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Viral microRNAs Involved in Cell Reprogramming

miRCURY LNA™ microRNA Inhibitors
 
Amy Hansen
Amy Hansen is a Ph.D student at the University College London . She works in the Cancer Research UK Viral Oncology Group lead by Professor Chris Boshoff, Director of the UCL Cancer Institute. This group has a long standing interest in Kaposi sarcoma herpes virus (KSHV) and its role in the development of Kaposi sarcoma. Recently, they published an excellent paper in Genes and Development showing that KSHV-encoded microRNAs cause a radical change in the transcriptional activity of infected lymphatic endothelial cells. One of the important results reported was achieved thanks to the exceptional potency of our miRCURY LNA™ microRNA Power Inhibitors .



1. Tell us about the background for this work and how come you became interested in the poorly characterized KSHV microRNAs?

Kaposi sarcoma (KS) is comprised of poorly differentiated spindle-shaped endothelial cells, of lymphatic endothelial origin, which express markers of the blood vessel and lymphatic endothelial lineage. Previously, our lab showed that KSHV is capable of reprogramming endothelial cell transcriptomes ( Wang et al. Nature Genetics ). KSHV infection of lymphatic endothelial cells induces transcriptional reprogramming towards a more blood vessel-like phenotype. However, the molecular mechanism underlying these transcriptional changes was unknown.

Lymphatic endothelial cells are derived from blood vessel endothelial cells during embryogenesis. The lymphatic identity is plastic and maintained through the continual expression of lymphatic endothelial cell-defining transcription factors. Mammalian miRNAs have been shown to mediate cellular differentiation and reprogramming. Therefore, we postulated that viral miRNAs may also affect cellular differentiation and that KSHV-encoded miRNAs could play a role in mediating the transcriptional reprogramming of endothelial cells.

2. Initially you had to determine which KSHV microRNAs are expressed in KS. How did you do this?

KSHV encodes 17 mature miRNAs, 14 of which are co-expressed as a cluster, the other three miRNAs are located further upstream. The KSHV miRNAs are transcribed during latency, which is the mode of gene expression associated with KSHV-mediated oncogenesis.

We began by characterizing the viral miRNA expression in AIDS-KS lesions performing miRNA microarray profiling. These data were then confirmed by qRT-PCR for all 17 mature KSHV miRNAs.
 

3. Once you had determined which microRNAs that were expressed in KS lesions the challenge was to identify mRNA targets that might explain KSHV-induced transcriptional reprogramming. Please explain to us the approach you used and what you found out?

We performed gene expression microarray analysis to determine lymphatic endothelial cell-specific transcripts which were de-regulated in the presence of the KSHV-miRNA cluster. Using the target prediction program PITA (Kertesz et al., 2007), we scanned the 3’UTRs of all genes showing decreased expression in the presence of the cluster for potential KSHV miRNA binding sites. For each down-regulated transcript the cumulative PITA score for all expressed viral miRNAs was calculated. The majority of down-regulated genes had a negative cumulative score, which is indicative of miRNA regulation.

4. Of three genes most likely to be regulated by KSHV microRNA you chose to work on MAF. Why was MAF an appealing target?

MAF is involved in lineage determination and is a lymphatic endothelial cell-specific transcription factor. It is expressed at higher levels in lymphatic endothelial cells compared with blood vessel endothelial cells. Therefore, it was feasible that miRNA down regulation of MAF might mediate the KSHV-induced transcriptional reprogramming of lymphatic endothelial cells.
 

5. How did you show that KSHV microRNAs are indeed capable of MAF regulation?

First we performed qRT-PCR analysis and Western blots of lymphatic endothelial cells transduced with KSHV miRNA cluster to confirm MAF down-regulation at the mRNA and protein level, respectively. Following this, a lentiviral screen of individual KSHV miRNAs identified K12-6 and K12-11 as individual MAF repressors. In parallel, MAF 3’UTR luciferase assays confirmed MAF silencing was by way of miRNA target sites located in the 3’UTR. Finally, transfection with K12-6 and K12-11 2’OMe inhibitors provoked an increase in MAF mRNA in a stable 293 cell line expressing the KSHV microRNA cluster.
 

6. You also looked at the effect of primary KSHV infection on MAF expression?

MAF was down-regulated early during primary KSHV infection of lymphatic endothelial cells, this down-regulation coincided with expression of the viral miRNAs. MAF silencing was maintained during infection.
 

7. You approached Exiqon for help with potent K12-6-5p, K12-6-3p and K12-11 inhibitors for use in your primary KSHV infection experiments. Tell us why these experiments were crucial and why they proved to be quite a challenge?

Inhibition of miRNA silencing is an essential experiment required to confirm miRNA-target regulation. We wanted to inhibit MAF-targeting by KSHV miRNAs in primary lymphatic endothelial cells, this presented multiple technical issues. Primary cells are difficult to transfect and sensitive to high concentrations of transfecting reagents. This was compounded by the fact that we were aiming to inhibit exogenous, highly expressed miRNAs. Previously, we had tried other inhibitor designs but these gave inconclusive results (Figure 1).
 

8. MAF is known as a transcriptional activator, but you show that part of the KSHV-induced reprogramming is due to activation of BEC specific genes that are normally repressed by MAF. You did this by a combination of sophisticated array data analysis and reverse genetics?

Using the pathway-orientated method “gene set enrichment analysis” ( Subramanian et al, 2005, PNAS ) we analyzed three gene expression matrix data sets in which MAF was down-regulated. This identified a concordant increase in blood vessel markers when KSHV miRNAs were expressed in lymphatic endothelial cells and when MAF was silenced specifically with siRNA. Exogenous MAF lacking a 3’UTR rescued the increase in blood vessel markers expression induced by the KSHV miRNAs. Furthermore, expression of MAF in blood vessel endothelial cells, which normally express low levels of this protein, repressed blood vessel marker expression. These data confirmed a role for MAF as a transcriptional repressor of BEC identity.
 

9. Can you in few words summarize the significance of the results presented in this paper?

Our work identifies a novel role for MAF in the maintenance of lymphatic identity, through the transcriptional repression of BEC genes. Furthermore, we show that by silencing MAF viral miRNAs play a pivotal role in the endothelial reprogramming observed upon KSHV infection.
 

 

Additional information




Figure 1
KSHV miRNAs contribute to LEC reprogramming. (Click to learn more)


Figure 2

Inhibition of miR-K12-6 and miR-K12-11 attenuates MAF silencing. (Click to learn more)
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