Regulation of neuropilin-1 in radiation-induced transformation of lung epithelial cells

Abstract

Objective \nTo investigate the effect of neuropilin-1 (NRP1) on radiation-induced epithelial-mesenchymal transition (EMT) by measuring the expressions of EMT-related transcription factors in the irradiated cells with different levels of NRP1. \n \n \nMethods \nHuman lung type II epithelial cells (A549) were transfected with NRP1 over-expression lentiviral vector and NRP1 inhibition vector to construct two cell models of NRP1high-A549 and NRP1low-A549. A NRP1 knock-down cell model was also constructed by transferring siNRP1 into normal mouse lung epithelial MLE-12 cells that was validated at both protein and mRNA levels. A single dose of 10 Gy X-ray was delivered to these cell models, then total protein and RNA were extracted at 0, 12, 24 and 48 h after irradiation. The expressions of EMT-related transcription factors (Twist and ZEB1) and EMT markers (β-catenin, N-cadherin, and Vimentin) in each cell model were detected by Western blot and qPCR. \n \n \nResults \nAfter 10 Gy irradiation, the expressions of NRP1 mRNA and protein were significantly increased in A549 and MLE-12 cells. The expressions of the mesenchymal markers (Vimentin and N-cadherin) and the transcription factors of ZEB1 and Twist were also significantly increased (A549: t=2.917, 7.361, 4.852, 9.278, P<0.01; MLE-12: t=9.652, 31.357, 30.985, 17.266, P <0.01). The expressions of Vimentin and N-cadherin were significantly decreased in NRP1low-A549 (t=10.077, 15.707, P<0.01) and siNRP1-MLE-12 cells (t=5.745, P<0.01), but the expression of epithelial marker (β-catenin) was significantly increased in these cells. The expressions of N-Cadherin and Vimentin were significantly elevated (t=16.055, 5.560, P <0.01), while β-catenin decreased significantly in NRP1high-A549 cells. After irradiation, the transcription factor of Twist in NRP1low-A549 group was significantly decreased (t=3.987, P<0.01), while the transcription factors of ZEB1 and Twist in the NRP1high-A549 group increased in a time-dependent manner (t=11.289, 2.903, P<0.01). After irradiation, the transcription factor of ZEB1 decreased significantly in siNRP1-MLE-12 cells (t=13.449, P<0.01), and the protein expressions of ZEB1 and Twist in siNRP1-MLE-12 cells were lower than those of control group in a time-dependent manner. \n \n \nConclusions \nNRP1 promotes radiation-induced EMT in human and mouse epithelial cells through up-regulation of transcription factors of ZEB1 and Twist. \n \n \nKey words: \nNeuropilin-1;\xa0Ionizing radiation;\xa0Epithelial-mesenchymal transition;\xa0Transcription factor