肿瘤抑制基因Tg737缺失介导肝干细胞恶性转化过程中的促增殖和抑分化作用及其分子机制研究

One of the most recognized mechanisms for liver cancer genesis,is from the origin of liver cancer stem cells (LCSCs). However, some of these LCSCs are suppsosed to come from malignant transformation of liver normal stem cells (LNSCs), which gain multiple genetic changes under the stimulation of cancer inductive factors. In order to explore similarities and differences between these two kinds of stem cells, we isolated both LCSCs and LNSCs using our established methods. By comparing the microRNA and genetic expression profiles, we found many distinctly expressed microRNAs and genes between LNSCs and LCSCs. Among a great number of dys-regulated microRNAs and genes, we selected the most down-regulated gene Tg737 (almost lack of expression) in LCSCs for later thorough exploration. On the support of our previous National Natural Science Foundation of China, we found that the inhibition of Tg737 gene could push LNSCs to proliferate at much fast speed, and in the mean time maintain a status of differentiation block. As a result, through the above mechanisms, the inhibition of Tg737 gene could finally induce LNSCs to go through malignant transformation. Although the above mentioned findings have been sequentially published in several high influence journals including Stem Cell Rev, the detailed molecular mechanism responsible for the malignant transformation of LNSCs induced by the Tg737 inhibition is still unclear. Based on multiple prediction softwares to forecast the relationships between microRNAs and target genes, we hypothesize some microRNAs, which highly expressed in LCSCs, may inhibit the expression of the Tg737 gene; while some literatures suggest that the Tg737 gene regulates multiple moleculars related to regualtion of cell proliferation or cell differentiation. This project will follow the preliminary findings, further confirm the upstream microRNAs that lead to the Tg737 inhibition, clear the downstream molecules which are affected by Tg737 inhibition, and eventually shed the molecular mechanisms that Tg737 inhibition leads to malignant proliferation of LNSCs.

肝癌干细胞起源是肝癌重要发生机制之一，而肝癌干细胞的来源之一被推测是正常肝干细胞在诱癌因素刺激下发生恶性转化所致。利用建立的干细胞分离法，我们前期富集了肝干细胞和肝癌干细胞，并建立了两种干细胞间microRNA和基因差异表达谱。在前一个国科金支持下，我们针对肝癌干细胞中几乎缺失表达的肿瘤抑制基因Tg737进行了重点研究。我们发现肝干细胞中抑制Tg737基因的表达会促进细胞增殖明显加快，并表现为分化受阻状态，最终导致肝干细胞发生恶性转化成为类似肝癌干细胞的细胞。虽然上述研究结果已在"Stem Cell Rev"等多篇高水平国际杂志发表，但是Tg737缺失介导恶性转化的促增殖和抑分化作用及其分子机制尚不清楚。本项目将延续前期研究，进一步鉴定导致Tg737缺失的上游调控性microRNAs，明确Tg737基因缺失后受影响的增殖和分化调控分子，最终阐明Tg737缺失后促增殖和抑分化的关键分子机制。

肝癌干细胞是导致肝癌治疗失败的重要原因。随着研究的深入，肝癌干细胞起源于肝干细胞恶性转化这一观点被广为接受，然而其中的机制仍有诸多未知。基于前期成果，通过对比正常肝干细胞（LSCs）和肝癌干细胞（LCSCs）基因及miRNA芯片，我们筛选出LCSCs中几乎缺失表达的基因Tg737并展开相应研究。结果表明Tg737不仅在LSCs向LCSCs恶性转化过程中起到重要作用，也对肝癌有诸多影响。缺失表达Tg737主要通过促进LSCs恶性增殖和抑制其正常分化两方面促进恶性转化，其中涉及广泛的相互调控作用。Tg737可抑制β-catenin入核，缺失表达Tg737可致其入核并激活Wnt/β-catenin通路，进而上调snail表达，引发EMT过程；激活的Wnt/β-catenin通路可增加Cyclin D1等的表达，引发恶性增殖，促进其恶性转化。HNF4α是促进分化的重要因子，其与Tg737在LSCs内有共定位，且总是伴随Tg737表达的变化而变化，提示两者有密切相互作用；Tg737缺失能激活snail表达，而snail可与HNF4α形成一个较为稳定的负调控环路。我们发现在这一环路里，Tg737起着调定点的作用：Tg737表达时，snail抑制而HNF4α活化，细胞维持上皮表型； Tg737缺失时，负调控环路失衡，LSCs发生恶性增殖，并导致细胞出现EMT。Tg737缺失引发的多因素综合作用，最终导致LSCs向LCSCs恶性转化。Tg737还可产生其他广泛的影响。Tg737在肝癌与癌旁组织出现了明显下调。抑制表达Tg737可促进肝癌细胞的增殖和迁移能力。低氧条件培养肝癌细胞促使Tg737表达下调，同时细胞黏附力降低，转移和侵袭能力增强，polycystin-1表达下调而IL-8、活化的TGFβ1及总TGFβ1上升。重新表达Tg737后，可恢复这一效应。另外，肝癌细胞中过表达Tg737，可激活Hedgehog通路，并使AKT下调，从而使肝癌细胞增殖、侵袭及迁移降低。我们通过生物信息学预测筛选鉴定了与Tg737密切相关的miRNAs，其中miR548a-5p与Tg737最为密切。结果表明miR548a-5p能够直接作用于Tg737的3’UTR区域，进而影响下游表达。这一机制的阐明，为今后靶向治疗肝癌提供了一个新的靶点。