Project Summary: Lung cancer continues to be a significant health burden in the U.S. Thus, a functional understanding of the critical molecules during NSCLC oncogenesis will enhance our knowledge of its malignant transformation, and allow novel therapeutic strategies. We have recently identified critical roles of protein function effector sncRNAs (pfeRNAs) in the tumorigenesis and differentiation of NSCLC, have described their functions in the basic biology of NSCLC, and have elucidated their structural and functional features. We have identified eight pfeRNAs as promising non-invasive biomarkers using sncRNAs deep sequencing by analyzing 108 biospecimens, including (i) plasma from healthy controls, (ii) plasma from patients with Stage I/II NSCLC with matched biopsy-proven NSCLC tissue as well as histologically normal adjacent lung tissue, and (iii) plasma from patients with both biopsy-proven benign and malignant lung nodules. We have already validated these biomarkers in 352 clinical biospecimens, including 77 healthy controls, 44 patients with benign disease, and 231 patients with malignant lung nodules. These eight pfeRNAs were able to: (1) to differentiate patients with or without pulmonary nodules, with a sensitivity and specificity up to 98.1% and 100%, respectively; (2) to differentiate patients who had malignant versus benign pulmonary nodules, with a sensitivity and a specificity up to 78% and 78.8%, respectively. Furthermore, we identified a pfeRNA is ADC-specific and two pfeRNAs are SCC-specific. Besides, we have confirmed functional analyses of these three pfeRNAs one by one and identified their roles in the primary biological activities of ADC, SCC, and HBE cells in vitro. In addition, we specified the cellular distribution of these three pfeRNAs by fluorescence in situ hybridization (FISH) assay. The data strongly suggest that these three pfeRNAs play critical roles in tumorigenesis and cancer development of NSCLC. Thus, in this project, we will identify the target proteins of these three pfeRNAs and investigate the mechanisms underlying the interaction with the target proteins. This project will provide new evidence on NSCLC oncogenesis.