Advertisement

Down-Regulation of SIRT1 Expression by mir-23b Contributes to Lipid Accumulation in HepG2 Cells

  • Mohammad Borji
  • Mitra NourbakhshEmail author
  • Sayed Mohammad ShafieeEmail author
  • Ali Akbar Owji
  • Zohreh Abdolvahabi
  • Zahra Hesari
  • Davod Ilbeigi
  • Parvaneh Seiri
  • Zeynab Yousefi
Original Article
  • 44 Downloads

Abstract

Non-alcoholic fatty liver disease is one of the main causes of chronic liver disease and therefore is currently considered a major public health problem. Sirtuin 1 (SIRT1) is an NAD-dependent deacetylase enzyme that contributes in the regulation of metabolic processes and protects against lipid accumulation in hepatocytes. Its expression is potentially regulated by microRNAs which attach to the 3′ untranslated region (3′-UTR) of their target mRNA. HepG2 cells were incubated by glucose to induce lipid accumulation and were subsequently transfected with mir-23b mimic and inhibitor. Real-time PCR was used for measuring the expression of mir-23b and SIRT1 mRNA. Cell survival assay and intracellular triglyceride measurement were performed using colorimetric methods. Determination of SIRT1 protein level and activity were done by western blot and fluorometric analysis, respectively. The interaction of miR-23b with 3′-UTR of SIRT1 mRNA was confirmed by dual luciferase. miR-23b mimic inhibited gene and protein expression of SIRT1, while the inhibitor of miR-23b significantly elevated the expression levels of SIRT1 mRNA and protein. The results showed that the 3′-UTR of SIRT1 mRNA is a direct target for miR-23b. The intracellular triglyceride level was increased following the inhibition of SIRT1 in transfected HepG2 cell by miR-23b mimic. Cell viability was decreased in response to miR-23b upregulation compared to control cells. miR-23b reduces the expression and activity of SIRT1 and therefore may be a causative factor in the enhancement of lipid accumulation in HepG2 cells.

Keywords

miR-23b-3p Sirtuins 1 Non-alcoholic fatty liver disease Luciferase assay 

Abbreviations

NAFLD

non-alcoholic fatty liver disease

NAD

nicotinamide adenine dinucleotide

SIRT1

silent information regulation homology 1

UTR

untranslated region

MRE

microRNA response elements

Notes

Acknowledgements

This research has been extracted from the Ph.D. thesis of Mohammad Borji and was supported by Grant Number 94-01-01-10596 from Vice-chancellor for Research Affairs of Shiraz University of Medical Sciences, Shiraz, Iran.

Compliance with Ethical standards

Conflict of Interest

The authors declare that they have no conflict of interest.

Ethical Approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Informed Consent

This article does not contain any studies with human participants and there is no need for informed consent.

Supplementary material

10528_2019_9905_MOESM1_ESM.docx (251 kb)
Supplementary file1 (DOCX 250 kb)

