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. 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.