Adenosine deaminase acting on RNA-1 regulates the radiosensitivity of lung adenocarcinoma cells

Abstract

Objective To investigate the effect of down-regulating adenosine deaminase acting on RNA-1(ADAR1) on the radiosensitivity of lung adenocarcinoma cells. Methods Lentiviral transfection was used to establish an ADAR1 knockdown cell line based on A549 cells.Then the cells were divided into negative control (shNC) and ADAR1 knockdown (shADAR1) groups, which were followed by a single-dose irradiation of 0 Gy and 6 Gy X-rays.Western blotting and RT-PCR were utilized to detect the expression of ADAR1 at protein and mRNA levels, respectively.CCK-8 assay, wound healing assay and Transwell migration assay were applied to measure cell proliferation and migration abilities.Meanwhile, clone formation assay was performed to detect the effect of down-regulating ADAR1 on the radiosensitivity of A549 cells.Flow cytometry and Western blotting were conducted to detect the expression levels of apoptosis and apoptosis-related proteins Bax and Bcl-2.Immunofluorescence assay and Western blotting were used to detect the expression level of γ-H2AX.Comet assay was performed to detect the level of cellular DNA damage.Twelve female nude mice (4~6 weeks old, weighing 16~18 g) were divided into shNC group, shADAR1 group, shNC+ionizing radiation (IR) group and shADAR1+IR group, with 3 mice in each group.The growth of tumor of different groups was observed with subcutaneous tumorigenesis assay. Results Western blotting and RT-qPCR showed that the protein and mRNA expression of ADAR1 were significantly reduced in A549 shADAR1 cells (P < 0.05).CCK-8 assay, wound healing assay and Transwell migration assay indicated that down-regulation of ADAR1 inhibited the proliferation and migration abilities of A549 cells, and this inhibition trend became more obvious (P < 0.01) after IR.Cell clone formation assay showed that the clone formation rate of both groups was decreased, with the increase of radiation dose.But the number of formed clones was lower in the shADAR1 group than the shNC group.Flow cytometry and Western blotting displayed that down-regulation of ADAR1 increased the apoptotic rate and Bax expression in A549 cells (P < 0.01) and decreased Bcl-2 expression (P < 0.05), and the apoptotic rate and Bax protein level were further increased in A549 shADAR1 cells after IR (P < 0.01), and the Bcl-2 protein level was further decreased (P < 0.01).The number of γ-H2AX foci and protein level in A549 shADAR1 cells were significantly increased after IR (P < 0.05), and the results of comet assay showed that the DNA damage was more obvious in A549 shADAR1 cells after IR (P < 0.01).Subcutaneous tumorigenesis assay in nude mice showed that the growth of subcutaneous tumour of A549 shADAR1 cells was significantly inhibited after IR (P < 0.01). Conclusion Down-regulation of ADAR1 significantly inhibits the proliferation and migration of A549 cells after IR and promotes apoptosis and DNA damage, and thereby increases the radiosensitivity of lung adenocarcinoma cells.

Keywords

• adar1
• radiosensitivity
• lung adenocarcinoma
• dna damage