The Role And Molecular Mechanism Of DAZL Gene In Lung Cancer

Background:Some genes are normally expressed only in the germ cells of the testis and/or ovary,but can be abnormally activated in various tumor tissues.These genes are collectively referred to as cancer-testis genes or cancer-germline genes.DAZL is only expressed in germ cells.It encodes an RNA-binding protein that promotes translation of its target mRNA.Many studies have shown that DAZL plays an important role for the development of sperm and eggs,but its role in cancer has not been reported.Methods:The expression of DAZL and RRM2 in lung cancer tissues was detected by tissue microarray immunohistochemistry.Stable cell lines overexpressing DAZL and interfering with DAZL were constructed using lentivirus-mediated expression system;RRM2 gene was interfered by adenovirus-mediated expression system;MTT assay,colony formation assay,EDU cell proliferation and flow cytometry were performed to analyze the effects of DAZL gene on the proliferation of lung cancer cells.Results:1)The expression of DAZL protein was not detected in normal lung tissues,but its expression in lung cancer tissues increased with tumor grade.2)Suppression of DAZL expression inhibited cell viability,colony formation and DNA synthesis of lung cancer cells,and led to cycle arrest in S phase.Moreover,knockdown of DAZL gene significantly increased the sensitivity of lung cancer cells to gemcitabine.In contrast,overexpression of DAZL gene promoted cell viability,colony formation,and DNA synthesis,resulting in reduced sensitivity to gemcitabine of the lung cancer cells.3)Bioinformatics analysis revealed that the 3'UTR region of RRM2 contains a binding site for DAZL.Luciferase assay and Western blotting showed that DAZL promoted the translation of RRM2,while the mutant with inactivated RNA binding activity(R135G)could not promote the translation of RRM2.Immunohistochemical analysis showed that there was a significant positive correlation between DAZL and RRM2 expression in lung cancer tissues(Pearson's correlation coefficient = 0.388,p value < 0.001).4)Based on the above results,we hypothesized that DAZL may affect cell proliferation and gemcitabine sensitivity by regulating the expression of RRM2.In order to validate our conjecture,we performed a rescue experiment to suppress RRM2 expression using adenovirus-mediated DAZL shRNA in DAZL-overexpressing cells.The results showed that knockdown of RRM2 gene significantly inhibited cell proliferation and increased the sensitivity to gemcitabine in cells overexpressing DAZL.Conclusion:1)DAZL is a novel cancer-testis gene;2)DAZL promotes the proliferation of lung cancer cells and reduces the sensitivity to gemcitabine of lung cancer cells by enhancing the translation of RRM2.