miR-34a通过端粒途径调控肝癌细胞衰老的作用及分子机制研究

Abnormal cellular senescence is an extremely important indicator of malignant transformation. The regulatory roles of telomere and telomerase in cellular senescence have gained widely concerns from current researches. However, as an important regulatory factor, whether miRNAs participated in the regulation of telomerase, which eventually influence tumor senescene is still unknown. Owing to our previous study in screening miRNAs by gene chips, we found that miR-34a was significantly increased during the process of induced senescence in hepatocellular carcinoma, with five potential targets that might regulate telomerase activation in significant steps, which were verified by bioinformatical prediction and tissue detection. Therefore, based on the supposition above, this project assumes: ① to demonstrate the impact of miR-34a on the senescence in hepatocellular carcinoma with the utilization of a variety of hepatocellular carcinoma cell lines and their models in mice; ② to validate the relationship between the miR-34a and the five target genes ( C-MYC, HDAC, FOXM1, PPARG and PNUTS ) in regulation, with the help of the technologies such as the luciferase reporter, miRNA transfection, and so on; ③ to explore their influence on telomere length, telomerase activity, and senescence pathways in hepatocellular carcinoma by further regulating expression level of miR-34a. A new theoretical mechanism in regulating hepatic senescence via telomere and telomerase regulation would be proposed with the accomplishment of this project, and new directions in targeted treatment of hepatocellular carcinoma would also be provided.

衰老逃逸是肿瘤细胞恶性转变的重要标志。端粒、端粒酶调控衰老是当前的研究热点。然而，miRNAs作为调控基因表达的重要分子，是否在肿瘤的发生发展中参与端粒酶的调控，并最终影响肿瘤衰老？本项目组前期通过基因芯片筛选发现，miR-34a在肝癌细胞衰老过程中表达显著提高，可能在调控肿瘤衰老中发挥关键作用。经过生物信息学预测和临床标本检测，提示其有5个潜在靶点是调控端粒酶活性的关键基因。基于上述设想，本项目拟：①利用多种肝癌细胞株及小鼠荷瘤模型，明确miR-34a对肝癌衰老的影响；②采用荧光素酶报告基因、miRNA表达调控等技术，验证miR-34a与5个靶基因（C-MYC、HDAC、FOXM1、PPARG和PNUTS）的调控关系；③进一步通过调控miR-34a表达，检测其对端粒长度、端粒酶活性及衰老通路的影响。本项目的完成将为探究肝癌衰老的端粒调控机制奠定新的理论基础，亦为肝癌靶向治疗提供新的思路。

衰老逃逸是肿瘤细胞恶性转变的重要标志，端粒、端粒酶调控衰老是当前的研究热点。肿瘤实质的端粒长度越长提示其恶性度越高、患者预后越差。近年来，miR-34家族（a，b和c）因其被确定为p53靶点并调节p53介导的细胞周期阻滞和凋亡而受到关注。先前的研究表明miR-34诱导的癌细胞衰老全部以非端粒依赖性细胞周期停滞的形式存在，而miR-34家族是否能以端粒依赖性方式诱导肝癌细胞衰老尚不清楚。在本研究中，确定了miR-34a在调控端粒酶活性中的作用，以及随后对细胞衰老和生存的影响。首先，本研究组发现年轻肝癌患者与老年肝癌患者预后存在显著差别，老年肝癌患者血清AFP水平显著低于年轻肝癌患者。其次，miR-34a在肝癌中低表达，与肝癌预后关系密切。利用60种肿瘤细胞特征的NCI-60数据，发现在不同肿瘤细胞中，miR-34a与端粒酶活性呈显著负相关。并且，在75对肝癌和癌旁组织中提取RNA进行Realtime PCR检测，进一步验证了miR-34a水平与端粒指数（端粒长度和端粒酶活性）之间呈负相关。本研究组发现，miR-34a抑制端粒酶活性并且促进端粒缩短。在HepG2，SMMC-7721，HHCC和SK-Hep-1细胞中，利用细胞转染技术调控miR-34a的表达水平，发现上调miR-34a水平可以显著抑制端粒酶的活性，并且促进端粒缩短。利用β半乳糖苷酶技术，发现上调miR-34a水平可以显著促进肝癌细胞衰老。本研究组发现，miR-34a可以有效靶向抑制c-Myc和FoxM1，两者参与端粒酶逆转录酶（hTERT）转录的激活，并且miR-34a诱导的肝癌衰老仍然受到p53的调控。裸鼠成瘤实验提示miR-34a的表达水平可以显著影响裸鼠的肿瘤活性。转染miR-34a的裸鼠肿瘤的ki-67及PCNA等肿瘤增殖相关指标明显减弱。因此，本研究结果表明，miR-34a可以通过调控细胞衰老中的端粒通路抑制肿瘤。此外，我们还发现miR-34a可以通过抑制SNAI1等影响肝癌细胞的侵袭和转移。作为衰老关键通路中的miR-34a及FoxM1等分子可以作为肿瘤预后的关键分子指标。