LIM-domain binding protein 2 regulated by m6A modification inhibits lung adenocarcinoma cell proliferation in vitro

Abstract

To investigate the role and expression pattern of LIM-domain binding protein 2 (LDB2) in lung adenocarcinoma. We studied the expression pattern of LDB2 in lung adenocarcinoma based on data from the online databases TCGA, GEO and CPTAC, and the results were verified in lung adenocarcinoma tissues and cells using immunohistochemistry, qRT-PCR and Western blotting. The relationship between LDB2 and the prognosis of patients with lung adenocarcinoma was analyzed using GEPIA and GEO databases. We further analyzed the role of LDB2 in regulating cell behaviors in a H1299 cell model over-expressing LDB2 using cell counting, soft agar colony forming assay and flow cytometry. The m6A binding sites on LDB2 were confirmed by bioinformatics analysis and MeRIP-qPCR assays. The effect of YTHDC2 on LDB2 was examined using qRT-PCR and Western blotting, and the binding of YTHDC2 to the transcript of LDB2 was verified with RIP-qPCR assays. Dual luciferase reporter assay was performed to verify YTHDC2 functioning via m6A sites. LDB2 expression was significantly decreased in lung adenocarcinoma in comparison with normal tissues based on data from TCGA, GEPIA and CPTAC, and the same results were obtained from 80 lung adenocarcinoma tissues and 17 adjacent normal tissues. Similarly, LDB2 expression was decreased in lung adenocarcinoma cells as compared with 16HBE cells. The data from Prognoscan and GEPIA suggested that a high LDB2 expression was positively correlated with a more favorable outcome of lung adenocarcinoma patients. LDB2-overexpressing H1299 cells showed a significant inhibition of proliferative activity with cell cycle arrest in S phage. Bioinformatics analysis and MeRIP-qPCR assay confirmed the presence of m6A sites on LDB2. The m6A reader YTHDC2 was positively related with LDB2 in lung adenocarcinoma based on data from GEPIA (r=0.22). Overexpression YTHDC2 significantly enhanced LDB2 expression in H1299 cells by about 19.35 folds. Dual luciferase reporter assay showed that YTHDC2 enhanced the promoter activity in the wild-type group but not in deletion group. LDB2 expression can be up-regulated by m6A reader YTHDC2 in lung adenocarcinoma to inhibit the proliferation of the tumor cells in vitro.