MicroRNA-137-3p Improves Nonalcoholic Fatty Liver Disease through Activating AMPKα

Figure 6

miR-137-3p activates AMPKα through downregulating PDE4D. (a) Graphic representation of the miR-137-3p binding motifs within the 3-UTR of PDE4D. (b) HEK293T cells were cotransfected the WT or TRU 3-UTR of PDE4D with miR-137-3p agomir (50 nmol/L) for 48 h, and then, the cells were collected and the luciferase activity was determined using the Dual-Luciferase Reporter Assay System (). (c–e) Mice were fed with a HFD for 24 weeks to establish NAFLD and were also intraperitoneally injected with the miR-137-3p agomir, antagomir, or respective controls (100 mg/kg weekly) at the last 6 consecutive weeks. Relative PDE4D mRNA and protein levels in HFD mice treated with miR-137-3p agomir, antagomir, or their respective controls (). (f, g) The cAMP level and PKA activity in HFD mice treated with miR-137-3p agomir, antagomir, or their respective controls (). (h) Relative AC activity (). (i) Primary hepatocytes were transfected with siPDE4D or siRNA for 24 h, and relative PDE4D mRNA level was detected (). (j) Primary hepatocytes were preincubated with miR-137-3p antagomir (50 nmol/L) for 24 h and then stimulated with PO for 24 h. To knock down PDE4D, cells were pretransfected with siPDE4D or siRNA for 24 h before miR-137-3p antagomir treatment. AMPKα phosphorylation in miR-137-3p antagomir-treated primary hepatocytes with or without PDE4D silence (). (k) Mice were fed with a HFD for 24 weeks to establish NAFLD and were also intraperitoneally injected with the miR-137-3p agomir, antagomir, or respective controls (100 mg/kg weekly) at the last 6 consecutive weeks. To inhibit PDE4D, mice were intraperitoneally injected with 0.5 mg/kg rolipram or an equal volume of vehicle every other day for 8 consecutive weeks before the mice were sacrificed. Serum ALT and AST levels in miR-137-3p antagomir-treated HFD mice with or without PDE4D inhibition (). Data were expressed as the , and was considered significant. n.s. indicated no significance.