Introduction: The study explored the effects of SHOX2 and RASSF1A DNA methylation in lung cancer (LC). Method: Bronchoalveolar lavage fluid (BALF) samples as well as LC and normal adjacent tissues were collected from 72 LC patients and 35 patients with benign pulmonary nodules. Quantitative analysis of SHOX2 and RASSF1A DNA methylation was performed in benign pulmonary nodules and different stages of LC. The diagnostic value of SHOX2 and RASSF1A DNA methylation in LC and benign pulmonary nodules was determined by receiver operating characteristics analysis. Gain/loss-of-function experiments were constructed in LC cells and mouse models of xenograft and pulmonary nodule metastasis. The levels of SHOX2 and transfer-associated genes were tested through quantitative reverse transcription polymerase chain reaction and Western blot. Malignant phenotype of LC cells was assessed by functional experiment. The tumor volume and weight of mice in xenograft models were measured. Pulmonary nodule metastasis was determined through HE staining assay. 5-azacytidine appeared as a positive control drug. Result: SHOX2 DNA methylation or RASSF1A DNA methylation had diagnostic efficiency in pulmonary nodules and early LC, with the two combined having better diagnostic value. SHOX2 expression was upregulated in LC. Similar to 5-azacytidine, SHOX2 knockdown inhibited LC cell viability, migration, and invasion in vitro as well as restrained LC tumorigenesis and pulmonary nodule metastasis in vivo, whereas overexpressed SHOX2 had the opposite effects. Conclusion: The combination of SHOX2 and RASSF1A DNA methylation had a diagnostic value in pulmonary nodules and early LC. SHOX2 positively modulated the tumorigenesis and metastasis of LC by regulating DNA methylation processes.

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