Knockdown of NADPH Oxidase 4 Affects Proliferation via Reducing STAT3 Activity in Human Melanoma Cells
CAI Tian-Chi1),2), WANG Yan-Ling1), ZHANG Chun-Hua1),3), ZHANG Zheng1),3), CHEN Long1), LI Yu-Qian1), ZHU Yue-Chun1)*
1) Department of Biochemistry and Molecular Biology, Kunming Medical University, Kunming 650500;
2) The Chinese People’s Liberation Army 65426 Hospital, Harbin 65426;
3) Yunnan Maternal and Children Health Hospital, Kunming 650051
Abstract：NADPH oxidase involves reactive oxygen species (ROS) generation process in cell, and ROS is closely related to tumor cell proliferation. Here we elucidated the mechanism of NADPH Oxidase influencing proliferation in human melanoma A375 cells. We first verified that NOX4 was the core subunit of NADPH oxidase using real-time PCR and Western blot. Then, three shRNA interference sequences and one control sequence were designed based on the human NOX4 gene, and were inserted into the pSuper-retro-puro, respectively, and transformed into E.coli DH5α competent cells. An efficient siRNA sequence targeting NOX4 gene was obtained and used to retrovirus packaging. After the A375-WT cells with the viral solution were infected and selected 10 days by puromycin, the stable A375 cell line with NOX4 knock-down (A375-NOX4Δ) was successfully established, which displayed by a 66.02% reduction of NOX4 mRNA (P＜0.05), 77.35% decrease of NOX4 protein (P＜0.01), accompanied by 79.17% descend of NADPH oxidase activity (P＜0.01) and 64-46% decline of ROS level, as those of the A375-WT cells, respectively. Meanwhile, MTT and EdU staining showed that A375-NOX4Δ cell displayed slower growth，prolonged doubling time，cell proliferation declined by 68-27%, and with arrest in the G1 to S phase, compared with that of the A375-WT cell. In comparison with the A375WT cells, the A375NOX4Δ cell was characterized by 55-7% (P＜0.01) and 64.8% (P＜0.01) decrease in the expressions of cyclin D1 and CDK4, and up to 6.89fold of P53 (P＜0.01) and 3.27fold of P21 (P＜0.01) increase, respectively. Furthermore, the electrophoretic mobility shift assay (EMSA) showed that the activity of STAT3 binding to DNA in the A375-NOX4Δ cell decreased, with 51.80% (P＜0.034) and 82.58% (P＜0.01) decline of STAT3 and P-STAT3, respectively, compared with that of the A375WT cell. These results suggest that the lower proliferation and G1 to S phase arrest in A375 cell induced by NOX4 knockdown may be mediated via the decrease of P-STAT3 and STAT3-DNA binding ability from lower ROS lever. This study might provide new clues and drug targets.
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