Enterovirus71 (EV71) utilise host microRNAs to mediate host immune system enhancing survival during infection

Hand, Foot and Mouth Disease (HFMD) is a self-limiting viral disease that mainly affects infants and children. In contrast with other HFMD causing enteroviruses, Enterovirus71 (EV71) has commonly been associated with severe clinical manifestation leading to death. Currently, due to a lack in understanding of EV71 pathogenesis, there is no antiviral therapeutics for the treatment of HFMD patients. Therefore the need to better understand the mechanism of EV71 pathogenesis is warranted. We have previously reported a human colorectal adenocarcinoma cell line (HT29) based model to study the pathogenesis of EV71. Using this system, we showed that knockdown of DGCR8, an essential cofactor for microRNAs biogenesis resulted in a reduction of EV71 replication. We also demonstrated that there are miRNAs changes during EV71 pathogenesis and EV71 utilise host miRNAs to attenuate antiviral pathways during infection. Together, data from this study provide critical information on the role of miRNAs during EV71 infection.

Elucidating the host-pathogen interactions between in vitro human cells and enterovirus 71 in Hand, Foot and Mouth Disease (HFMD)

This study has provided further understanding of the pathogenesis of EV71, one of the major etiological agents associated with significant mortality in Hand, Foot and Mouth disease. Elucidating the host-pathogen interaction and the mechanism that the virus uses to bypass host defence systems to establish infection will aid in the development of potential antiviral therapeutics against EV71.

ΔNp63 REGULATES A COMPLEX NETWORK OF TARGET GENES IN LIMB AND EPIDERMAL DEVELOPMENT

The skin is composed of two major compartments, the dermis and epidermis. The epidermis forms a barrier to protect the body. The stratified epithelium has self-renewing capacity throughout life, and continuous turnover is mediated by stem cells in the basal layer. p63 is structurally and functionally related to p53. In spite of their structural similarities, p63 is critical for the development and maintenance of stratified epithelial tissues, unlike p53. p63 is highly expressed in the epidermis and previously has been shown to play a critical role in the development and maintenance of the epidermis. The study of p63 has been complicated due to the existence of multiple isoforms: those with a transactivation domain (TAp63) and those lacking this domain (ΔNp63). Mice lacking p63 cannot form skin, have craniofacial and skeletal defects and die within hours after birth. These defects are due to the ability of p63 to regulate multiple processes in skin development including epithelial stem cell proliferation, differentiation, and adherence programs. To determine the roles of these isoforms in skin development and maintenance, isoform specific p63 conditional knock out mice were generated by our lab. TAp63-/- mice age prematurely, develop blisters, and display wound-healing defects that result from hyperproliferation of dermal stem cells. That results in premature depletion of these cells, which are necessary for wound repair, that indicates TAp63 plays a role in dermal/epidermal maintenance. To study the role of ΔNp63, I generated a ΔNp63-/- mouse and analyzed the skin by performing immunofluorescence for markers of epithelial differentiation. The ΔNp63-/- mice developed a thin, disorganized epithelium but differentiation markers were expressed. Interestingly, the epidermis from ΔNp63-/- mice co-expressed K14 and K10 in the same cell suggesting defects in epidermal differentiation and stratification. This phenotype is reminiscent of the DGCR8fl/fl;K14Cre and Dicerfl/fl;K14Cre mice skin. Importantly, DGCR8-/- embryonic stem cells (ESCs) display a hyperproliferation defect by failure to silence pluripotency genes. Furthermore, I have observed that epidermal cells lacking ΔNp63 display a phenotype reminiscent of embryonic stem cells instead of keratinocytes. Thus, I hypothesize that genes involved in maintaining pluripotency, like Oct4, may be upregulated in the absence of ΔNp63. To test this, q-RT PCR was performed for Oct4 mRNA with wild type and ΔNp63-/- 18.5dpc embryo skin. I found that the level of Oct4 was dramatically increased in the absence of ΔNp63-/-. Based on these results, I hypothesized that ΔNp63 induces differentiation by silencing pluripotency regulators, Oct4, Sox2 and Nanog directly through the regulation of DGCR8. I found that DGCR8 restoration resulted in repression of Oct4, Sox2 and Nanog in ΔNp63-/- epidermal cells and rescue differentiation defects. Loss of ΔNp63 resulted in pluripotency that caused defect in proper differentiation and stem cell like phenotype. This led me to culture the ΔNp63-/- epidermal cells in neuronal cell culture media in order to address whether restoration of DGCR8 can transform epidermal cells to neuronal cells. I found that DGCR8 restoration resulted in a change in cell fate. I also found that miR470 and miR145 play a role in the induction of pluripotency by repressing Oct4, Sox2 and Nanog. This indicates that ΔNp63 induces terminal differentiation through the regulation of DGCR8.

INVESTIGATING THE ROLES OF THE p63 ISOFORMS IN THE MICRORNA BIOGENESIS PATHWAY

MicroRNAs play roles in various biological processes like development, tumorigenesis, metastasis and pluripotency. My thesis work has demonstrated roles for p63, a p53 family member, in the upstream regulation of microRNA biogenesis. The p63 gene has a complex gene structure and has multiple isoforms. The TAp63 isoforms contain an acidic transcription activation domain. The ΔNp63 isoforms, lack the TA domain, but have a proline rich region critical for gene transactivation. To understand the functions of these isoforms, the Flores lab generated TAp63 and ΔNp63 conditional knock out mice. Using these mice and tissues and cells from these mice we have found that TAp63 transcriptionally regulates Dicer while ΔNp63 transcriptionally regulates DGCR8. TAp63 -/- mice are highly tumor prone. These mice develop metastatic mammary adenocarcinomas, squamous cell carcinomas, and lung adenocarcinomas to distant sites including the liver, lungs, and brain. I found that TAp63 suppresses metastasis by transcriptionally activating Dicer. TAp63 and Dicer levels were very low or lost in high grade human tumors like mammary adenocarcinomas, squamous cell carcinomas, and lung adenocarcinomas. Expression of Dicer in these tumor cell lines reduced their invasiveness. Using ΔNp63 -/- mice, I found that ΔNp63 transcriptionally activates DGCR8, resulting in a miRNA profile that is critical to reprogram cells to pluripotency. Analysis of epidermal cells derived from ΔNp63 -/- mice revealed that these cells expressed markers of pluripotency, including Sox2, Oct 4 and Nanog; however, genome-wide analysis revealed a novel profile of genes that are common between ΔNp63 -/- epidermal cells and embryonic stem cells. I also found that mouse cells depleted of ΔNp63 form chimeric mice and teratomas in SCID mice, demonstrating that ΔNp63 deficient cells are pluripotent. Further, I found that restoration of DGCR8 in ΔNp63 -/- epidermal cells reduces their pluripotency and induces terminal differentiation. I also demonstrated that iMS (induced multipotent stem) cells could be generated using human keratinocytes by knockdown of ∆Np63 or DGCR8. Taken together, my work has placed p63 and its isoforms at a critical node in controlling miRNA biogenesis.