The PLIN4 variant rs8887 modulates obesity related phenotypes in humans through creation of a novel miR-522 seed site

Abstract

PLIN4 is a member of the PAT family of lipid storage droplet (LSD) proteins. Associations between seven single nucleotide polymorphisms (SNPs) at human PLIN4 with obesity related phenotypes were investigated using meta-analysis followed by a determination if these phenotypes are modulated by interactions between PLIN4 SNPs and dietary PUFA. Samples consisted of subjects from two populations of European ancestry. We demonstrated association of rs8887 with anthropometrics. Meta-analysis demonstrated significant interactions between the rs8887 minor allele with PUFA n3 modulating anthropometrics. rs884164 showed interaction with both n3 and n6 PUFA modulating anthropometric and lipid phenotypes. In silico analysis of the PLIN4 3'UTR sequence surrounding the rs8887 minor A allele predicted a seed site for the human microRNA-522 (miR-522), suggesting a functional mechanism. Our data showed that a PLIN4 3'UTR luciferase reporter carrying the A allele of rs8887 was reduced in response to miR-522 mimics compared to the G allele. These results suggest variation at the PLIN4 locus, and its interaction with PUFA as a modulator of obesity related phenotypes, acts in part through creation of a miR-522 regulatory site.

Figure 1. LD Plot of PLIN4 SNPs in FOS & GOLDN.

LD plots were generated in the Haploview program using unrelated individuals from the corresponding studies. The r² LD estimate was used for both populations and is reported in the figure above.

Figure 2. The rs8887 minor A allele creates a novel miR-522 MRE in the PLIN4 3′UTR.

Diagram of the miR-522:PLIN4 3′UTR sequences with the A or G allele. The miR-522 seed site is highlighted in gray, and the rs8887 variants are in bold.

Figure 3. The PLIN4 3′UTR with the A allele creates a miR-522 MRE.

A) Relative miR-522 expression across indicated cell types, hek293T (293T), Cos7 (c7), hepG2 (hG2), pre-adpocytes (pre-Ad) and adipocytes(Ad). B) Luciferase expression of pmiR-LucPLIN4-G or A constructs with miR-522 (522) or control mimic (CM). Data are expressed as relative luciferase activity to control samples. C) Luciferase expression of pmiR-LucPLIN4-A constructs with increasing concentration of miR-522 compared to control mimic. All data represent experiments performed in triplicate. Statistical Analysis: P values for the difference between luciferase activity obtained for LucPLIN4-A in the presence of mir-522 or control mimic (P = .0169) or LucPLIN4-G in the presence of mir-522 or control mimic (P = .0584) were determined using the student's paired t-test.

Figure 4. Evolutionary history of the PLIN4 3′UTR.

A) The evolutionary history of PLIN4 was inferred using the Maximum Parsimony method. The bootstrap consensus tree inferred from 10000 replicates is taken to represent the evolutionary history of the taxa analyzed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test are shown next to the branches. The MP tree was obtained using the Close-Neighbor-Interchange algorithm with search level 3 in which the initial trees were obtained with the random addition of sequences (100 replicates). B) Clustal W was used to align the PLIN4 3′UTR sequences from H. sapiens, H. neanderthalensis, P. troglodytes, G. gorilla, P. pygmaeus, M. murinis, C. familiaris, B. Taurus, S. scrofa, and M. musculus. The last 100 bases of the alignment are show here, with the position harboring the rs8887 SNP outlined.