RNA Journal Club 7/15/10
Yujing Zhang, Danqing Liu, Xi Chen, Jing Li, Limin Li, Zhen Bian, Fei Sun, Jiuwei Lu, Yuan Yin, Xing Cai, Qi Sun, Kehui Wang, Yi Ba, Qiang Wang, Dongjin Wang, Junwei Yang, Pingsheng Liu, Tao Xu, Qiao Yan, Junfeng Zhang, Ke Zen, and Chen-Yu Zhang
Molecular Cell 39, 133–144, 9 July 2010.
This week’s summary and gloves-off analysis by Anonymous:
This group had previously examined microRNA (miRNA) profiles in the serum samples of patients with certain cancers and diabetes, and found them to be able to serve as biomarkers for these diseases (Chen et al, 2008). In that study, they also found that serum miRNAs were resistant to RNase A digest and this study follows up on that. Exosomes/microvesicles (MVs) are small vesicles shed from many cell types of endocytic origin. These are delimited by a lipid bilayer and have been found to contain proteins, mRNAs and miRNAs. MVs can deliver their contents to recipient cells and while it has been shown previously that delivered proteins can alter cellular functions in recipient cells (Skog et al, 2008; Valadi et al, 2007), there has been no direct evidence of miRNAs being delivered to alter target gene expression in recipient cells. This study thus set out to fill that gap.
Briefly, the group first shows that MVs generated by THP-1 cells (a human macrophage/monocytic cell line) contained miRNAs that were resistant to RNase A digest by virtue of the protection afforded by the MV membrane. Next, the authors attempted to show that upon treatment by various stimuli, cellular miRNAs are selectively packaged into MVs such that the miRNA profile in MVs differs from that in the origin cells. However, the evidence was not convincing. The entire study uses quantitative real-time PCR (qRT-PCR) to measure miRNA expression levels. Aside from concerns that qRT-PCR measurements of miRNAs can be wildly noisy, this study is also handicapped by the fact that a reliable internal control that can be found in both cells and MVs is hard to find (it is unclear which control was used in this study, if any). Although the authors attempted to get around this issue by measuring absolute levels of miRNAs normalized to the total protein content in MVs, the miRNA levels in the “no-treatment control” for three different sets of stimuli are not very comparable (even though they should be if absolute levels were measured), underscoring the noise inherent in the miRNA qRT-PCR and/or normalization method. As such, it cannot be said conclusively that miRNAs are selectively packaged into MVs upon different stimulation. It would have been better if the authors had used deep sequencing to quantify miRNA expression instead.
It is, however, fair to say that MVs from THP-1 cells contain high levels of miR-150, which can be delivered to recipient HMEC-1 cells (an endothelial cell line). Upon incubation with THP-1 MVs, miR-150 levels (originally low in HMEC-1 cells) were increased in the recipient cells. The authors also checked that this was not because interactions with the MVs caused the HMEC-1 cells themselves to upregulate expression of miR-150 by checking the levels of pre-miR-150 (which were unaltered) in the HMEC-1 cells. The delivered miR-150 was shown to repress the protein levels of c-Myb, a known miR-150 target, in HMEC-1 cells, and this downregulation enhanced the migration capability of the HMEC-1 cells. Numerous controls were done here to demonstrate that this effect could only be seen when the donor MVs came from cells with high levels of miR-150, which is perhaps the redeeming factor in this paper. Although the authors showed that miR-150 repressed c-Myb protein expression via the 3′ untranslated region (3′UTR), they did not mutate the miR-150 target sites in the 3′UTR to show direct targeting definitively. The paper ends by showing that MVs that were intravenously injected into mouse tail veins can be taken up by the endothelium of mouse blood vessels. Interestingly, the authors also found that MVs from the plasma of patients with atherosclerosis have high levels of miR-150 and that incubation of recipient HMEC-1 cells with these MVs replicated the effects seen (repressed c-Myb protein levels, increased cellular migration) when HMEC-1 cells were incubated with THP-1 MVs.
Several questions remain. As the evidence for selective packaging of miRNAs into MVs is tenuous, it remains to be determined if this is indeed true. If this is true, the mechanism of miRNA packaging would be a natural question to address and miRNAs that are processed differently might behave differently in this respect. In the immunology field, MVs are thought to be “zipcoded” by having different combinations of markers/receptors on their surface (Théry et al, 2002). This paper only tested HMEC-1 cells as the recipient cells and it would be interesting to see if monocytic MVs can be targeted to different cell types and thus modulate the cellular environment differently. In the paper, the delivered miR-150 appeared to repress c-Myb protein levels by ~4-fold, which seems rather high, even after taking into account that the c-Myb 3′UTR has two conserved 8mer seed matches to miR-150. It would have been nice if the authors had determined the concentration reached by miR-150 in the recipient cells, relative to endogenous miRNA concentrations, to see if this could explain the strong repression. Alternatively, as monocytic MVs (of a different cell line) were previously found to be enriched in GW182 (Gibbings et al, 2009), it would be interesting to see if this enrichment also occurs in THP-1 MVs and had somehow contributed to the strong repression observed. At the end of the paper, the authors suggest that finding high levels of miR-150 in the plasma MVs of atheroschlerotic patients may indicate that a contributing factor to atherosclerosis might be the secretion of MVs with high levels of miR-150 by stimulated macrophages, which then cause target endothelial cell migration. However, the cellular origin(s) of these plasma MVs was not determined. This hypothesis thus remains to be tested.
Chen et al (2008) Characterization of microRNAs in serum: a novel class of biomarkers for diagnosis of cancer and other diseases. Cell Res 18: 997-1006
Gibbings et al (2009) Multivesicular bodies associate with components of miRNA effector complexes and modulate miRNA activity. Nat Cell Biol 11:1143-1149
Skog et al (2008) Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers. Nat Cell Biol 10: 1470-1476
Théry et al (2002) Exosomes: composition, biogenesis and function. Nat Rev Immunol 2:569-579
Valadi et al (2008) Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol 9:654-659
Citation for researchblogging.org:
Zhang Y, Liu D, Chen X, Li J, Li L, Bian Z, Sun F, Lu J, Yin Y, Cai X, Sun Q, Wang K, Ba Y, Wang Q, Wang D, Yang J, Liu P, Xu T, Yan Q, Zhang J, Zen K, & Zhang CY (2010). Secreted monocytic miR-150 enhances targeted endothelial cell migration. Molecular cell, 39 (1), 133-44 PMID: 20603081