Effect of knock-down of circASH2 on gene expression in HCC cells

Metastasis, especially intrahepatical, is a major challenge for hepatocellular carcinoma (HCC) treatment. Cytoskeleton remodeling has been identified as a vital process mediating intrahepatic spreading. Previously we reported HCC tumor adhesion and invasion were modulated by circular RNA (circRNA), which has emerged as important regulators of various cellular processes and has been implicated in the cancer progression. Here we uncovered a novel nuclear circRNA, circASH2, which is preferentially lost in HCC tissues, and inhibits HCC metastasis by altering tumor cytoskeleton structure. Tropomyosin 4 (TPM4), a critical binding protein of actin, turned out to be the major target of circASH2 and was post-transcriptionally suppressed. Such regulation is based on mRNA/pre-mRNA splicing and degradation process. Furthermore, liquid-liquid phase separation (LLPS) of nuclear Y-box binding protein 1 (YBX1) enhanced by circASH2 augments TPM4 transcripts decay. Together, our data have revealed a novel tumor suppressive circRNA and more importantly, uncovered a fine regulation mechanism for HCC progression. Overall design: To investigate the function of circASH2-silence in the regulation of HCC meatastasis, we established HCCLM3 and Huh7 cell lines with/without circASH2 knock-down. Then, we performed gene expression profiling analysis using data obtained from RNA-seq of 2 different HCC cells.

转移，尤其是肝内转移，是肝细胞癌（hepatocellular carcinoma, HCC）治疗的重大挑战。细胞骨架重塑已被证实是介导肝内扩散的关键过程。此前我们曾报道，肝细胞癌的肿瘤黏附与侵袭受环状RNA（circular RNA, circRNA）调控；环状RNA已被证实是多种细胞过程的重要调控因子，并与癌症进展密切相关。本研究中我们发现了一种新型细胞核环状RNA circASH2，其在肝细胞癌组织中表达显著下调，并通过改变肿瘤细胞骨架结构抑制肝细胞癌转移。肌动蛋白的关键结合蛋白原肌球蛋白4（Tropomyosin 4, TPM4）被证实是circASH2的主要靶标，其转录后表达受到抑制。该调控机制基于mRNA/前mRNA的剪接与降解过程。此外，circASH2增强的细胞核Y盒结合蛋白1（Y-box binding protein 1, YBX1）液液相分离（liquid-liquid phase separation, LLPS）可促进TPM4转录本的降解。综上，本研究揭示了一种新型抑癌环状RNA，更为重要的是，阐明了肝细胞癌进展的精细调控机制。实验设计：为探究circASH2沉默在肝细胞癌转移调控中的作用，我们构建了分别带有circASH2敲低与未敲低的HCCLM3及Huh7细胞系。随后，我们对两种不同的肝细胞癌细胞系的RNA-seq数据进行了基因表达谱分析。