7 or 14 days (*P<0.05).</p

Live/Dead assay for USCs on β-TCP scaffold.

<p>(a) 1 day; (b) 7 days. Scale bar = 100 μm.</p

Surgical procedures and in vivo experiment.

<p>(a) β-TCP scaffold; (b) femoral defect with a length of 6 mm was created; (c) the bone defect was implanted with USCs/β-TCP, β-TCP or nothing.</p

X-ray and micro-CT analysis of femoral defects.

<p>The groups are listed on the upper side of image. The times are listed on the right side of the image. (a): X-ray images of the femoral defect shows callus formation and bridging the defect in USCs/β-TCP group. (b): Representative micro-CT slice and reconstructed image show the continuous callus that bridged the femoral defect in USCs/β-TCP group; scaffold only group exhibited a reparative callus but did not show bridging of the defect; and blank control group showed limited callus formation. (c) Bone mineral density (BMD) and (d) bone volume were quantified within a standard ROI placed concentrically over the defect site (*P<0.05).</p

Biological characteristics of USCs.

<p>(a) Primary USCs exhibited a spindle-shaped morphology. Osteogenic differentiation was confirmed using Alizarin Red (b) and von Kossa (c) staining, (d) Toluidine blue staining revealed chondrogenic differentiation, and (e) adipogenic differentiation was verified using Oil red O staining. (f) Flow cytometric analysis demonstrates that USCs expressed markers associated with mesenchymal stem cells and did not express HLA-DR. Black traces indicate isotype controls and gray traces show the level of cell-surface expression. Scale bar = 400 μm.</p

Scanning electron micrographs of USCs on β-TCP scaffold after different incubation time.

<p>(a) 12 h, (b) 24 h, (c) 3 days, and (d) 5 days.</p

Histological analysis reveals bone formation within empty control, β-TCP and USCs/β-TCP at 12 weeks.

<p>(a) HE and Masson’s trichrome staining showing the repair of bone formation in the critical size femoral defect model. Arrows indicate the edges of host bone. (b) The percentage of the new bone area was calculated from the image of Masson’s Trichrome sections (*P<0.05). Scale bar = 100 μm (HE 200×).</p