The Influence Of NADPH Oxidase 1 On The Proliferation Andapoptosis In A549 Cells

BackgroundMore and more evidence had showed that ROS played an important role in the progression of lung cancer, and NADPH oxidases are the only enzymes in non-phagocytic cells whose primary function is generate cellular ROS. RO S generated by NOX have been implicated play an important role in regulatio n of cytoskeletal remodeling, gene expression, proliferation, differentiation, migr ation and cell death. Recently, the expression of NOX has been found in sever al cancer cells including NSCLC cells, and NOX1 was implicated play an imp ortant role in cancer pathogenesis. JNK was belong to mitogen-activated protei n kinase(MAPK) family, which can be activated by stress and regulate apopto sis signaling. Some studies had showed that JNK can be activated by NOX1.To explore the influence of NOX1 on the proliferation and apoptosis in A549 Cells, we inhibited NOX1 by transfect NOX1 si RNA into A549 cells. Then we established an oxidase stress model of A549 cells by using tumor necrosis factor TNF-α. Cell viability of each group was tested by MTT assay. ROS expression and cell apoptosis rate of different group was tested by flow cytometry. The expression level of NOX1 and p-JNK were tested by Western blot. We try to explore the role of NOX1 and ROS produced by NOX1 on proliferation and apoptosis in A549 cells. Methods1 The experimental methods1.1 In order to explore the proliferation and apoptosis in A549 cells after down-regulate NOX1 expression, we set mock group,the negative control group and The NOX1 si RNA group. The mock group was treated with Lipofectamine 2000 only. The negative control group was transfected with NC-si RNA. The NOX1 si RNA group was transfected with NOX1 si RNA. The NOX1 si RNAs were transfected into A549 cells by Lipofectamine 2000. The effect of suppression of NOX1 to A549 cells: After transfection for twenty-four hours,the expression levels of NOX1 m RNA was measured with Real-Time PCR. Then we used TNF-αto set an oxidase stress model which include blank control group,TNF- group and TNF –αgroup+ NOX1 si RNA group. The blank control group was normal group, the TNF-αgroup was,and the TNF–αgroup+ NOX1 si RNA group was transfected with NOX1 si RNA before stimulated with TNF-α.1.2 MTT assay for cell viability: Cells of mock group、the negative control group and The NOX1 si RNA group were used to do MTT assay. The OD492 was detected at 12 h, 24 h, 36 h and 48 h after transfecttion.1.3 Flow cytometry for ROS expression: ROS expression of each group we re detected by flow cytometry after forty-eight hours.1.4 Western blot for protein expression: Forty-eight hours after treatment of each group, cells were collected for extracted protein and quantified all protein samples. The proteins expression of each group was detected by Western blot and analyzed with Quantity One software. We Use β-actin as a loading control to check the integrity of each group.1.5 Flow cytometry for apoptosis of each group: Forty-eight hours after treatment, all cells were harvested to prepare single cell suspension, and treated with Annexin-V-FITC and PI. Then apoptosis ratio was tested by flow cytometry.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. Significant level is 0.05 Results1 The silencing effect of si RNA of NOX1 in A549 cells: After transfected NOX1 si RNA in A549 cells, NOX1 m RNA expression was decreased by 78%. NOX1 protein expression was decreased by 70%.2 Change of ROS levels of each group: Compared with Mock group, ROS level was decreased in NOX1 si RNA group. Compared with blank control group,ROS level was increased in TNF-αgroup(P<0.05). Compared with TNF-αgroup,ROS level was decreased in TNF-α+NOX1si RNA group(P<0.05).3 The cell viability changes after inhibit the expression of NOX1: Compared with the Mock group, the cell viability of NOX1 si RNA group was decreased at 24 h(P<0.05), 36h(P<0.05) and 48 h(P<0.05) after transfection.4 The protein level changes after inhibit the expression of NOX1: The expression level of NOX1 was reduced in NOX1 si RNA group compared with Mock group(P<0.05) and negative control group(P<0.05). The expression of NOX1 and p-JNK was increased in TNF-αgroup compared with blank control group(P<0.05). The expression of NOX1 and p-JNK was decreased in TNF-α+NOX1si RNA group compared with TNF-αgroup(P<0.05).5 The apoptosis ratio changes after inhibit the expression of NOX1: There was no significance difference in apoptosis rate between Mock group、NC group and NOX1 si RNA group. The ratio of apoptosis was increased in TNF- α group compared with blank control group(P<0.05). And compared with TNF-αgroup, the ratio of apoptosis was decreased in TNF-α+NOX1si RNA group(P<0.05). ConclusionsSuppress the expression of NOX1 by si RNA can inhibit the proliferation of A549 cells; the expression level of NOX1 was increased by TNF-αstimu lation. Suppress the expression of NOX1 by si RNA can suppress the apoptosi s rate stimulate by TNF-αin A549 cells accompanying with change of ROS and p-JNK.