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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%(P<0.01),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%(P<0.01)和64.8%(P<0.01),而P53、P21分别增加了6.89倍(P<0.01)和3.27倍(P<0.01),STAT3、P-STAT3分别下降了51.80%(P<0.05)和82.58%(P<0.01);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.
NADPH oxidase is involved in the generation of reactive oxygen species (ROS), and ROS is closely related to the proliferation of tumor cells.To elucidate the molecular mechanism of NADPH oxidase affecting the proliferation of melanoma A375 cells, we first confirmed the expression of NOX4 by fluorescence quantitative PCR and Western blot Then, 3 interference and control sequences were designed according to NOX4 gene and ligated to p Super-retro-puro vector. After identification, the transformed E. coli DH5α competent cells were screened A375 cells (A375-NOX4Δ) with NOX4 deficiency were constructed, and the mRNA and protein expression of NOX4 decreased by 66.02 % And 77.35%, respectively. The levels of NADPH oxidase and ROS decreased by 79.17% and 64.16%, respectively. The MTT assay and Ed U assay showed that the proliferation of A375-NOX4Δ cells was significantly lower than that of A375-WT cells, The number of cells decreased by 68.27% (P <0.01), showing G1 → S phase arrest. Western blot showed that cyclin D1 and CDK4 in A375-NOX4Δ cells decreased by 55.7% (P <0.01) and 64 respectively. P53 and P21 increased by 6.89-fold (P <0.01) and 3.27-fold (P <0.01), while STAT3 and P-STAT3 decreased by 51.80% and 82.58% P <0.01). The electrophoretic mobility shift assay (EMSA) showed that the STAT3-DNA binding activity of A375-NOX4Δ cells was significantly decreased.The above results suggest that knockdown of NOX4 expression in A375 cells may reduce STAT3 phosphorylation by ROS generation and The decrease of DNA binding activity leads to the decrease of the proliferation of A375-NOX4Δ cells and the G1 → S phase arrest, which provides a new idea and possible drug targets for the study of the pathogenesis of melanoma.