Quantitative Analyses of miRNAs in Clinical Samples and Receiver Operating Characteristic (ROC) Curve Analysis. (A) Candidate endogenous references selected from the discovery set (n = 12) RNA-seq data. The scatter plot shows expression level distributions normalized to TPM (transcript per million) and the coefficient of variation (CV) values. The y-axis represents the mean miRNA expression levels and is displayed as log10 (mean TPM). The x-axis represents the dispersion degree of these miRNAs and is described by the CV. The most stably expressed miRNAs with a moderate expression are indicated with red dots (n = 8). The blue dotted line represents the maximum CV value of these 8 miRNAs (0.236). (B) A subset of 9 differentially upregulated miRNAs identified from the RNA-seq data were further examined using an independent validation set (50 LSCC patients and 25 HCs), with expression levels visualized using box plots. MiRNA expression levels were detected using qRT-PCR, with selected endogenous references (miR-30a-5p, miR-532-5p and U6) used as controls. The Y-axis displays the expression level as log10(2-ΔCt). *** P < 0.001, ** P < 0.01. (C) ROC curves assessing LSCC miR-941(blue line) and miR-27a-5p (green line), with area under the curve (AUC) values also determined. (D) Kaplan-Meier survival curves examining miR-941 expression in head and neck squamous cell carcinoma (HNSCC) tissue sample data obtained from the TCGA database. MiR-941 was significantly correlated with a poor outcome (log rank test, P = 0.0016). (E) Examination of HNSCC primary solid tumor data obtained from the YM500v3 database showed increased miR-941 expression. (F) Expression levels of miR-941 in different tumors from the YM500v3 database. The Y-axis represents the relative expression level in the tumors as compared to normal tissues. The X-axis displays the different tumor types. BLCA, Bladder urothelial carcinoma; BRCA, Breast invasive carcinoma; CESC, Cervical squamous cell carcinoma and endocervical adenocarcinoma; COAD, Colon adenocarcinoma; ESCA, Esophageal carcinoma; HNSC, Head and Neck squamous cell carcinoma; KICH, Kidney chromophobe; KIRC, Kidney renal clear cell carcinoma; KIRP, Kidney renal papillary cell carcinoma; LIHC, Liver hepatocellular carcinoma; LUAD, Lung adenocarcinoma; LUSC, Lung squamous cell carcinoma; PAAD, Pancreatic adenocarcinoma; PRAD, Prostate adenocarcinoma; READ, Rectum adenocarcinoma; SKCM, Skin cutaneous melanoma; STAD, Stomach adenocarcinoma.