Let-7b Inhibits Human Cancer Phenotype by Targeting Cytochrome P450 Epoxygenase 2J2

BackgroundMicroRNAs (miRNAs) are small, noncoding RNA molecules of 20 to 22 nucleotides that regulate gene expression by binding to their 3′ untranslated region (3′UTR). Increasing data implicate altered miRNA participation in the progress of cancer. We previously reported that CYP2J2 epoxygenase promotes human cancer phenotypes. But whether and how CYP2J2 is regulated by miRNA is not understood. Methods and ResultsUsing bioinformatics analysis, we found potential target sites for miRNA let-7b in 3′UTR of human CYP2J2. Luciferase and western blot assays revealed that CYP2J2 was regulated by let-7b. In addition, let-7b decreased the enzymatic activity of endogenous CYP2J2. Furthermore, let-7b may diminish cell proliferation and promote cell apoptosis of tumor cells via posttranscriptional repression of CYP2J2. Tumor xenografts were induced in nude mice by subcutaneous injection of MDA-MB-435 cells. The let-7b expression vector, pSilencer-let-7b, was injected through tail vein every 3 weeks. Let-7b significantly inhibited the tumor phenotype by targeting CYP2J2. Moreover, quantitative real-time polymerase chain reaction and western blotting were used to determine the expression levels of let-7b and CYP2J2 protein from 18 matched lung squamous cell cancer and adjacent normal lung tissues; the expression level of CYP2J2 was inversely proportional to that of let-7b. ConclusionsOur results demonstrated that the decreased expression of let-7b could lead to the high expression of CYP2J2 protein in cancerous tissues. These findings suggest that miRNA let-7b reduces CYP2J2 expression, which may contribute to inhibiting tumor phenotypes.背景 微小RNA（MicroRNAs, miRNAs）是一类长度为20至22个核苷酸的小型非编码RNA分子，可通过结合靶基因的3′非翻译区（3′UTR）调控基因表达。越来越多的研究表明，miRNA的异常表达参与了癌症的发生发展进程。我们此前的研究发现，CYP2J2环氧酶可促进人类癌症表型，但目前尚不清楚miRNA是否以及如何调控CYP2J2的表达。 方法与结果 通过生物信息学分析，我们在人类CYP2J2基因的3′UTR区域发现了miRNA let-7b的潜在结合位点。双荧光素酶报告基因实验与蛋白质印迹（Western blot）实验证实，let-7b可调控CYP2J2的表达。此外，let-7b可降低内源性CYP2J2的酶活性。进一步研究发现，let-7b可通过转录后抑制CYP2J2的表达，从而抑制肿瘤细胞增殖并促进其凋亡。 我们通过皮下注射MDA-MB-435细胞构建裸鼠异种移植瘤模型，每3周通过尾静脉注射let-7b过表达载体pSilencer-let-7b。实验结果显示，let-7b可通过靶向CYP2J2显著抑制肿瘤生长表型。此外，我们对18对配对的肺鳞状细胞癌组织及癌旁正常肺组织进行了实时荧光定量聚合酶链式反应与蛋白质印迹检测，结果显示CYP2J2的表达水平与let-7b的表达水平呈负相关。 结论 本研究结果证实，癌组织中let-7b的低表达可导致CYP2J2蛋白的高表达。上述研究结果表明，miRNA let-7b可通过下调CYP2J2的表达，进而发挥抑制肿瘤表型的作用。