Long non-coding RNA MALAT1 promotes steatosis in HepG2 cells by modu- lating target gene expression in presence of Exendin-4

Abstract

Background: Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease worldwide in part due to the obesity epidemic and insulin resistance. The mechanisms underlying the non-alcoholic steatosis are poorly understood and little information is available on the pathways responsible for the progressive hepatocellular damage that follows lipids accumulation. NAFLD research is incredibly important because its diagnosis requires the conduction of a biopsy. Accordingly, more recently biomarkers including noncoding RNAs have been investigated for their involvement in the disease. There are several types of noncoding RNAs associated with NAFLD. Long noncoding RNAs (LncRNAs), which include more than 200 nucleotides, have a role in a range of biological processes including cell proliferation, death, and differentiation. MALAT1 (metastasis associated with lung adenocarcinoma transcript 1) is a well-conserved lncRNA that has been linked to a variety of illnesses, including cancer. However, its role in hepatic steatosis and lipid accumulation is unknown. MALAT1 has recently attracted attention for its role in the evolution of diabetic complications, since it has been demonstrated that MALAT1 dysregulation contributes to the pathogenesis of diabetes and liver disease. Objective: The purpose of this study was to investigate MALAT1's effects on hepatic lipid accumulation and potential targets in presence of the GLP-1R agonist Exendin-4 (Ex-4), which has the potential to improve steatosis and even steatohepatitis. Methods: A steatosis cell model was established using HepG2 cells by treating them overnight with 400 mM oleic acid (OA). We determined the optimal concentration of OA needed to obtain saturating levels of triglycerides. Then starved the cells for 6h in DMEM containing 1% fatty-acid-free bovine serum albumin. Following the starvation, a 16h incubation in DMEM containing increasing concentrations of OA (0–500μM) at 37 °C was performed, and steatosis was quantified by triglycerides quantification, Bodipy staining, and quantification of the expression of perilipin genes family. The transcriptomic profiling was performed using total RNA extracted from untreated, steatotic and Ex-4-treated steatotic, cells. We validated a subset of differentially expressed LncRNAs with qRT-PCR and identified the most significantly enriched cellular functions associated with the relevant LncRNAs, among them MALAT1. Finally, CD36 was silenced, and lipogenesis genes expression were quantified using q-PCR and/or western blots. Results: Two distinct populations of stem cells were detected including pluripotent, very small (2-6 μm) embryonic-like stem cells (VSELs) that expressed nuclear OCT-4A and pluripotent transcripts (Oct- 4A, Sox2, Nanog, Stella) and slightly bigger progenitors, pancreatic stem cells (PSCs) that expressed cytoplasmic OCT-4B and PDX-1. Streptozotocin treated diabetic pancreas showed an increase in numbers of VSELs (2-6 μm, 7AAD-, LIN-CD45-SCA1+ cells) and up-regulation of transcripts specific for stem/ progenitor cells. Diabetic mice were further subjected to partial pancreatectomy to study involvement of VSELs/ PSCs during regeneration. VSELs/ PSCs were mobilized in large numbers, were observed in the lumen of blood vessels and PCNA expression suggested their proliferation. Initially, new acini assembled to regenerate the exocrine pancreas and later by Day 30, neogenesis of islets was observed in the vicinity of the blood vessels and pancreatic ducts by the differentiation of endogenous VSELs/ PSCs which may be targeted to regenerate diabetic pancreas in clinical settings. Conclusion: In conclusion, this study suggested inhibition of MALAT1 has potential for the treatment of NAFLD. Our findings imply that MALAT1 may play important role in the processes driving steatosis improvement in response to GLP-1R agonists and call for more functional research.

Keywords MALAT1, NAFLD, Steatosis, GLP-1R