METTL3 and METTL14-mediated N ⁶-methyladenosine modification of SREBF2-AS1 facilitates hepatocellular carcinoma progression and sorafenib resistance through DNA demethylation of SREBF2

Abstract

As the most prevalent epitranscriptomic modification, N ⁶-methyladenosine (m ⁶A) shows important roles in a variety of diseases through regulating the processing, stability and translation of target RNAs. However, the potential contributions of m ⁶A to RNA functions are unclear. Here, we identified a functional and prognosis-related m ⁶A-modified RNA SREBF2-AS1 in hepatocellular carcinoma (HCC). The expression of SREBF2-AS1 and SREBF2 in HCC tissues and cells was measured by RT-qPCR. m ⁶A modification level of SREBF2-AS1 was measured by methylated RNA immunoprecipitation assay. The roles of SREBF2-AS1 in HCC progression and sorafenib resistance were investigated by proliferation, apoptosis, migration, and cell viability assays. The regulatory mechanisms of SREBF2-AS1 on SREBF2 were investigated by Chromatin isolation by RNA purification, RNA immunoprecipitation, CUT&RUN, and bisulfite DNA sequencing assays. Our findings showed that the expression of SREBF2-AS1 was increased in HCC tissues and cells, and positively correlated with poor survival of HCC patients. m ⁶A modification level of SREBF2-AS1 was also increased in HCC and positively correlated with poor prognosis of HCC patients. METTL3 and METTL14-induced m ⁶A modification upregulated SREBF2-AS1 expression through increasing SREBF2-AS1 transcript stability. Functional assays showed that only m ⁶A-modified, but not non-modified SREBF2-AS1 promoted HCC progression and sorafenib resistance. Mechanistic investigations revealed that m ⁶A-modified SREBF2-AS1 bound and recruited m ⁶A reader FXR1 and DNA 5-methylcytosine dioxygenase TET1 to SREBF2 promoter, leading to DNA demethylation at SREBF2 promoter and the upregulation of SREBF2 transcription. Functional rescue assays showed that SREBF2 was the critical mediator of the oncogenic roles of SREBF2-AS1 in HCC. Together, this study showed that m ⁶A-modified SREBF2-AS1 exerted oncogenic roles in HCC through inducing DNA demethylation and transcriptional activation of SREBF2, and suggested m ⁶A-modified SREBF2-AS1 as a prognostic biomarker and therapeutic target for HCC.