circRNA_0046366 inhibits hepatocellular steatosis by normalization of PPAR signaling

AIM To investigate micro(mi)R-34 a-antagonizing circular(circ)RNA that underlies hepatocellular steatosis.METHODS The effect of circ RNA on mi R-34 a was recognized by the mi RNA response element(MRE), and validated by the dual-luciferase reporter assay. Its association with hepatocellular steatosis was investigated in Hep G2-based hepatocellular steatosis induced by free fatty acids(FFAs; 2:1 oleate:palmitate) stimulation. After normalization of the steatosis-related circRNA by expression vector, analysis of mi R-34 a activity,peroxisome proliferator-activated receptor(PPAR)α level, and expression of downstream genes were carried out so as to reveal its impact on the mi R-34 a/PPARα regulatory system. Both triglyceride(TG) assessment and cytopathological manifestations uncovered the role of circRNA in miR-34 a-dependent hepatosteatogenesis.RESULTS Bioinformatic and functional analysis verified circRNA_0046366 to antagonize the activity of mi R-34 a via MRE-based complementation. In contrast to its lowered level during FFA-induced hepatocellular steatosis, circ RNA_0046366 up-regulation abolished the mi R-34 a-dependent inhibition of PPARα that played a critical role in metabolic signaling pathways. PPARα restoration exerted transcriptional improvement to multiple genes responsible for lipid metabolism. TGspecific lipolytic genes [carnitine palmitoyltransferase 1 A(CPT1 A) and solute-carrier family 27 A(SLC27 A)] among these showed significant increase in their expression levels. The circ RNA_0046366-related rebalancing of lipid homeostasis led to dramatic reduction of TG content, and resulted in the ameliorated phenotype of hepatocellular steatosis.CONCLUSION Dysregulation of circ RNA_0046366/mi R-34 a/PPARα signaling may be a novel epigenetic mechanism underlying hepatocellular steatosis. circ RNA_0046366 serves as a potential target for the treatment of hepatic steatosis.

KMT2D deficiency enhances the anti-cancer activity of L48H37 in pancreatic ductal adenocarcinoma

BACKGROUND Novel therapeutic strategies are urgently needed for patients with a delayed diagnosis of pancreatic ductal adenocarcinoma(PDAC)in order to improve their chances of survival.Recent studies have shown potent anti-neoplastic effects of curcumin and its analogues.In addition,the role of histone methyltransferases on cancer therapeutics has also been elucidated.However,the relationship between these two factors in the treatment of pancreatic cancer remains unknown.Our working hypothesis was that L48H37,a novel curcumin analog,has better efficacy in pancreatic cancer cell growth inhibition in the absence of histonelysine N-methyltransferase 2D(KMT2D).AIM To determine the anti-cancer effects of L48H37 in PDAC,and the role of KMT2D on its therapeutic efficacy.METHODS The viability and proliferation of primary(PANC-1 and MIA PaCa-2)and metastatic(SW1990 and ASPC-1)PDAC cell lines treated with L48H37 was determined by CCK8 and colony formation assay.Apoptosis,mitochondrial membrane potential(MMP),reactive oxygen species(ROS)levels,and cell cycle profile were determined by staining the cells with Annexin-V/7-AAD,JC-1,DCFH-DA,and PI respectively,as well as flow cytometric acquisition.In vitro migration was assessed by the wound healing assay.The protein and mRNA levels of relevant factors were analyzed using Western blotting,immunofluorescence and real time-quantitative PCR.The in situ expression of KMT2D in both human PDAC and paired adjacent normal tissues was determined by immunohistochemistry.In vivo tumor xenografts were established by injecting nude mice with PDAC cells.Bioinformatics analyses were also conducted using gene expression databases and TCGA.RESULTS L48H37 inhibited the proliferation and induced apoptosis in SW1990 and ASPC-1 cells in a dose-and time-dependent manner,while also reducing MMP,increasing ROS levels,arresting cell cycle at the G2/M stages and activating the endoplasmic reticulum(ER)stress-associated protein kinase RNA-like endoplasmic reticulum kinase/eukaryotic initiation factor 2α/activating transcription factor 4(ATF4)/CHOP signaling pathway.Knocking down ATF4 significantly upregulated KMT2D in PDAC cells,and also decreased L48H37-induced apoptosis.Furthermore,silencing KMT2D in L48H37-treated cells significantly augmented apoptosis and the ER stress pathway,indicating that KMT2D depletion is essential for the anti-neoplastic effects of L48H37.Administering L48H37 to mice bearing tumors derived from control or KMT2Dknockdown PDAC cells significantly decreased the tumor burden.We also identified several differentially expressed genes in PDAC cell lines expressing very low levels of KMT2D that were functionally categorized into the extrinsic apoptotic signaling pathway.The KMT2D high-and low-expressing PDAC patients from the TCGA database showed similar survival rates,but higher KMT2D expression was associated with poor tumor grade in clinical and pathological analyses.CONCLUSION L48H37 exerts a potent anti-cancer effect in PDAC,which is augmented by KMT2D deficiency.