Inhibition of NIPBL enhances the chemosensitivity of non-small-cell lung cancer cells via the DNA damage response and autophagy pathway

摘要

Background: Previously, we reported that high expression of nipped-B-like protein (NIPBL) was strongly correlated with poor prognosis, tumor differentiation, and lymph node metastasis. Survival analysis indicated that NIPBL expression was a potential prognostic factor for non-small cell lung cancer (NSCLC). Moreover, loss of NIPBL decreased lung cancer cells proliferation, migration, invasion and promoted apoptosis as well as sensitivity to chemotherapeutic agents. However, the deep mechanisms were not explored. Purpose: The objective of this study was to identify the role of NIPBL in DNA damage response, as well as autophagy pathway, so as to interpret the mechanisms of how NIPBL knockdown enhances the chemosensitivity of lung cancer cell. Methods: Cells (NCI-H1299 and NCI-H1650) were transfected by specific siRNAs before immunofluorescence and single-cell gel electrophoresis, which were mainly used to observe the differences of DNA damage in different groups. Additionally, protein were obtained and then analyzed by western blot and mass spectroscopy. Results: In this study, we found that knockdown of NIPBL resulted in accumulation of phosphorylated H2AX (γ-H2AX) foci and higher levels of DNA damage, as revealed by comet assay. Western blot assay revealed that loss of NIPBL decreased expression of ATM/ATR, Rad3-related protein and Ku70/Ku80, but increased expression of LC3-B and depletion of p62. Using mass spectroscopy, we identified eight proteins that were significantly differentially expressed upon NIPBL knockdown. Gene Ontology analysis revealed that these proteins are mainly involved in DNA repair, mismatch repair, and binding to damaged DNA. The expression changes in two of the proteins, MSH2 and STAT1, were verified by Western blotting in NIPBL-knockdown cells. Conclusions: In summary, these results reflected that loss of NIPBL impairs the DNA damage response and promotes autophagy. And NIPBL suppression may represent a novel strategy for preventing chemotherapy resistance in lung cancer.