The National Institutes of Health earmarked more than $1.2 million in grant money in January to support three separate research projects investigating the roles of microRNAs in herpes infection, pulmonary hypertension, and placental-maternal communication.
The first grant was awarded to University of Florida researcher David Bloom to fund his efforts to uncover the role of miRNAs in herpes simplex virus type 1 infection.
HSV-1 typically infects oral mucosa, then establishes latent infection within sensory neurons, according to Bloom's grant abstract. Latency is characterized by the repression of viral lytic genes, during which “only one transcript is abundantly transcribed, the latency associated transcript,” or LAT.
During intermittent episodes of recurrence, viral particles are produced and transported to the original site of infection, the abstract notes. And while antiviral drugs can control the severity of recurrences, there is no cure for HSV infection.
As such, “understanding the molecular basis of how HSV regulates the lytic and latent phases of infection could provide new therapeutic approaches,” Bloom wrote.
Eight miRNAs have been shown to be encoded within and adjacent to the HSV-1 LAT region, and in vitro analyses have shown that at least two of these regulate HSV-1 gene expression.
In light of these data, Bloom and his colleagues aim to investigate the role of these miRNAs in HSV-1 pathology in vivo.
Specifically, they plan to construct HSV-1 recombinants containing inactivating mutations in the eight HSV-1 miRNAs, and then assess these mutant viruses for changes in viral gene expression and replication in vitro.
Moving in vivo, the recombinants will be analyzed in the mouse and rabbit models for changes in replication, spread, establishment of latency, and ability to reactivate. Those recombinants that show altered phenotypes will be rescued and examined for the molecular basis underlying the phenotypic change.
Bloom's team also plans to use a photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation approach to “directly identify mRNA target sites occupied by HSV-1 miRNAs in cells infected with wild-type HSV-1 or with HSV-1 variants lacking specific miRNAs,” according to the abstract.
Viral or cellular mRNA targets identified by these analyses will be confirmed through RT-PCR, and their functional role during the viral infection cycle elucidated by knock-down and over-expression analyses.
Bloom noted that this work with be done in collaboration with Duke University's Bryan Cullen. His grant runs from Jan. 1 until the end of 2016, and is worth $386,302 in its first year.
The second grant went to Vijay Kalra of the University of Southern California to support his research into the role of miRNAs in sickle cell anemia-associated pulmonary hypertension.
According to the grant's abstract, pulmonary hypertension occurs in roughly 30 percent of sickle cell patients, and is in part triggered by hemolysis-induced impaired nitric oxide bioavailability; chronic thrombo-embolism from a pro-coagulant state; and increased levels of the vasoconstrictor endothelin-1.
“Our studies have shown that placenta growth factor, an angiogenic growth factor produced in high amounts by sickle erythroid cells, induces expression of … ET-1, and a procoagulant [called] plasminogen activator inhibitor-1 from human pulmonary microvascular endothelial cells,” the abstract states. “PlGF increases ET-1 and PAI-1 expression via induction of hypoxia-inducible factor-11. PHT can be induced experimentally by ectopic PlGF expression in normal mice characterized by increased ET-1, as is observed in transgenic sickle [cell] mice and in [sickle cell anemia] patients.”
Kalra and his colleagues have reported that PIGF-mediated induction of HIF-11 and PAI-1 in human pulmonary microvascular endothelial cells is regulated by the miRNAs, and hypothesize that “cytoplasmic RNA-binding proteins and miRNAs alter the stability of HIF-11, ET-1, and PAI-1 mRNAs, and are directly involved in the development” of pulmonary hypertension, the abstract states.
To test this idea, the investigators will try to determine the post-transcriptional mechanisms regulating PlGF-mediated expression of HIF-1a, ET-1, and PAI-1, and then identify RNA-binding proteins and the specific miRNAs that regulate the translation of their mRNA, according to the grant's abstract.
They will also look to see whether the miRNAs that regulate PAI-1 expression are co-synthesized from the primary transcripts of the genes in which they are located or if they are independently transcribed from a smaller, pre-miRNA transcription unit.
Lastly, Kalra and his colleagues will try to demonstrate whether key miRNAs are essential for the regulation of HIF-11, ET-1, and PAI-1 in genetic mouse models that over-express PlGF and develop pulmonary hypertension. Plasma levels of the miRNAs will be compared with levels of PlGF, ET-1, and PAI-1 in sickle cell anemia patients with and without pulmonary hypertension symptoms.
Kalra's grant runs from Jan. 1 until Dec. 31, 2016, and is worth $635,054 in its first year.
The last grant was awarded to Yoel Sadovsky of the Magee-Women's Research Institute to fund his investigation into epigenetic signals involved in the communication between a fetus and a mother's body.
“We posit that microRNA and other small RNA signals have an extraordinary significance in regulation of placental and maternal gene expression and consequently, successful pregnancy,” he wrote in his grant's abstract.
Noting that the placenta expresses miRNAs not normally present elsewhere in the body, he and his team aim to use deep-sequencing technology to “comprehensively interrogate microRNA and small RNA species in the human maternal blood and the placenta, and deploy novel computational tools to analyze dynamic changes in microRNA and small RNA expression in normal pregnancies or pregnancies complicated by clinically relevant placental injury,” the abstract states.
They will specifically focus on small RNAs assembled in vesicular exosomes, which may be responsible for trafficking such molecules between mother and fetus.
“Information gleaned from our data may not only illuminate non-coding RNA pathways that influence human embryonic development, but may also suggest new, clinically relevant signals, designed to inform the risk of placental injury and substandard pregnancy outcome,” the abstract notes.
Sadovsky's grant runs from Jan. 16 until the end of 2013, and is worth $210,187 this year.