新型神经营养因子MANF对肝细胞癌的抑制作用及其机制研究

MANF is a new neurotrophic factor. ER stress up-regulates MANF expression and secretion. However, whether MANF play a role in carcinogenesis is unknown. On screening the proteins interacting with MANF by using of yeast two-hybrid assay, we found MANF directly interacts with p65 (a subunit of NF-kB). It was well known NF-kB pathway is involved in carcinogenesis. Therefore, it is reasonable to assume that MANF may be associated with carcinogenesis. To test it, we detected the cancer tissues from 150 pateints by using of immunohistochemistry assay. We found that the level of MANF was reduced in the hepatocelluler cancer (HCC) in large part of patients（74.7%）. In small number of patients with high MANF level, the rate of survival was significantly increased, compared with the control (p=0.022). This result suggests that MANF probably is a tumor suppressor, and MANF may inhibit carcinogenesis and development by regulating NF-kB pathway. To prove the hypothesis, we firstly detect the levels of MANF in liver tissues and serum of 200 patients by using immunohistochemistry and ELISA, respectively. The change trends of MANF were compared in the liver tissues and serum. Based on this result, we will figure out whether MANF becomes a serum biomarker. Secondly, we will investigate the effects of MANF on the carcinogenesis and the malignant biological behavior, including migration, invasion, and metastasis in hepatoma cell lines. Thirdly, we will verify the tumor suppressive effect of MANF in the specific liver MANF knockout mice. In the MANF knockout mice, liver cancer was induced by diethylnitrosamine and CCl4. Finally, we will explore the mechanisms by which MANF suppresses the carcinogenesis and development via interacting with p65. This study will find a new tumor suppressor and discover a novel mechanism in liver carcinogenesis.

MANF是一新型神经营养因子，内质网应激上调其表达与分泌，但它与肿瘤的关系未见报道。我们在筛选与MANF相互作用的蛋白时，发现MANF可与p65（NF-kB的一个亚单位）相互作用，而后者与肿瘤的发生密切关系，提示MANF可能参与了肿瘤的调控。我们的前期研究也发现，MANF的表达水平与肝细胞癌（HCC）患者的存活有关，推测MANF可能是一个抑癌因子，通过与p65的相互作用来抑制肿瘤的发生发展。为此，我们拟在200例HCC中检测MANF的表达，并比较与血清中MANF的水平是否一致，以此评估MANF是否可以作为血清学诊断指标；在肝癌细胞株上通过过表达和敲低MANF来证明MANF对肝癌细胞恶性生物学行为的抑制；在肝脏特异性敲除MANF基因的小鼠模型上，用化学剂诱导肝癌，通过与野生型小鼠肿瘤诱导时间和程度的比较，来验证MANF的抑癌作用；从MANF对NF-kB信号通路的调节来探讨MANF的抑癌机制。

我们在对150例肝癌和136例癌旁对照进行研究中发现，在肝癌组织中MANF的mRNA和蛋白水平均呈现低表达。高表达MANF的病人5年生存期明显延长。MANF在肝癌患者血清中的低水平与肝癌组织中的低水平一致，因此MANF可以作为肝癌的血清学诊断及预后评价指标。在体外肝癌细胞株上我们证实MANF对肝癌细胞的增殖、迁移和侵袭均有抑制作用。将携带MANF shRNA干扰片段的慢病毒载体稳定转染人肝癌细胞并接种裸鼠，然后在瘤内注射重组人MANF蛋白，观察MANF对肿瘤生长的影响。结果显示MANF敲低促进肿瘤形成和生长，而重组MANF蛋白明显抑制肿瘤生长。采用肝细胞特异性敲除MANF鼠（MANF fl/fl Alb-Cre-T），于出生后第15天腹腔注射DEN诱导肝癌模型，4-12周后处死动物取肝组织。DEN诱导8个月的结果发现，与野生型相比，肝细胞特异性敲除MANF鼠瘤体数量，体积明显增大，重量增加，提示MANF有抑制肝癌作用。进一步研究发现，肝癌细胞中MANF与p65在核内共定位，而低分化肝细胞癌中MANF与p65在胞浆共定位。ER应激可促进MANF和p65在核内相互作用，并抑制p65与DNA结合，从而抑制NF-κB下游基因表达。MANF、p65、SUMO1或SUMO2/3在核内能形成p65-MANF-SUMO复合物。SUMO1能增加缺氧诱导的MANF和p65入核，SUMO2/3能稳定MANF和p65表达，提示SUMO化修饰能调节MANF和p65的相互作用，从而参与调控NF-κB通路。为了进一步了解MANF的作用机制，我们对酵母双杂交筛选出来的与MANF相互作用的蛋白进行进一步验证，例如，泛素连接酶RNF2 (ring finger protein 2, RNF2)、过氧化物酶PRDX6 (Peroxiredoxin 6)和剪接因子SRSF3 (serine/arginine-rich splicing factor 3, SRSF3)等。在后续研究中我们将研究上述功能蛋白与MANF相互作用在肝脏病理机制中可能发挥的作用。