Opposing roles of C/EBPα and eEF1A1 in Sp1-regulated miR-122 transcription

We previously showed that miR-122 was frequently downregulated in hepatocellular carcinoma (HCC) and C/EBPα transactivated miR-122 expression. In this study, we found that Sp1 bound to the miR-122 promoter at two different sites. Interestingly, either inhibition or overexpression of Sp1 could decrease the miR-122 promoter activity and the cellular miR-122 level in hepatoma cells. Further investigations disclosed that Sp1 cooperated with C/EBPα to induce miR-122 transcription by binding to the positive regulatory site D in the miR-122 promoter, whereas eEF1A1 interacted with Sp1 to bind to the negative regulatory site E and inhibit miR-122 transcription. Significantly, both Sp1 and eEF1A1 levels were enhanced, but C/EBPα and miR-122 expression were reduced in HCC tissues. Knockdown of eEF1A1 enhanced miR-122 level and inhibited cell growth, and these effects were abrogated when Sp1 was silenced. Consistently, the promoter activity enhanced by site E deletion was attenuated by silencing Sp1. Moreover, reduction of miR-122 resulted from Sp1 overexpression was rescued by coexpressing C/EBPα. These data suggest that C/EBPα and eEF1A1 may play opposing roles in Sp1-regulating miR-122 transcription, and the eEF1A1 upregulation accompanied by C/EBPα downregulation in HCC may switch the regulatory functions of Sp1 and led to reduced miR-122 transcription. These findings highlight the complex regulatory network of miR-122 expression and its significance in hepatocarcinogenesis. <b>Abbreviations:</b> MiRNA: microRNA; HCC, hepatocellular carcinoma; eEF1A1: eukaryote translation elongation factor 1A1; siRNA: small interfering RNA; qPCR: real-time quantitative RT-PCR; EMSA: electrophoretic mobility shift assay; ChIP: chromatin immunoprecipitation; TSS: transcription start site.

我们此前的研究证实，miR-122在肝细胞癌（hepatocellular carcinoma, HCC）中频繁下调，且C/EBPα可反式激活miR-122的表达。本研究中，我们发现特异性蛋白1（Sp1）可通过两个不同位点结合至miR-122启动子。有趣的是，无论是抑制Sp1的表达还是过表达Sp1，均会降低肝癌细胞中miR-122的启动子活性以及细胞内miR-122水平。进一步研究显示，Sp1可与C/EBPα协同，通过结合miR-122启动子中的正调控位点D来诱导miR-122转录；而真核翻译延伸因子1A1（eukaryotic translation elongation factor 1A1, eEF1A1）则可与Sp1相互作用，结合至负调控位点E并抑制miR-122转录。值得注意的是，在HCC组织中，Sp1与eEF1A1的表达水平均升高，而C/EBPα与miR-122的表达则出现下调。敲低eEF1A1可提升miR-122的水平并抑制细胞增殖，而当Sp1被沉默后，上述效应会被消除。与之相符的是，位点E缺失所增强的启动子活性，会因Sp1的沉默而被减弱。此外，过表达Sp1所导致的miR-122水平降低，可通过共表达C/EBPα得以挽救。上述数据表明，C/EBPα与eEF1A1在Sp1调控miR-122转录的过程中可能发挥相反的作用；而在HCC中，eEF1A1的上调伴随C/EBPα的下调，可能会改变Sp1的调控功能，最终导致miR-122转录水平降低。这些发现凸显了miR-122表达的复杂调控网络，及其在肝细胞癌变过程中的重要意义。<b>缩写：</b> MiRNA：微小RNA（microRNA）；HCC：肝细胞癌（hepatocellular carcinoma）；eEF1A1：真核翻译延伸因子1A1（eukaryotic translation elongation factor 1A1）；siRNA：小干扰RNA（small interfering RNA）；qPCR：实时定量逆转录聚合酶链反应（real-time quantitative RT-PCR）；EMSA：电泳迁移率变动分析（electrophoretic mobility shift assay）；ChIP：染色质免疫沉淀（chromatin immunoprecipitation）；TSS：转录起始位点（transcription start site）。