HOXB7 Promoting Epithelial-mesenchymal Transformation And Migration Of Lung Adenocarcinoma Cells Induced By Ionizing Radiation

Objective: To investigate the influence of ionizing radiation on HOXB7 expression in lung adenocarcinoma A549 cells and the molecular mechanism of HOXB7-induced epithelial-mesenchymal transition and migration by ionizing radiation in lung adenocarcinoma A549 cells.Methods: 1.The influence of ionizing radiation on the expression of HOXB7,PIK3R1 and Snail protein were detected by Western blot analysis in A549 cells.2.HOXB7 shRNAs and and their matched negative controls(shRNAs)were used to infect the cells according to the manufacturer’s instructions.2Gy X-ray was used to irradiate A549 cells before and after infection.Western blotting was used to detect the expression of HOXB7,PIK3R1,p-AKT,AKT,Snail,E-cadherin and Vimentin proteins after exposing A549 cells of 2Gy X-ray for three days 3.PIK3R1 shRNAs and shRNAs were infected the A549 cells.A549 cells were treated with 2Gy X-ray before and after infection.After three days irradiating,the expression of HOXB7,PIK3R1,p-AKT,AKT,Snail,E-cadherin and Vimentin proteins in A549 cells were examined.4.A549 cells were treated with PI3 K inhibitor or AKT inhibitor and exposed of 2Gy X-ray for three days.Western blotting was used to detect the expression of HOXB7,PIK3R1,p-AKT,AKT,Snail,E-cadherin and Vimentin proteins.5.Migration and wound healing assays were conducted to detect the migration ability of those cells in the all groups as described previously.Results: 1.A549 cells were treated with a wide range of dose(0,1,2,5,10 Gy)of X-rays for three days.The results showed that the expression of HOXB7,PIK3R1,Snail proteins were upregulated.The expression of HOXB7,PIK3R1 and Snail proteins were the most markedly up-regulated after exposing A549 cells of 2Gy X-ray.The expression of various proteins were detected at different time points(0h,5h,1d,2d,3d)after irradiating.Western blotting showed significantly highest HOXB7,PIK3R1,Snail proteins expression were verified after exposing A549 cells of 2Gy X-ray for three days.The results showed that the expression levels of HOXB7,PIK3R1,Snail proteins were the most markedly upregulated after exposing A549 cells of X-ray(2Gy)for three days.Migration and wound healing assays showed that ionizing radiation led to promotion of migration in A549 cells.2.Infection of cells with HOXB7 shRNAs,which significantly silenced the level of HOXB7,resulted in suppression of PIK3R1,p-AKT and Snail protein levels.It also led to a significant increase in expression of E-cadherin protein,whereas the expression of interstitial phenotype marker Vimentin protein was decreased.Migration and wound healing assays also confirmed decreased the level of endogenous HOXB7,led to repression of migration in A549 cell lines.2Gy of X-ray was used to treat with the lung adenocarcinoma A549 cells for three days after HOXB7 shRNAs infection,and we identified that infection of cells with HOXB7 shRNAs,which decreased the level of endogenous HOXB7,led to a no obviouly decrease expression of PIK3R1,Snail,E-cadherin,Vimentin protein proteins in A549 cell lines.Similarly,migration ability of cells which HOXB7 shRNAs infected had no marked change after exposing with 2Gy X-ray for three days.3.Infection with PIK3R1 shRNAs,which significantly decreased the level of PIK3R1,resulted in suppression of p-AKT、Snail and Vimentin at protein levels,but led to a significant increase in expression of E-cadherin protein,whereas the expression of HOXB7 protein in A549 cells had no obviously change.PIK3R1 shRNAs-infected cells were exposed with 2Gy X-ray for three days,and we futher found the expression of PIK3R1,Snail,E-cadherin,Vimentin proteins had no obvious changes.Similarly,migration ability of cells which PIK3R1 shRNAs infected had no marked change after exposing with 2Gy X-ray for three days.4.The lung adenocarcinoma A549 cells were treated with PI3 K inhibitor or AKT inhibitor.Western blotting showed that Suppression of PI3K/AKT signaling pathway also resulted in an attenuation of Vimentin and the cell migration ability was ihibited,however,the expression of Snail and E-cadherin were up-regulated.Cells which treated with PI3 K inhibitor or AKT inhibitor were exposed with 2Gy X-ray for three days,and we further found HOXB7,PIK3R1 proteins were upregulated,whereas the expression of Snail,E-cadherin,Vimentin proteins and the migration ability of cells had no marked change.Conclusion: Low dose of ionizing radiation could up-regulate the expression of HOXB7 and then activates PI3K/AKT signaling pathway to promote the epithelial-mesenchymal transformation and migration of lung adenocarcinoma cells.