m(6)A-modified CTC-297N7.9 inhibits hepatocellular carcinoma metastasis via epigenetically downregulating CCL2 and CD47.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of most common malignancies with poor prognosis, largely due to its high propensity for metastasis and recurrence. As the most common internal RNA modification, N(6)-methyladenosine (m(6)A) plays critical and diverse roles in HCC progression. However, the mechanisms by which m(6)A regulates HCC metastasis remain incompletely understood. Here, we aimed to identify key m(6)A modification events during HCC metastasis. METHODS: The expression of CTC-297N7.9. was measured by qPCR. m(6)A modification level of CTC-297N7.9 was measured by methylated RNA immunoprecipitation (MeRIP) and single-base elongation- and ligation-based qPCR amplification method (SELECT). The roles of m(6)A-modified CTC-297N7.9 in HCC were investigated by in vitro cell viability, proliferation, migration and phagocytosis assays, and in vivo liver metastasis and lung metastasis assays. The underlying mechanisms of m(6)A-modified CTC-297N7.9 were dissected by chromatin isolation by RNA purification (ChIRP), assay for transposase accessible chromatin (ATAC) and cleavage under target & tagmentation (CUT&Tag) assays. RESULTS: The m(6)A modification level of CTC-297N7.9 is decreased in HCC tissues and correlated with microvascular invasion and poor prognosis. CTC-297N7.9 suppresses HCC metastasis in an m(6)A-dependent manner. m(6)A-modified CTC-297N7.9 attenuates tumor-associated macrophages (TAMs) infiltration and M2 polarization through downregulating CCL2 expression and secretion. Additionally, m(6)A-modified CTC-297N7.9 promotes phagocytosis of HCC cells by macrophages through downregulating the phagocytosis checkpoint CD47. Mechanistic investigations revealed that m(6)A-modified CTC-297N7.9 binds the m(6)A reader YTHDC1, which further binds and recruits the histone H3K9me3 methyltransferase SETDB1 and H3K27me3 methyltransferase EZH2 to the promoters of CCL2 and CD47, leading to the upregulation of H3K9me3 and H3K27me3 modifications at CCL2 and CD47 promoters, and transcriptional silencing of CCL2 and CD47. CONCLUSIONS: Our study demonstrates that m(6)A-modified CTC-297N7.9 acts as a metastasis suppressor in HCC, and highlights its potential as a prognostic biomarker and therapeutic target for HCC.