Association Between the Deletion Allele of Ins/Del Polymorphism (Rs145204276) in the Promoter Region of GAS5 with the Risk of Atherosclerosis

Cell Physiol Biochem. 2018;49(4):1431-1443. doi: 10.1159/000493447. Epub 2018 Sep 11.

Abstract

Background/aims: LncRNA is a growth arrest-specific transcript 5 (GAS5) with tumor suppressor activities in some cancers, but its role in atherosclerosis is unclear.

Methods: Bioinformatics algorithm analysis was utilized to search the target of GAS5 and miR-21, followed by luciferase assay to confirm these targets. Real-time PCR and western-blot were utilized to verify the connection among GAS5, miR-21 and Programmed cell death 4 (PDCD4). MTT assay and flow cytometry analysis were performed to explore the mechanism of GAS5 in the regulation of atherosclerosis.

Results: GAS5 directly targets miR-21 and functions as a competing endogenous RNA to suppress miR-21 expression. We also observed that rs145204276 polymorphism, including INS/INS and DEL/DEL, on GAS5 promoter increased transcription activity of GAS5, but the presence of rs145204276 DEL/DEL allele significantly promoted the transcription of GAS5 promoter compared with rs145204276 INS/INS allele. PDCD4 was predicted as a direct target gene of miR-21 with a binding site on PDCD4 3'UTR. It was further confirmed by luciferase assay that miR-21 significantly reduced the luciferase activity of wild-type PDCD4 3'UTR but not that of mutant PDCD4 3'UTR. In addition, high glucose significantly inhibited the growth rate of EC genotyped as DEL/DEL or INS/ INS, and apparently promoted the apoptotic rate of either DEL/DEL or INS/INS genotype ECs. Furthermore, the effect of high glucose was stronger in the INS/INS group, while the expression of GAS5 was dramatically upregulated with the presence of GAS5 DEL/DEL, while GAS5 positively regulated PDCD4 expression via inhibiting miR-21 expression. GAS5 siRNA and miR-21 mimics significantly decreased GAS5 and PDCD4 expressions, and the inhibitory effects of GAS5 siRNA or miR-21 mimics on GAS5 and PDCD4 expressions in the INS/INS group was stronger. Moreover, GAS5 siRNA and miR-21 mimics remarkably triggered cells proliferation and suppressed cell apoptosis, and the inhibition effects of GAS5 siRNA or miR-21 mimics on either cell viability and apoptosis in the INS/INS group was stronger. In this study, we enrolled 1,306 subjects with or without atherosclerosis and found that the INS/DEL or DEL/DEL genotypes significantly decreased the risk of atherosclerosis compared with the ins/ins genotype (adjusted odds ratio: 0.74 and 0.40, respectively).

Conclusion: In summary, rs145204276 was associated with the risk of atherosclerosis by affecting the proliferation and apoptosis of endothelial cells via regulating the GAS5/miR-21/PDCD4 signaling pathway.

Keywords: Atherosclerosis; GAS5; MiR-21; PDCD4; Rs145204276.

MeSH terms

  • 3' Untranslated Regions
  • Aged
  • Antagomirs / metabolism
  • Apoptosis Regulatory Proteins / chemistry
  • Apoptosis Regulatory Proteins / genetics
  • Apoptosis Regulatory Proteins / metabolism
  • Atherosclerosis / genetics
  • Atherosclerosis / pathology*
  • Cell Proliferation / drug effects
  • Female
  • Gene Deletion
  • Glucose / pharmacology
  • Human Umbilical Vein Endothelial Cells
  • Humans
  • Male
  • MicroRNAs / antagonists & inhibitors
  • MicroRNAs / genetics
  • MicroRNAs / metabolism
  • Middle Aged
  • Polymorphism, Genetic
  • Promoter Regions, Genetic
  • RNA Interference
  • RNA, Long Noncoding / antagonists & inhibitors
  • RNA, Long Noncoding / genetics*
  • RNA, Long Noncoding / metabolism
  • RNA, Small Interfering / metabolism
  • RNA-Binding Proteins / chemistry
  • RNA-Binding Proteins / genetics
  • RNA-Binding Proteins / metabolism
  • Risk

Substances

  • 3' Untranslated Regions
  • Antagomirs
  • Apoptosis Regulatory Proteins
  • GAS5 long non-coding RNA, human
  • MIRN21 microRNA, human
  • MicroRNAs
  • PDCD4 protein, human
  • RNA, Long Noncoding
  • RNA, Small Interfering
  • RNA-Binding Proteins
  • Glucose