Lin-28B表現會引起肝細胞癌的轉形及侵犯

Abstract

許多microRNA (miRNA) 於人類癌症中常受抑制，如let-7 miRNA 家族。let-7 miRNA 家族除了調節癌細胞的自體更新(self-renewal)及腫瘤形成能力(tumorigenecity)外，於胚胎發育中也扮演重要角色。最近研究發現RNA結合蛋白(RNA-binding protein) Lin-28及Lin-28B會透過let-7的前趨物(precursor)於末端之尿嘧啶化(uridylation)並使之分解，以致let-7生合成減少。在肝癌細胞中，我們發現只有Lin-28B會高度表現，而Lin-28並沒有此現象。此外，Lin-28B較常表現於高度表現α胎兒蛋白並且分級(grade)較高的肝癌。於肝癌細胞株中，Hep3B及HCC36所表現的Lin-28B量最多，因此我們選擇這兩株細胞株為研究之用。我們利用RNA干擾的方式將其Lin-28B 敲減(knockdown)。利用轉殖Lin-28B載體，使Lin-28B過度表現於HA22T。Lin-28B被knockdown的細胞株Hep3B及HCC36在體外增生的速度降低。Lin-28B敲減的Hep3B細胞株在免疫不全小鼠(NOD/SCID mice)生成腫瘤的速率也下降。相反地，Lin-28B過度表現的HA22T細胞生長速度較快，且生成腫瘤的能力也較高。我們也發現過度表現Lin-28B的HA22T會引起上皮-間質轉化(Epithelial-mesenchymal transition)，而使其侵犯(invasion)及移動(migration)能力皆提高。由於Lin-28B與let-7 miRNA的密切關係，我們以大規模即時PCR陣列(Large-scale real-time PCR array)進一步研究，分析顯示Lin-28B所調節最主要的miRNA是let-7/mir-98家族。Lin-28B過度表現會使let-7所抑制的基因c-myc及HMGA2表現量增加； Lin-28B過度表現也會使受let-7控制的第一型類胰島素成長因子受體(IGF1R)表現量上升。我們的研究結果顯示，透過共同抑制let-7/mir-98家族及許多致癌基因途徑，Lin-28B於肝癌腫瘤形成及轉移扮演著重要的角色。

In human cancers, multiple members of microRNAs (miRNA) are often repressed, such as the let-7 family miRNA. The let-7 family regulates self-renewal and tumorigenicity of cancer cells, and plays critical roles in embryonic development. The RNA binding proteins Lin-28 and Lin-28B are recently demonstrated to negatively regulate let-7 biogenesis by inducing terminal uridylation and degradation of the let-7 precursors. In this study, we showed that in hepatocellular carcinoma (HCC), only Lin-28B but not Lin-28 was highly expressed. Lin-28B expression in HCC was more frequently found in high grade tumors with high α-fetoprotein levels. Among the HCC cell lines, Hep3B and HCC36 expressed higher levels of Lin-28B than the other cell lines, and were chosen for gene knockdown of Lin-28B. HA22T cell line which had very low level or undetectable Lin-28B, was chosen for ectopic expression of Lin-28B. Knockdown of Lin-28B by RNA interference in the Hep3B and HCC36 led to suppressed proliferation in vitro and reduced in vivo tumor growth in NOD/SCID mice. In contrast, overexpression of Lin-28B in HA22T enhanced proliferation and tumorigenecity. Moreover, overexpression of Lin-28B induced epithelial-mesenchymal transition in HA22T cells, accompanied by increased invasion and migration capacity. To further delineate the role of Lin-28B in the expression of miRNA family, we did a large-scale real-time PCR array analysis. The results revealed that only let-7/mir-98 family members were regulated by Lin-28B. Lin-28B overexpression enhanced the expression of the known let-7 targets c-myc and HMGA2. In this study, we also found that Lin-28B enhanced the expression of type 1 insulin-like growth factor receptor (IGF1R) in a let-7-dependent manner. These findings highlight the important role for Lin-28B in tumor formation and invasion in HCC, through coordinated repression of the let-7/mir-98 family and induction of multiple oncogenic pathways.