低氧微环境下circRNF13–miR-135b–Bmal1轴重调胰腺生物钟对胰腺癌发生发展的影响

Pancreatic cancer (PC) is a highly malignant digestive neoplasm characterized by local hypoxia. Our previous study has shown that endogenous hypoxia signaling is a strong resetting cue for mammalian circadian clocks and can significantly cripple the circadian rhythmicity and intensity. Given that circadian misalignment has been closely linked to tumor pathologies, we investigated the role of the pancreatic timing system in human PC. For the first time, we demonstrated that the aberrantly elevated miR-135b level in PC directly inhibits expression of the core circadian clock gene Bmal1, and thereby disrupts the whole pancreatic clockwork and promotes cancer progression. In addition, we also identified that circRNF13, a significantly downregulated circular RNA in PC cells under hypoxia, may function as a miR-135b sponge to regulate tumorigenicity. We thus speculate that the intratumor hypoxia-mediated dysregulation of circRNF13–miR-135b–Bmal1 axis reprograms the pancreatic oscillator and represents an important mechanism of PC development. In this study, we aim to explore the relationship of the hypoxic environment, the circRNF13-regulated signaling axis, the pancreatic clock, and the tumorigenesis of PC by using in vitro cell biology experiments, PDX models, orthotopic PC-bearing animal models, and clinicopathological and prognostic investigations. Our work will help unravel the mechanism by which tumor hypoxia resets the pancreatic clock as well as its influence on PC growth. What’s more, establishment of the circRNF13–miR-135b–Bmal1 axis holds translational values and may provide new insights into the clinical management of PC.

胰腺癌（PC）是恶性程度极高的消化系肿瘤，局部低氧是其显著特征。课题组前期发现内源低氧信号可以重调定哺乳动物生物钟，致其节律性受损。鉴于生物钟异常已被证明与人类肿瘤发生密切相关，我们对其与PC的关联进行探讨，并首次证明miR-135b抑制核心钟基因Bmal1表达导致胰腺生物钟网络紊乱以及肿瘤演进；而低氧下PC中显著下调的circRNF13可能以miRNA sponge方式直接调控miR-135b。由此我们推测，肿瘤低氧微环境通过circRNF13–miR-135b–Bmal1信号轴介导胰腺生物钟发生重置或是引起PC进展的重要机制。本课题拟从细胞实验、PDX模型、原位PC荷瘤模型、临床病理及预后研究等多个层面详细阐述低氧、circRNF13功能轴、胰腺生物钟与PC间的联系，以明确低氧信号重调胰腺生物钟的机制和对PC生长的影响，以及circRNF13–miR-135b–Bmal1轴的临床意义。

胰腺癌是恶性程度极高的消化系肿瘤，局部低氧是其显著特征。生物钟直接影响着人类健康，生物节律紊乱被证明可导致组织恶变的发生。胰腺是外周生物钟的重要组成部分，我们前期研究发现低氧可以重调定哺乳动物胰腺生物钟，引起Bmal1在内的核心钟基因表达抑制以及节律强度减弱；而Bmal1作为潜在的抑癌基因，其表达降低致p53 通路抑制加速PC生长。另一方面，人类PC微环境中存在大量具有特定生物学功能的非编码RNA异常表达。在本课题中，我们首先通过乏氧TME中PC细胞内circRNA测序以及生物信息学手段发现circRNF13可能在人类胰腺癌中发挥重要作用，并以多种分子生物学手段对circRNF13的定位和基因功能进行鉴定。在低氧微环境中HIF1a结合至RNF13启动子诱导circRNF13转录剪切，后者可促进PC细胞增殖、迁移和侵袭，并具有促进肿瘤血管形成的作用。circRNF13在活体模型中同样可以加速PC生长并促进肿瘤的远处脏器包括肝肺转移。随后我们利用RNA pull-down实验对circRNF13的miRNA靶点进行分析，结果示miR-135可与circRNF3形成miRNA sponge工作模型，而miR-135a在人PC组织中低表达。我们接着通过一系列gain and loss实验验证了circRNF13–miR-135a失调对PC的促进作用。机制研究发现miR-135可以靶向钟基因Nr1d1，后者在PC组织细胞中异常活化并通过生物钟TTFL对Bmal1形成直接转录抑制，最终导致Bmal1表达降低。DMBA和CDX两种模型均证实了circRNF13异常表达可导致胰腺固有节律丧失。因此，肿瘤微环境中circRNF13–miR-135–Nr1d1–Bmal1轴介导了低氧信号对生物钟系统以及肿瘤演进的调控。最后我们利用临床标本验证了circRNF13–Bmal1轴的临床意义，circRNF13高表达/Bmal1低表达与患者不良预后相关。此外我们还研究了另一个核心钟基因Per1在胰腺癌中的作用。PC中Per1以m6A-YTHDF2依赖性方式受到RNA去甲基化酶ALKBH5的调控，后者在PC中的失表达导致Per1丰度降低从而促进肿瘤的侵袭性行为。本课题论证了胰腺癌低氧微环境–生物钟紊乱–肿瘤演进的科学假说，并阐明了调控该途径的分子通路以及靶向circRNA的巨大应用前景。

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