Background: N6-methyladenosine (m6A) is the most abundant reversible methylation modification of eukaryotic mRNA, and it plays vital roles in tumourigenesis. This study aimed to explore the role of the m6A demethylase ALKBH5 in pancreatic cancer (PC). Methods: The expression of ALKBH5 and its clinicopathological impact were evaluated in PC cohorts. The effects of ALKBH5 on the biological characteristics of PC cells were investigated on the basis of gain-of-function and loss-of-function analyses. Subcutaneous and orthotopic models further uncovered the role of ALKBH5 in tumour growth. mRNA and m6A sequencing and assays of m6A methylated RNA immunoprecipitation-qPCR (MeRIP-qPCR) were performed to identify the targeted effect of ALKBH5 on PER1. P53-binding sites in the ALKBH5 promoter were investigated by ChIP and luciferase assays to reveal the interplay between ALKBH5 and PER1-activated ATM-CHK2-P53/CDC25C signalling. Results: ALKBH5 loss characterized the occurrence and poor clinicopathological manifestations in patients with PC. Overexpression of ALKBH5 reduced tumoural proliferative, migrative, invasive activities in vitro and ameliorated tumour growth in vivo, whereas ALKBH5 knockdown facilitated PC progression. Mechanistically, ALKBH5 posttranscriptionally activated PER1 by m6A demethylation in an m6A-YTHDF2-dependent manner. PER1 upregulation led to the reactivation of ATM-CHK2-P53/CDC25C signalling, which inhibited cell growth. P53-induced activation of ALKBH5 transcription acted as a feedback loop regulating the m6A modifications in PC. Conclusion: ALKBH5 serves as a PC suppressor by regulating the posttranscriptional activation of PER1 through m6A abolishment, which may highlight a demethylation-based approach for PC diagnosis and therapy.

Hepatic steatosis reflects the miRNA‐related pathological disorder with triglyceride accumulation and lipid peroxidation, which leads to nonalcoholic steatohepatitis, liver fibrosis/cirrhosis, and even hepatocellular carcinoma. Circular RNA (circRNA)/miRNA interaction reveals a novel layer of epigenetic regulation, yet the miRNA‐targeting circRNA remains uncertain in hepatic steatosis. Here, we uncover circRNA_0046367 to be endogenous modulator of miR‐34a that underlies hepatic steatosis. In contrast to its expression loss during the hepatocellular steatosis in vivo and in vitro, circRNA_0046367 normalization abolished miR‐34a’s inhibitory effect on peroxisome proliferator‐activated receptor α (PPARα) via blocking the miRNA/mRNA interaction with miRNA response elements (MREs). PPARα restoration led to the transcriptional activation of genes associated with lipid metabolism, including carnitine palmitoyltransferase 2 (CPT2) and acyl‐CoA binding domain containing 3 (ACBD3), and then resulted in the steatosis resolution. Hepatotoxicity of steatosis‐related lipid peroxidation, being characterized by mitochondrial dysfunction, growth arrest, and apoptosis, is resultantly prevented after the circRNA_0046367 administration. These findings indicate a circRNA_0046367/miR‐34a/PPARα regulatory system underlying hepatic steatosis. Normalized expression of circRNA_0046367 may ameliorate the lipoxidative stress on the basis of steatosis attenuation. circRNA_0046367, therefore, is suggested to be potential approach to the therapy of lipid peroxidative damage.

Activation of hepatic stellate cells (HSCs), which is regulated by multiple signal transduction pathways, is the key event in liver fibrosis. Moreover, members of these pathways are important targets for microRNAs (miRNAs). To better understand the critical pathways of HSC activation, we performed comprehensive comparative bioinformatics analysis of microarrays of quiescent and activated HSCs. Changes in miRNAs associated with HSC activation status revealed that 13 pathways were upregulated and 22 pathways were downregulated by miRNA. Furthermore, mitochondrial integrity, based on highly upregulated Bcl-2 and downregulated caspase-9, was confirmed in HSCs and fibrotic livers by immnofluorescence assay, quantitative RT-PCR, and western blot analysis. These findings provide in vitro and in vivo evidence that the mitochondrial pathway of apoptosis plays a significant role in the progression of liver fibrogenesis via HSC activation.

CircRNA_0046366 inhibits hepatocellular steatosis by normalization of PPAR signaling

Circular RNAs (circRNAs) exhibit a wide range of physiological and pathological activities. To uncover their role in hepatic steatosis, we investigated the expression profile of circRNAs in HepG2-based hepatic steatosis induced by high-fat stimulation. Differentially expressed circRNAs were subjected to validation using QPCR and functional analyses using principal component analysis, hierarchical clustering, target prediction, gene ontology (GO), and pathway annotation, respectively. Bioinformatic integration established the circRNA-miRNA-mRNA regulatory network so as to identify the mechanisms underlying circRNAs’ metabolic effect. Here we reported that hepatic steatosis was associated with a total of 357 circRNAs. Enrichment of transcription-related GOs, especially GO: 0006355, GO: 004589, GO: 0045944, GO: 0045892, and GO: 0000122, demonstrated their specific actions in transcriptional regulation. Lipin 1 (LPIN1) was recognized to mediate the transcriptional regulatory effect of circRNAs on metabolic pathways. circRNA-miRNA-mRNA network further identified the signaling cascade of circRNA_021412/miR-1972/LPIN1, which was characterized by decreased level of circRNA_021412 and miR-1972-based inhibition of LPIN1. LPIN1-induced downregulation of long chain acyl-CoA synthetases (ACSLs) expression finally resulted in the hepatosteatosis. These findings identify circRNAs to be important regulators of hepatic steatosis. Transcription-dependent modulation of metabolic pathways may underlie their effects, partially by the circRNA_021412/miR-1972/LPIN1 signaling.