NOB1通过UPP途径调控肝癌细胞放射线敏感性分子机制研究

Currently，Clinical evidence has shown that about 40% patients with liver cancer are not sensitive to this treatment, which leading to poor therapeutic effects. The mechanism underlying this sensitivity differentiation remains unknown. Our preliminary experiments demonstrated that ionizing radiation treatment could down-regulate NOB1 protein expression by increasing microRNA;oncoprotein NOB1 was able to modulate the sensitivity of liver cancer to ionizing radiation, whereas the molecular mechanism has yet to be investigated. Therefore, we hypothesized that NOB1 modulated by microRNA1972 could promote p53 and telomerase (TER) protein degradation through ubiquitin proteosome pathway(UPP) which results in the insensitivity of liver cancer to ionizing radiotherapy. To further validate this hypothesis, this project will use cultured liver cancer cell lines and xenograft liver cancer mice model, and explore Western blot, RNA interfere and adenovirus vector transfection to verify the crucial roles of NOB1 in regulating the sensitivity of liver cancer cells to ionizing radiotherapy at different levels including molecule, cell and animal model. Our study will clarify the molecular mechanism of NOB1-mediated UPP regulating tumor suppressor gene p53 and TER. This study will provide solid evidence by revealing a novel mechanism underlying radiotherapy sensitivity. Our findings hope to give useful clues for clinical trials to decide whether the patients with liver cancer are suitable to be treated with radiotherapy.

临床发现约35%的肝癌患者对放射线敏感性差，治疗效果不明显，这种敏感性差异的分子机理仍不明确。预实验结果提示放射线诱导microRNA上调和NOB1下调，NOB1能够介导体外培养肝癌细胞放射敏感性，但其作用机制仍不清楚。为此，我们提出假说：推测受microRNA调控的NOB1在放射敏感性调节中发挥重要作用，miRNA调控的NOB1可能通过泛素-蛋白酶体途径(UPP)促进抑癌蛋白p53和端粒酶（TER）降解导致肝癌细胞对放射线产生抗性。为验证这一假说，本项目拟通过人肝癌细胞系和小鼠模型，采用免疫印迹，RNA干扰等方法，从分子到动物整体水平等多个角度探讨miRNA调节NOB1表达分子机制，NOB1调控P53和TER表达在肝癌细胞放射线敏感性调节机理。明确miRNA-NOB1-UPP-p53-TER调控影响放疗效果的重要作用。以期对临床肝癌患者是否可以接受放射线治疗提供新的理论依据和有益线索。

NOB1（NIN/RPN12 binding protein 1）与多种恶性肿瘤的发生发展密切相关，NOB1通过泛素-蛋白酶体途径（ubiquity-proteasome pathway，UPP）参与调控细胞周期。UPP途径是细胞内诸多重要蛋白的降解途径。我们认为阐明NOB1通过UPP途径调控肝癌放射线敏感性的分子机制，从中找出NOB1作为新的治疗靶点的理论依据，将极大的推动肝癌的临床治疗。本研究的目的是确定短发夹状RNA(ShRNA)抑制NOB1是否抑制人肝细胞癌(HCC)细胞的生长。构建携带NOB1的重组慢病毒shRNA表达载体，转染人肝癌细胞系SMMC-7721。用MTT法、BrdU掺入法和流式细胞仪检测SMMC-7721/pGCSIL-GFP-shNC和pGCSIL-GFP-shNob1细胞的生长特性。此外，还检测了NOB1在裸鼠体内的集落形成和肿瘤生长能力，以明确NOB1在细胞转化和肿瘤发生中的作用。结果表明，SMMC-7721/pGCSIL-GFP-shNob1细胞的生长和增殖明显低于SMMC-7721/pGCSILGFP-shNC。此外，抑制NOB1后，SMMC-7721细胞的集落形成受到抑制。在体内，SMMC-7721/pGCSIL-GFP-shNob1细胞的成瘤能力明显低于对照组。我们的数据支持NOB1是人肝癌致瘤特性的重要调节因子，可以作为人肝癌的候选治疗靶点。在此基础上 ，我们将进一步研究NOB1通过UPP途径调控肝癌细胞放射线敏感性的分子机制，并深入探讨NOB1作为肝癌治疗靶点的可能性。