References

  1. Adlakha Y et al (2013) Pro-apoptotic miRNA-128-2 modulates ABCA1, ABCG1 and RXRα expression and cholesterol homeostasis. Cell Death Dis 4(8):e780CrossRefGoogle Scholar
  2. Albhaisi S, Sanyal A (2018) Recent advances in understanding and managing non-alcoholic fatty liver disease. F1000Research, vol. 7Google Scholar
  3. Alisi A et al (2011) Mirnome analysis reveals novel molecular determinants in the pathogenesis of diet-induced nonalcoholic fatty liver disease. Lab Invest 91(2):283CrossRefGoogle Scholar
  4. Arner P, Kulyté A (2015) MicroRNA regulatory networks in human adipose tissue and obesity. Nat Rev Endocrinol 11(5):276CrossRefGoogle Scholar
  5. Bala S, Marcos M, Szabo G (2009) Emerging role of microRNAs in liver diseases. World J Gastroenterol 15(45):5633CrossRefGoogle Scholar
  6. Chalasani N et al (2018) The diagnosis and management of nonalcoholic fatty liver disease: practice guidance from the American Association for the Study of Liver Diseases. Hepatology 67(1):328–357CrossRefGoogle Scholar
  7. Cohen HY et al (2004) Calorie restriction promotes mammalian cell survival by inducing the SIRT1 deacetylase. Science 305(5682):390–392CrossRefGoogle Scholar
  8. Ding R-B, Bao J, Deng C-X (2017) Emerging roles of SIRT1 in fatty liver diseases. Int J Biol Sci 13(7):852CrossRefGoogle Scholar
  9. Esau C et al (2006) miR-122 regulation of lipid metabolism revealed by in vivo antisense targeting. Cell Metab 3(2):87–98CrossRefGoogle Scholar
  10. Feng YY et al (2014) Aberrant hepatic microRNA expression in nonalcoholic fatty liver disease. Cell Physiol Biochem 34(6):1983–1997CrossRefGoogle Scholar
  11. Feng T et al (2018) SIRT1 activators and their effects on atherosclerosis progression. Cardiol Res Cardiovasc Med.  https://doi.org/10.29011/CRCM-138.000038 Google Scholar
  12. Fernández-Hernando C (2013) Emerging role of microRNAs in the regulation of lipid metabolism. Hepatology 57(2):432–434CrossRefGoogle Scholar
  13. Ferrante SC et al (2015) Adipocyte-derived exosomal miRNAs: a novel mechanism for obesity-related disease. Pediatr Res 77(3):447–454CrossRefGoogle Scholar
  14. Frescas D, Valenti L, Accili D (2005) Nuclear trapping of the forkhead transcription factor FoxO1 via sirt-dependent deacetylation promotes expression of glucogenetic genes. J Biol Chem 280(21):20589–20595CrossRefGoogle Scholar
  15. Grossi I et al (2017) Clinical and biological significance of miR-23b and miR-193a in human hepatocellular carcinoma. Oncotarget 8(4):6955–6969CrossRefGoogle Scholar
  16. Guarente L, Picard F (2005) Calorie restriction—the SIR2 connection. Cell 120(4):473–482CrossRefGoogle Scholar
  17. He J et al (2016) Analysis of miRNAs and their target genes associated with lipid metabolism in duck liver. Sci Rep 6:27418CrossRefGoogle Scholar
  18. Hou X et al (2008) SIRT1 regulates hepatocyte lipid metabolism through activating AMP-activated protein kinase. J Biol Chem 283(29):20015–20026CrossRefGoogle Scholar
  19. Kane AE, Sinclair DA (2018) Sirtuins and NAD+ in the development and treatment of metabolic and cardiovascular diseases. Circ Res 123(7):868–885CrossRefGoogle Scholar
  20. Kemper JK et al (2009) FXR acetylation is normally dynamically regulated by p300 and SIRT1 but constitutively elevated in metabolic disease states. Cell Metab 10(5):392–404CrossRefGoogle Scholar
  21. Lazo M et al (2013) Prevalence of nonalcoholic fatty liver disease in the United States: the Third National Health and Nutrition Examination Survey, 1988–1994. Am J Epidemiol 178(1):38–45CrossRefGoogle Scholar
  22. Li Y et al (2011) Hepatic overexpression of SIRT1 in mice attenuates endoplasmic reticulum stress and insulin resistance in the liver. FASEB J 25(5):1664–1679CrossRefGoogle Scholar
  23. Li X (2013) SIRT1 and energy metabolism. Acta Biochim Biophys Sin 45(1):51–60CrossRefGoogle Scholar
  24. Liu Y et al (2008) A fasting inducible switch modulates gluconeogenesis via activator/coactivator exchange. Nature 456:269CrossRefGoogle Scholar
  25. Lu T-M et al (2014) Downregulation of Sirt1 as aging change in advanced heart failure. J Biomed Sci 21(1):57–57CrossRefGoogle Scholar
