缺氧肿瘤微环境中TUFT1促进肝细胞癌生长和转移的机制研究

Hypoxia is a core element of tumor microenvironment, and a crucial factor for promoting tumor growth and metastasis including hepatocellular carcinoma (HCC). However, the underlying mechanism remains largely unknown. We previously found that hypoxia induced TUFT1 overexpression in HCC cells, and HIF-1α, TGF-β1 and miR-671-5p exerted a possible regulatory role in this process. High TUFT1 expression in HCC tissues indicated a poor prognosis of HCC patients. TUFT1 interacted with E3 ubiquitin ligase TRIM27, which mediated PTEN ubiquitination, in HCC cells. TUFT1 knockdown resulted in the inactivation of PI3K/AKT pathway and inhibited the growth and metastasis of HCC. Thus, clinical and biological experiments will be performed to verify the following scientific hypothesis that HIF-1α, TGF-β/SMAD pathway and miR-671-5p cooperatively promote TUFT1 expression in hypoxia-related tumor microenvironment. TUFT1 induces PTEN ubiquitination and inactivation via interacting with TRIM27 and subsequently activates PI3K/AKT pathway, and finally promotes the growth and metastasis of HCC. Thus, this project will reveal the role and regulatory mechanism of TUFT1 in the growth and metastasis of HCC, and explore the anti-HCC effect by targeting TUFT1, providing a basis for the clinical development of new HCC targeted therapies.

缺氧是肿瘤微环境的核心特征，是促进肝细胞癌（HCC）等肿瘤生长和转移的关键因素，但具体机制仍不清楚。我们前期发现①缺氧诱导HCC细胞高表达TUFT1，HIF-1α、TGF-β1及miR-671-5p在此过程中可能发挥调控作用；②TUFT1高表达提示HCC患者预后不良；③TUFT1与PTEN泛素化相关E3连接酶TRIM27结合；④敲低TUFT1导致PI3K/AKT通路失活并抑制HCC生长和转移。因此，我们拟采用临床和功能学实验证实如下科学假说：缺氧肿瘤微环境中HIF-1α、TGF-β/SMAD通路及miR-671-5p协同促进TUFT1表达，TUFT1通过结合TRIM27诱导PTEN泛素化失活并激活PI3K/AKT通路，从而促进HCC生长和转移。本项目研究将揭示TUFT1在HCC生长和转移中的功能及调控机制，探索靶向干扰TUFT1的抗HCC作用，从而为临床上开发新HCC靶向治疗策略提供依据。

由于肝癌具有发病隐匿、进展快、易复发和耐药的特点，目前整体预后仍很不理想。缺氧是肝癌微环境的核心特征之一，也是导致肿瘤恶性进展和治疗抵抗的关键因素，但缺氧驱动肝癌恶性进展的精确分子机制很大程度上仍然未知。因此，进一步阐明该机制对于肝癌治疗很重要。（1）本项目发现LINC01123在肝癌组织和细胞系中表达明显上调，LINC01123高水平与肝癌不良预后有关。 LINC01123促进肝癌细胞增殖、迁移和侵袭，LINC01123沉默抑制体内肝癌生长。LINC01123充当miR-34a-5p的分子海绵，TUFT1被鉴定为miR-34a-5p的靶基因。LINC01123通过靶向miR-34a-5p来正向调节TUFT1水平，TUFT1可以消除miR-34a-5p诱导的对肝癌细胞增殖、迁移和侵袭的抑制作用。（2）本项目研究表明缺氧或缺氧诱导因子（HIF）诱导剂（DMOG）可上调肝癌细胞中TM4SF1-AS1表达，HIF-1α敲低消除了缺氧上调的TM4SF1-AS1表达。TM4SF1-AS1在肝癌组织和细胞系中的表达升高，TM4SF1-AS1沉默显着抑制肝癌细胞增殖、迁移和侵袭能力。干扰TM4SF1-AS1可显着降低肝癌细胞中TM4SF1水平，但不影响TM4SF1 mRNA稳定性。缺氧增强了TM4SF1 mRNA表达，而TM4SF1-AS1敲低阻断这一过程。肝癌标本中TM4SF1 mRNA和TM4SF1-AS1表达正相关，TM4SF1显著逆转了TM4SF1-AS1耗竭对Hep3B细胞的抑制作用。（3）本项目揭示了ALKBH3-AS1在肝癌组织和细胞系中的过度表达，ALKBH3-AS1高表达与肝癌患者的生存率降低显着相关。ALKBH3-AS1增强肝癌细胞侵袭和增殖，ALKBH3-AS1沉默限制了体内肝癌生长。肝癌组织中ALKBH3-AS1和ALKBH3 mRNA表达呈显著正相关。ALKBH3-AS1沉默通过降低mRNA稳定性来减少ALKBH3表达。ALKBH3沉默对肝癌细胞的影响与ALKBH3-AS1敲低的影响相似，ALKBH3恢复显着减弱了肝癌细胞中ALKBH3-AS1沉默引起的抑制作用。HIF-1α在缺氧肝癌细胞中转录激活ALKBH3-AS1表达，ALKBH3-AS1敲低显着减弱了缺氧肝癌细胞增殖和侵袭。本项目成果将为筛选抗肝癌新靶点提供理论依据。