Abstract
Since proprotein convertase subtilisin kexin 9 (PCSK9) discovery, a gene involved in LDL metabolism regulation and cardiovascular diseases (CVD), many therapeutic strategies have been introduced for direct targeting of PCSK9. The main goal of these strategies has been to reduce PCSK9 protein level either by application of antibodies or inhibition of its production. In this study, we have tried to discover microRNAs (miRNAs) which can target, and hence regulate, PCSK9 expression. Using bioinformatics tools, we selected three microRNAs with binding sites on 3′-UTR of PCSK9. The expression level of these miRNAs was examined in three different cell lines using real-time RT-PCR. We observed a reciprocal expression pattern between expression level of miR-191, miR-222, and miR-224 with that of PCSK9. Accordingly, the expression levels were highest in Huh7 cells which expressed the lowest level of PCSK9, compared to HepG2 and A549 cell lines. PCSK9 mRNA level also showed a significant decline in HepG2 cells transfected with the vectors overexpressing the aforementioned miRNAs. Furthermore, the miRNAs target sites were cloned in psiCHECK-2 vector, and a direct interaction of the miRNAs and the PCSK9 3′-UTR putative target sites was investigated by means of luciferase assay. Our findings revealed that miR-191, miR-222, and miR-224 can directly interact with PCSK9 3′-UTR and regulate its expression. In conclusion, our data introduces a role for miRNAs to regulate PCSK9 expression.
Highlights
An estimated 17.5 million people died from cardiovascular diseases (CVDs) in 2012, representing ∼31% of all global deaths
We employed various target prediction programs to predict miRNAs targeting 3′-UTR of the proprotein convertase subtilisin kexin 9 (PCSK9) transcript. These programs predicted several miRNAs, and according to common predicted data miR-191, miR222, and miR-224 were selected for further investigation. miR-191 and miR-222 have one target site, whereas miR-224 possess two target sites on PCSK9 3′-UTR (Figure 1)
We examined miR-191, miR-222, miR-224, and PCSK9 expression levels in three different cell lines including HepG2, Huh7, and A549
Summary
An estimated 17.5 million people died from cardiovascular diseases (CVDs) in 2012, representing ∼31% of all global deaths. Of these death numbers, roughly 7.4 million were due to coronary heart disease and 6.7 million were due to stroke (WHO, 2016). LDL particles are removed from the circulation mainly by hepatic uptake via the LDL receptor (LDLR). PCSK9 Regulation by miR-191, miR-222, and miR-224 to the LDLR and the LDL/LDLR complex is internalized via clathrin-coated vesicles. LDL is separated from its receptor in the endosomes and the LDLR is recycled back to cell surface, while LDL is degraded (Kwiterovich, 2000; Goldstein and Brown, 2009)
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