  26. Matteoni CA et al (1999) Nonalcoholic fatty liver disease: a spectrum of clinical and pathological severity. Gastroenterology 116(6):1413–1419CrossRefGoogle Scholar
  27. Najafi-Shoushtari SH et al (2010) MicroRNA-33 and the SREBP host genes cooperate to control cholesterol homeostasis. Science 328(5985):1566–1569CrossRefGoogle Scholar
  28. Nassir F, Ibdah JA (2016) Sirtuins and nonalcoholic fatty liver disease. World J Gastroenterol 22(46):10084CrossRefGoogle Scholar
  29. Purushotham A et al (2009) Hepatocyte-specific deletion of SIRT1 alters fatty acid metabolism and results in hepatic steatosis and inflammation. Cell Metab 9(4):327–338CrossRefGoogle Scholar
  30. Reznick RM et al (2007) Aging-associated reductions in AMP-activated protein kinase activity and mitochondrial biogenesis. Cell Metab 5(2):151–156CrossRefGoogle Scholar
  31. Rogler CE et al (2017) Knockdown of miR-23, miR-27, and miR-24 alters fetal liver development and blocks fibrosis in mice. Gene Expr 17(2):99–114CrossRefGoogle Scholar
  32. Satapathy SK, Sanyal AJ (2015) Epidemiology and natural history of nonalcoholic fatty liver disease. In: Seminars in liver disease. Thieme Medical Publishers, StuttgartGoogle Scholar
  33. Schueller F et al (2018) The role of miRNAs in the pathophysiology of liver diseases and toxicity. Int J Mol Sci 19(1):261CrossRefGoogle Scholar
  34. Stefanowicz M et al (2018) Adipose tissue, but not skeletal muscle, sirtuin 1 expression is decreased in obesity and related to insulin sensitivity. Endocrine 60(2):263–271CrossRefGoogle Scholar
  35. Sun Y et al (2007) Downregulation of sirt1 by antisense oligonucleotides induces apoptosis and enhances radiation sensitization in A549 lung cancer cells. Lung Cancer 58(1):21–29CrossRefGoogle Scholar
  36. Sun YN et al (2015) Inhibition of microRNA-9-3p reduces lipid accumulation in HepG2 cells by targeting the expression of sirtuin type 1. Mol Med Rep 12(5):7742–7748CrossRefGoogle Scholar
  37. Vickers KC et al (2013) The complexity of microRNA function and the role of isomiRs in lipid homeostasis. J Lipid Res 54(5):1182-1191CrossRefGoogle Scholar
  38. Vickers MH (2014) Early life nutrition, epigenetics and programming of later life disease. Nutrients 6(6):2165–2178CrossRefGoogle Scholar
  39. Xiong W et al (2017) Circulatory microRNA 23a and microRNA 23b and polycystic ovary syndrome (PCOS): the effects of body mass index and sex hormones in an Eastern Han Chinese population. J Ovarian Res 10:10CrossRefGoogle Scholar
  40. Yang Z, Cappello T, Wang L (2015) Emerging role of microRNAs in lipid metabolism. Acta Pharm Sin. B 5(2):145–150CrossRefGoogle Scholar
  41. Zang M et al (2006) Polyphenols stimulate AMP-activated protein kinase, lower lipids, and inhibit accelerated atherosclerosis in diabetic LDL receptor–deficient mice. Diabetes 55(8):2180–2191CrossRefGoogle Scholar
  42. Zhang P et al (2017) Beraprost sodium, a prostacyclin analogue, reduces fructose-induced hepatocellular steatosis in mice and in vitro via the microRNA-200a and SIRT1 signaling pathway. Metab Clin Exp 73:9–21CrossRefGoogle Scholar
  43. Zhang Y et al (2017) Emerging roles for microRNAs in diabetic microvascular disease: novel targets for therapy. Endocrinol Rev 38(2):145–168CrossRefGoogle Scholar
  44. Zhao S et al (2016) miR-23b-3p induces the cellular metabolic memory of high glucose in diabetic retinopathy through a SIRT1-dependent signalling pathway. Diabetologia 59(3):644–654CrossRefGoogle Scholar
  45. Zullo A et al (2018) Sirtuins as mediator of the anti-ageing effects of calorie restriction in skeletal and cardiac muscle. Int J Mol Sci 19(4):928CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Biochemistry, School of MedicineShiraz University of Medical SciencesShirazIran
  2. 2.Department of Biochemistry, School of MedicineIran University of Medical SciencesTehranIran
  3. 3.Laboratory Sciences Research CenterGolestan University of Medical SciencesGorganIran
  4. 4.Neuroscience Researcher CenterTorbat Heydarieh University of Medical SciencesTorbat HeydariehIran
  5. 5.Department of Biochemistry, School of MedicineMashhad University of Medical SciencesMashhadIran

Personalised recommendations