Background: The aim of the present study was to find diagnostic and prognostic biomarkers for lung squamous cell carcinoma (LUSC) and to validate key biomarkers in vitro. Methods: RNA sequencing was used to identify differentially expressed mRNAs (DEmRNAs) and differentially expressed long non-coding RNAs (DElncRNAs) in LUSC tissues. RNA sequencing results were validated using a published dataset. Diagnostic and prognostic values of candidate genes were evaluated by receiver-operating characteristic (ROC) curve analysis and survival analysis, respectively. To determine the effect of MIR205HG in LUSC, MIR205HG expression was knocked down in NCI-H520 cells. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and Transwell assay were used to respectively detect the effect of MIR205HG on cell proliferation and migration. Results: In total, 1,946 DEmRNAs and 428 DElncRNAs were identified in LUSC compared with normal tissues. A total of 851 DElncRNA-DEmRNA co-expression pairs were obtained. With the exception of NEAT1, MCM2, SERPINB5, ITGB8, CASC19, and MIR205HG were upregulated in LUSC. ROC curve analysis indicated that MCM2, SERPINB5, ITGB8, CASC19, and MIR205HG could predict LUSC. Survival analysis suggested that SERPINB5, NEAT1, and MIR205HG had potential prognostic value for LUSC. MIR205HG knockdown inhibited cell proliferation and migration, and significantly reduced the expression of ITGB8. Conclusions: The findings of the present study could help determine the pathogenesis of LUSC and provide new and accurate therapeutic targets for its treatment.