A single nucleotide polymorphism located in microRNA-499a causes loss of function resulting in increased expression of osbpl1a and reduced serum HDL level

: Atherosclerosis is the main pathological process that induces CVD (cardiovascular diseases), and the objective of our study was explore whether miR‑499a rs3746444 polymorphism was associated with the HDL level, one of the risk factors of atherosclerosis. Online public miRNA database was utilized to predict the miR‑499a target, and luciferase assay was conducted to confirm that miR‑499a targeted osbpl1a, then western blot analysis and real-time PCR were performed to verify miRNA-mRNA regulatory relationship between miR‑499a and osbpl1a. Based on results of bioinformatics algorithms, osbpl1a was predicted as a candidate target gene of miR‑499a, luciferase reporter was generated, and it was found that the luciferase activity of cells was substantial downregulated following co-transfection with wild osbpl1a 3'UTR and miR‑499a compared to that in scramble control, while the inhibitory effect of miR‑499a was abolished after transfection of mutant osbpl1a 3'UTR. Then, miRNA-mRNA regulatory relationship between miR‑499a and osbpl1a was detected, a concentration-dependent effect of miR‑499a on the miR‑499a expression was observed, and both osbpl1a mRNA and protein levels of cells transfected with miR‑449a (30 and 60 nM) or osbpl1a siRNA were markedly reduced, while notably improved subsequent to transfect with anti‑miR‑449a (30 and 60 nM) in comparison with NC groups, moreover, the inhibitory effect among 30 or 60 nM miR‑499a, osbpl1a siRNA was similar, the improved effect of 30 or 60 nM anti‑miR‑499a showed no significant change. The influence of rs3746444 A allele on expression level of miR‑499a represented a recessive pattern in high-grade group with a higher level of miR‑499a in AA group, and HDL level in AA group was significantly reduced related to those in AG and GG groups. This study validated that rs3746444 polymorphism influenced the expression of miR‑499a, its target gene, osbpl1a, and thereby associated with the HDL level, which makes it a potential factor involved in the mechanism of atherosclerosis.