A key major finding of this study is that HDL transports endogenous miRNAs. Although the classic view of HDL is that it is a delivery vehicle for the return of excess cellular cholesterol to the liver for excretion, a wide variety of biological functions, outside of reverse cholesterol transport, have recently been ascribed to HDL32, 33
. Furthermore, it is now recognized that HDL is much more complex in terms of differential lipids and proteins that it transports34
. This study, therefore, has revealed yet another constituent transported by HDL, namely miRNAs, and a possible new mechanism whereby some of the biological effects of HDL could be mediated.
The exact process of how HDL is loaded with miRNAs and what proteins if any facilitate this association are not known; however, small RNAs (25nt) have previously been shown to complex with zwitterionic liposomes, specifically PC35
. Previous biophysical studies suggest that HDL could simply bind to extracellular plasma miRNAs through divalent cation bridging9, 12, 14, 15, 35
. In these studies, interactions between DNA molecules and zwitterionic PCs resulted in conformational shifts in PC head groups and subsequent altered orientations of aliphatic chains, thus facilitating the incorporation of the DNA molecules into the protected space12
. In the case of HDL this could lead to a tighter association with miRNAs, and possibly shield bound miRNAs from external RNases8
. Furthermore, Langmuir monolayer model systems have shown that nucleic acids easily penetrate lipid monolayers at low surface pressures12
. Due to its high radius of curvature, HDL has a much lower surface tension than other lipoproteins, which is known to facilitate its binding to several of its other constituents36
. Results presented here suggest that nSMase2 and most likely the ceramide pathway repress miRNA export to HDL. Overexpression of nSMase2 and activation of the ceramide pathway have previously been shown to induce exosome release from cells and trigger cellular export of miRNAs25, 37
. These results suggest that the export of specific miRNAs through the exosomal pathway and the HDL pathway may be distinct mechanisms, possibly opposing, although both pathways are likely regulated by nSMase2 activity and ceramide synthesis.
One implication of this study is that HDL-miRNAs could potentially serve as novel diagnostic markers in much the same way that exosome miRNAs have been used38–42
. HDL could simply be a depot or carrier for circulating miRNAs and their presence on HDL may not necessarily relate to the function of HDL in atherosclerosis or lipid metabolism. If so, miRNAs on HDL could perhaps be used diagnostically for a wide variety of diseases besides atherosclerosis. Nonetheless, many of the genes significantly altered in response to atherosclerotic HDL-miRNA delivery have a role lipid metabolism, inflammation, and atherosclerosis; including NDST143
, NR1D244, 45
, and FLT146
Recently, apoA-I containing liposomes have been used to deliver siRNAs to the liver, with functional targeting and gene expression changes13, 47
; however, the exact mechanism of siRNA transfer from apoA-I liposomes to hepatocytes was not determined. Furthermore, HDL loaded with cholesterol-conjugated siRNAs was shown to be 8-15X more effective in delivering siRNA-mediated silencing than cholesterol-conjugated siRNAs alone29
. Similar to our observations in this study for HDL-miRNAs, the delivery of HDL-associated lipophilic-conjugated siRNAs was also determined to be SR-BI-dependent29
. SR-BI-mediated transfer of HDL-miRNAs would likely prevent the delivery of miRNAs into the lysosomal pathway and instead divert it into the cytoplasm where it would be expected to be more stable with increased functional integrity and potential to alter gene expression. Overall these results suggest that artificial HDL delivery strategies that incorporate lipophilic-siRNA conjugates may likely depend on a naturally occurring lipoprotein-RNA delivery pathway.
One of the most abundant miRNAs that was found in both human and mouse HDL, and increased with atherosclerosis, was the highly conserved miR-22348
. Expression of miR-223 in various diseases and its effect on biological processes has been extensively investigated6, 22, 40, 49–51
. For example, miR-223 has been found to be involved in the regulation of glucose metabolism in cardiomyocytes, cell cycle auto-regulatory loops, ischemia-reperfusion injury, granulopoiesis, and osteoclast differentiation50, 52–55
. Multiple targets of miR-223 have been experimentally validated, including RhoB26
. EFNA1 has been shown to promote hepatocellular carcinoma and is a marker for liver cancer27, 56
. In our study, the delivery of miR-223 by HDL significantly reduced EFNA1 and RhoB mRNA levels in hepatocytes. Taken together, increases in specific intracellular miRNA levels, reduction of specific mRNA targets, and the direct repression of luciferase 3′UTR reporters establish that HDL can deliver miRNAs to cells and directly alter gene expression. Furthermore, our results suggest that the delivery of endogenous miRNAs are sufficient to directly target mRNAs and alter gene expression, although some of the observed gene changes from FH HDL treatment could also be related to protein or lipid compositional differences between normal and FH HDL preparations.
In summary, a possible new intercellular communication pathway, involving the transport and cellular delivery of miRNAs by HDL, has been described. This novel role for HDL potentially elevates lipoproteins to mediators of systemic gene expression apart from their role in cholesterol dynamics. The discovery of the HDL-mediated miRNA delivery pathway raises many intriguing possibilities for better understanding the pathogenesis of atherosclerosis and possible new treatment strategies, involving modifications to the miRNA content of HDL for altering gene expression.