缺氧诱导的miR-495靶向P4HA2调控细胞外基质重构参与肝癌进展的作用及分子机制研究

Hypoxia and ECM consist of the tumor microenvironment and play a crucial role in the tumor development by promoting the invasion and metastasis of tumor cells. Our previous study found that the expression of P4HA2, a key enzyme involved in ECM remodeling, was increased in HCC, and was closely associated with clinical prognosis. By in vitro and in vivo experiments, we also found that P4HA2 could regulate the proliferation and invasion of HCC cells, and had influence on the ECM remodeling. Moreover, P4HA2 might be regulated by miR-495 via target binding. In addition, hypoxia and HIF1a could regulate miR-495/P4HA2 pathway. However, the role of P4HA2 in HCC progression has not been reported. Thus, we speculate that hypoxia induced miR-495/P4HA2 pathway involved in the ECM remodeling. In this study, we utilized CRISPR/Cas9 and in vivo imaging to investigate the function and mechanism of P4HA2, and upstream regulatory mechanism of P4HA2 in HCC progression by clinical tissue specimens, cell experiments and mice tumor-burdened model, and then illustrate that the miR-495/P4HA2 pathway induced by hypoxia promoted the proliferation and invasion of HCC cells by regulate the ECM remodeling, which would provide novel ideas and strategies for the targeting treatment of HCC.

缺氧和细胞外基质(ECM)构成的肿瘤微环境在肿瘤侵袭和转移的过程中发挥了重要作用。我们前期研究发现：调控ECM的关键酶P4HA2在肝癌中高表达并且和临床预后密切相关；P4HA2能够影响肝癌细胞增殖和侵袭，同时具有调控ECM重构的功能；miR-495能够靶向结合P4HA2；缺氧和HIF-1α能够调控miR-495/P4HA2轴。目前未见关于P4HA2参与肝癌进展的研究报道。据此提出科学假设：缺氧调控的miR-495/P4HA2通路参与了肝癌细胞外基质的重构过程。本课题拟基于CRISPR/Cas9和活体成像技术，从分子、细胞、组织以及动物水平多层次证明P4HA2调控ECM重构促进肝癌增殖和侵袭的作用；明确缺氧和HIF-1α对 miR-495/P4HA2通路的调控；阐明miR-495/P4HA2轴在肝癌中的表达以及调控肝癌侵袭转移的作用和机制，以期为临床治疗及新药研发提供新的理论依据和作用靶点。

肝细胞肝癌( Hepatocellular carcinoma，HCC) 是目前全世界发病率和死亡率极高的恶性肿瘤之一。由于肝癌容易发生转移，对化疗和放疗均不敏感，即使目前肝癌唯一有效的靶向治疗药物索拉非尼（Sorafenib），也只能使患者的平均生存期延长6月左右，这些因素导致HCC的临床治疗依然面临巨大挑战。因此，亟待对肝癌的发生转移的机制进行深入的研究，预防HCC转移的发生，研究新型、有效的治疗靶点进行早期干预，提高HCC的总体生存率。细胞外基质重构在肿瘤的增殖和转移过程中发挥了重要作用，其中胶原蛋白是细胞外基质的主要成分。.本项目主要开展了研究工作，取得了重要成果：.1)阐明了胶原合成的关键酶P4HA2在肝癌中异常高表达并且和临床预后密切相关；体内、外实验证实P4HA2能够调控肝癌细胞的增殖和侵袭；证实缺氧能够导致肝癌细胞的miR-495的表达下调和P4HA2的表达上调；解析了miR-495靶向调控P4HA2的生物学功能.2)N1-甲基腺苷(M1A)是一种广泛存在的RNA修饰，广泛影响RNA的结构稳定性、折叠和与蛋白质的相互作用。最近，关于M1A调节因子在肿瘤中的作用的报道越来越多。然而，它们的机制和临床相关性仍不清楚。申请人利用生物信息学方法系统地评价了M1A调节子的表观遗传学特征和临床相关性。结果显示，M1A调节剂的基因表达发生了广泛的变化，这与致癌途径的激活和抑制以及整体患者的生存有关。总体而言，这项研究为评估肿瘤预后和靶向治疗提供了新的见解。.该项目的开展为肝癌的靶向治疗提供新思路、新策略