The Influence Of NADPH Oxidase 4 On A549 Cells Migration

BackgroundLung cancer is one of the highest incidence and mortality cancers currently in China and the world, which leads many researchers working on lung cancer prevention and treatment. Studies have shown that the expression of NOX1 and NOX4 within NOX family elevated in lung cancer cells. That NOX family is NADPH oxidase family, which is the main source of reactive oxygen species(ROS) in human body, and many tumors happened with thechange of produced ROS level, such aspancreatic cancer, colorectal cancer and non-small cell lung cancer. NOX family plays an important role in the process of tumor genesis, and NOX4 may be involved in metastasis of tumor cells, the main cause of lung cancer deaths are lung cancer metastasis. So it is very important to study the role of NOX4 and the mechanism of lung cancer migration.EMT is Epithelial-Mesenchymal Transition, whichacquire the ability of migration and is a basic process of embryonic development.It makes up special part in the epithelial cells isolated from epithelial tissue and into the organization of the other, and it is the basis of wound healing and malignant tumor. The tumor cells obtained the ability of migration and progression in the process of tumor malignant transformation by EMT.which are visible in various pathological staging of the tumor cells.The TGF- β plays an important role in the EMT process model, while TGF- β can be produced by NADPH oxidase ROS, which also play a regulatory role in TGF- β signaling pathways.In order to investigate whether NOX4 affect the migration of A549 cells by TGF-β-induced EMT and Providing theoretical basis for the targeted therapy of lung cancer. firstly,we use different concentrations of TGF- β stimulus A549 cells and observe the changes of intracellular ROS,NOX expression level, and A549 cell migration by scratches experimental.Then inhibiting the expression of NOX by DPI, we observe intracellular ROS and NOX expression level、the change of cell migration, and snail and E- cadherin protein level by western blot. Methods1The experimental methods1.1 Experimental groups: according to the TGF- β concentration from low to high, we set up different groups of 1、0 ng/ml;2. 2.5 ng/ml;3、5ng/ml;4、10 ng/ml. Then we select a suitable concentration of TGF- β stimulation A549 cells, the experiment were divided into the following five groups: 1、 TGF-β stimulation group;2、 normal control group(ck);3、 DPI group(inhibitors);4、 TGF- β + DPI group;5、 DMSO group(solvent control group).1.2 Cell scratch test was used to detect the change of the A549 cell migration, according to the healing rate of cells to represent scratch experiment of mobility.1.3 The determination of intracellular ROS: DCFH-DA mark 20 min after stim ulate A549 cells 24h, and then using flow cytometry instrument to detect the ROS expression though the fluorescent value, according to the strength of the fluorescent value level of ROS expression.1.4 MRNA level of detection: nucleic acid analyser was used to detect the purity of RNA; agarose gel electrophoresis was used to detect the integrity of the RNA, then reverse transcription PCR and fluorescence quantitative PCR for c DNA.1.5 The detection of NOX1, NOX4, snail, E- cadherin protein levels: after testing the concentration and purity of extracted proteins, protein expression levels was detected by using western blot method.2 Statistical methodsAll data were analyzed by SPSS 21.0 with One-Way ANOVA. The difference among each group was analyzed by LSD or Dunnett. Two variables correlation analysis to select testing Pearson correlation coefficient,Significant level is 0.05 Results1. In different concentrations of TGF-β stimulus A549 cells, the healing rate of scratch test became higher and migration ability of A549 cell were enhanced(P < 0.001)with the increase of concentration of TGF-β. Fluorescent quantity of ROS detected by flow cytometry instrumentincrease showed the levels of intracellular ROS expression higher than before(P < 0.001);Compared with NOX1, NOX4 protein m RNA and protein expression levels were both elevated(P < 0.001), and the trend of the expression of NOX4 was the same as ROS levels.2.After NOX4 inhibitors DPI was added, NOX4 m RNA and protein of DPI group was significantly decreased compared to normal control group, and there was statistically significant difference basing on statistical tests(P < 0.001);NOX4m RNA and protein of TGF β+ DPI group expression levels were significantly decreased compared with the TGF- β group,and there was statistically significant difference basing on statistical tests(P < 0.001), which indicates that DPI could inhibits the expression of NOX4 A549 cell.3. When NOX4 was suppressed, there are no difference between control group and DMSO group, and there was no statistically significant difference(P > 0.05); when DPI group compared with normal control group, TGF- β + DPI group compared with the TGF- β group, the levels of ROS were significantly lower, and the statistical results show that the difference was statistically significant(P < 0.001).4.According to the results of Scratches tests,when DPI group compared with normal control group, the TGF- β + DPI group compared with the TGF- β group, scratches tests show that the healing rate is reduced,which illustrated that DPI could inhibit TGF-β induced A549 cell migration.5.The proteinchanges of snail and E- cadherin : DMSO group was as the same as normal control group, while TGF- β group compared with DMSO group, snail levels increased and E- cadherin expression decreased;DPI group compared with DMSO group, and TGF- β + DPI group compared with the TGF- β group, snail expression was reduced and E- cadherin expression increased.6. Cell morphology showed that there were not difference between normal control group and DMSO group.While TGF- β group compared with normal control group and DMSO group, the intercellular adhesion become low, cells become more slender, ectomesenchymal cells tend to form, and when TGF-β + DPI group compared with TGF-β group, the TGF-β stimulation induced cell morphology change was no longer apparent. ConclusionsNOX4 can promote the migration of A549 cells by EMT pathway, and the mechanisms may be that NOX4 promote the expression of snail, thereby inhibiting the expression of E-cadherin and inducing EMT occurs.