Dynamic gene expression in response to lifestyle and environmental changes is a hallmark of diabetic pathophysiology. Although altered DNA methylation is implicated during diabetes, upstream regulatory events remain poorly understood. Expression and activity of H19, DNMTs, Hk2, and Eno3 were evaluated in diabetic db/db mouse models and Hepa 1–6, HepG2 cells using qRT-PCR, Western blotting and enzyme activity assays. Functional studies were performed through siRNA knockdown and overexpression in cells. MeDIP and ChIP were performed to assess promoter methylation and DNMT3B occupancy. Glycolytic flux was measured using Seahorse analyser. Expression of H19, DNMTs, Hk2, and Eno3 was assessed in scramble/H19 siRNA-injected mice and chow/HFD-fed mice. H19 knockdown induced DNMT3B expression, which was accompanied by repressed expression of key glycolytic genes, Hk2 and Eno3. DNMT3B knockdown was sufficient to upregulate their expression, which also prevented H19 siRNA-mediated inhibition of Hk2 and Eno3 levels. Overexpression of DNMT3B alone was adequate to decrease the transcript levels of these two glycolytic genes in hepatic cells. Promoters of both Hk2 and Eno3 possess major CpG methylation marks, and H19 inhibition significantly enhanced CpG methylation and DNMT3B occupancy on these genes, which possibly drive their transcriptional repression during H19 inhibition. In vivo inhibition of H19 significantly increased hepatic DNMT3B expression and downregulated Hk2 and Eno3 levels. This study reveals the novel regulatory role of a lncRNA-epigenetic axis in hepatic metabolism and establishes DNMT3B as a downstream effector of H19, offering a potential therapeutic strategy for intervening in aberrant hepatic metabolism during diabetes.
Rizvi et al. (Thu,) studied this question.