辐射诱导肺上皮细胞GATA3表达及其RNA甲基化修饰机制

Radiation-induced GATA3 expression in lung epithelial cells and mechanism of RNA methylation

目的探讨肺上皮细胞的辐射反应中GATA3的表达和m^(6)A修饰的调控机制,为放射性肺损伤的发生机制与临床防治提供新的治疗靶点。方法首先,分别给予人肺上皮细胞系(A549)和小鼠肺上皮细胞系(MLE-12)单次10 Gy(剂量率为1 Gy/min)和6 Gy(剂量率为0.75 Gy/min)X射线辐照;采用慢病毒感染法在A549细胞转染shRNAVIRMA质粒和在MLE-12中转染siRNA-VIRMA干扰片段抑制VIRMA基因(RNA甲基化酶)的表达;利用比色法定量检测m^(6)A RNA甲基化水平;采用qRT-PCR法检测受照的A549和MLE-12细胞中VIRMA、GATA3和EMT标志物的mRNA的表达变化,利用Western Blots法检测受照的A549和MLE-12细胞中VIRMA、GATA3和EMT标志物蛋白的表达变化。结果结果显示,辐射诱导A549和MLE-12细胞中甲基化酶VIRMA上调,进而增强总RNA的m^(6)A水平,同时上调GATA3基因和蛋白水平表达,进而诱导上皮—间质转化(EMT)过程的发生。进一步在A549和MLE-12细胞内,干扰VIRMA基因时,明显降低GATA3基因和蛋白的表达水平,显著抑制EMT相关分子的表达。结论辐射诱导肺上皮细胞发生m^(6)A修饰,通过正向调控GATA3基因表达,诱导EMT的发生,进而对放射性肺损伤进程起到重要作用。

Objective To investigate GATA3 expression and the regulatory mechanism of m^(6)A modification in the response of alveolar epithelial cells to radiation,and to provide a new therapeutic target for radiation-induced lung injury based on its pathogenesis.Methods Human lung epithelial cell line(A549)and mouse lung epithelial cell line(MLE-12)were exposed to X-ray irradiation with a single dose of 10 Gy(dose rate 1 Gy/min)and 6 Gy(dose rate 0.75 Gy/min),respectively.The expression of VIRMA gene(RNA methylase)was inhibited by lipofection of A549 cells and MLE-12 cells with shRNA-VIRMA plasmid and siRNA-VIRMA interfering fragment,respectively.Quantification of m^(6)A RNA methylation was performed by colorimetry.Changes in the expression of mRNAs of VIRMA,GATA3,and epithelial-mesenchymal transition(EMT)markers in irradiated A549 and MLE-12 cells were determined by qRT-PCR.Changes in the expression of VIRMA,GATA3,and EMT marker proteins in irradiated A549 and MLE-12 cells were determined by Western blot.Results Radiation up-regulated the expression of methylase VIRMA in A549 and MLE-12 cells,which in turn enhanced the m^(6)A of total RNA and the expression of GATA3 gene and protein,resulting in EMT.Furthermore,in A549 and MLE-12 cells,interference of the VIRMA gene significantly reduced the expression of GATA3 gene and protein and the expression of EMT-related molecules.Conclusion Radiation induces m^(6)A modification in alveolar epithelial cells,which up-regulates the expression of GATA3 gene and induces EMT,thus playing an important role in the process of radiation-induced lung injury.