Abstract
Background: Cell-free microRNAs (miRNAs) exist in body fluid. Previous studies showed that cell-free mi-RNAs are partly bound in microvesicles, and could transfer between cells via fusion with cell membrane. Methods: We quantified the amount of a panel of mi-RNA targets in and outside microvesicles in human proximal tubular epithelial cell (HK2) medium by microarray and real-time quantitative polymerase chain reaction (RT-QPCR). Intercellular miRNA transfer was explored by medium transfer experiments. Results: We identified a portion of cell-free miRNAs that exists as non-vesicle bound, truly naked form. More importantly, these non-vesicle bound free miRNA could transfer between cells and exert biological effects. By miRNA microarray, we showed that the expression of many miRNA targets in HK-2 cells were altered, either up- or down-regulated, after exposure to extrinsic free miRNAs. The miRNA-200 family was the most affected in our model, with a corresponding alteration in the messenger RNA expression of down-stream targets including ZEB1 and vimentin. Conclusion: Our results suggest that free miRNA may serve as an intercellular messenger, a phenomenon that needs further exploration.
Highlights
MicroRNAs are small, non-coding, singlestranded, endogenous RNA molecules that regulate gene expression by promoting degradation of messenger RNA targets, inhibition of translation by binding to the 3’untranslated region (3’UTR), or altering mRNA metabolism by acting as molecular decoys for RNA-binding proteins [1,2,3]
We examine whether free miRNA in culture medium could gain entrance into the cytosol
After CyTM3abeled miRNA was added into the culture medium, fluorescence could be detected in human kidney-2 (HK-2) cells as early as 5 minutes, and it continued to accumulate in the following 24 hours (Figure 2)
Summary
MicroRNAs (miRNAs) are small, non-coding, singlestranded, endogenous RNA molecules that regulate gene expression by promoting degradation of messenger RNA targets, inhibition of translation by binding to the 3’untranslated region (3’UTR), or altering mRNA metabolism by acting as molecular decoys for RNA-binding proteins [1,2,3]. Previous studies showed that these miRNAs are largely enclosed in microvesicles and could transfer between cells, possibly playing a role in intercellular communication ([8,9,10,11]). Previous studies showed that cell-free miRNAs are partly bound in microvesicles, and could transfer between cells via fusion with cell membrane. Methods: We quantified the amount of a panel of miRNA targets in and outside microvesicles in human proximal tubular epithelial cell (HK2) medium by microarray and real-time quantitative polymerase chain reaction (RT-QPCR). Results: We identified a portion of cell-free miRNAs that exists as non-vesicle bound, truly naked form. These non-vesicle bound free miRNA could transfer between cells and exert biological effects. Conclusion: Our results suggest that free miRNA may serve as an intercellular messenger, a phenomenon that needs further exploration
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