敲减NADPH氧化酶4能降低STAT3活性抑制人黑色素瘤A375细胞的增殖

NADPH氧化酶参与细胞活性氧族(ROS)的生成过程,而ROS与肿瘤细胞增殖密切相关.为了阐明NADPH氧化酶影响黑色素瘤A375细胞增殖的分子机制,本文首先应用荧光定量PCR和Western印迹证实NOX4为人黑色素瘤A375细胞的NADPH氧化酶功能核心亚基;随后根据NOX4基因设计3条干扰序列和对照序列并连接到p Super-retro-puro载体,经鉴定后转化E.coli DH5α感受态细胞、筛选有效干扰序列并用于逆转录病毒包装,病毒液感染A375细胞并经嘌呤霉素筛选10d,构建了NOX4缺陷的A375稳转细胞珠(A375-NOX4Δ),其NOX4的mRNA和蛋白表达分别下降了66.02%和77.35%,伴随NADPH氧化酶活性和ROS水平分别下降了79.17%和64.16%;MTT、Ed U法检测显示,A375-NOX4Δ细胞的增殖能力比A375-WT细胞明显降低、倍增时间延长,增殖细胞数量下降了68.27%(P0.01),呈现G1→S期阻滞;Western blot检测表明A375-NOX4Δ细胞的cyclin D1、CDK4分别下降了55.7%(P0.01)和64.8%(P0.01),而P53、P21分别增加了6.89倍(P0.01)和3.27倍(P0.01),STAT3、P-STAT3分别下降了51.80%(P0.05)和82.58%(P0.01);电泳迁移率变动分析(EMSA)表明,A375-NOX4Δ细胞的STAT3-DNA结合活性明显降低.上述结果提示,敲减A375细胞的NOX4表达可能通过减少ROS生成使得STAT3磷酸化水平及其结合DNA的活性下降,最终导致A375-NOX4Δ细胞增殖减少、呈现G1→S期阻滞,这为黑色素瘤发病机制研究提供了新思路及可能的药物作用靶点.

Knockdown of NADPH Oxidase 4 Affects Proliferation via Reducing STAT3 Activity in Human Melanoma Cells

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 A375 WT cells, the A375 NOX4Δ 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 6.89 fold of P53 (P＜0.01) and 3.27 fold 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 A375 WT cell. These results suggest that the lower proliferation and G1 to S phase arrest in A375 cell induced by NOX4 knock down 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.