MicroRNA-148a信号网络调控肝癌恶性表型的继续研究

MicroRNA-148a is a liver-abundant miRNA and an inducer of hepatocytic differentiation. Compared with normal livers, miR-148a was signicantly decreased in hepatocellular carcinoma (HCC) tissues, especially in those with the portal vein tumor thrombus. An in vitro transwell assay and an in vivo orthotopic liver xenograft model showed that the restoration of miR-148a expression signicantly repressed the migration and pulmonary metastasis of hepatoma cells. In addition, miR-148a suppressed the level of lactate dehydrogenase (LDH) which is the key metabolic enzyme involved in anaerobic glycolysis. Higher level of preoperative serum LDH has been associated with metastasis and the worse prognosis in HCC. These results suggested that miR-148a may regulate anaerobic glycolysis. However, the mechanisms responsible for the downregulation of miR-148a in HCC and the role of anaerobic glycolysis in metastasis remain obscure. In this study, we plan to identify the promoter, transcription factors and targets of miR-148a by way of luciferase reporter system, electrophoretic mobility shift, chromatin immunoprecipitation and other molecular biologic assays, as indicated. The relationship between the transcription factors and targets of miR-148a would be elucidated. The roles, the corresponding mechanisms, and the clinic significances of the transcription factors and targets of miR-148a in aerobic glycolysis and metastasis would be explored. This study would identify a novel miR-148a mediated regulatory circuitry whose dysfunction may contribute to the development of aerobic glycolysis, metastasis and HCC, providing a promising novel target for anti-HCC therapy.

我们发现：miR-148a在肝癌组织中的表达显著下调、可抑制肝癌细胞转移，及乳酸脱氢酶（LDH，糖酵解的关键酶）的分泌。血清高水平的LDH与肝癌易发生转移及较差的预后密切相关。提示：miR-148a可能通过抑制糖酵解及转移来抑制肝癌进展，但miR-148a在肝癌中下调的机制、对糖酵解的调控作用尚不清楚。我们拟鉴定 miR-148a 的核心启动子区、转录因子及其调控糖酵解的靶标，阐明miR-148a的上游转录因子及下游靶标对肝癌糖酵解和转移的影响及调控机制，及其在信号通路上的联系；利用裸鼠肝原位成瘤和DEN诱发miR-148a基因敲除小鼠肝癌模型，明确miR-148a信号网络在体内对糖酵解及转移的影响；分析miR-148a信号网络的临床意义。本研究是在申请人负责的青年科学基金项目基础上的继续研究，研究结果将揭示miR-148a信号网络在肝癌恶性表型中的调控机制，可为肝癌防治提供新的分子靶点

MicroRNA-148a-3p（简写为“miR-148a”）在正常肝组织中高表达，并促进肝脏发育。我们的前期研究发现：miR-148a 在肝癌组织中的表达显著下调、可通过下调其靶标Met（肝细胞生长因子受体）的表达，减弱 AKT 的磷酸化，继而抑制肝癌细胞上皮间质转化及转移。但miR-148a的表达调控，其调控的信号网络及临床意义并不清楚。本研究中，我们发现 miR-148a 受DNA甲基化调控，并可诱导血清饥饿的SK-Hep1细胞发生caspase 3的剪切，为揭示miR-148a促进细胞凋亡的分子机制作了新的补充。机制研究发现：MiR-148a 可抑制靶标WNT1和SMAD2的表达，并可负调节b-catenin的蛋白水平。结合肝癌组织标本中MiR-148a的表达水平与肿瘤转移分析，发现在血清AFP高阳性组中，发生肝内微转移组miR-148a的表达水平显著低于未发生肝内微转移组。研究结果揭示了 miR-148a的上游表达调控机制，及其调控的下游信号网络；阐明了miR-148a对肝癌凋亡的调控作用及miR-148a的临床意义。相关研究结果可为肝癌的进展提供新的理论基础，并可为临床防治肝癌提供新的思路和靶标。