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Dicer ablation in keratinocytes impairs wound re-epithelialization by elevation of p21

Research Scholar

Subhadip Ghatak, Surgery - Davis Heart & Lung Research Institute (India)
Yuk Cheung Chan, Co-Reseacher
Savita Khanna, Faculty Mentor
Sashwati Roy, Faculty Mentor
Chandra Sen, Primary Faculty Mentor

Biography

Subhadip Ghatak was born in Durgapur, India. He received his master's in physiology from University of Calcutta, India in 2003. In 2011, he completed his PhD research in biochemistry from Center for Liver Research at the West Bengal University of Health Sciences, India. His thesis work mainly focused on the mechanisms of liver injury in arsenic induced toxicity. He joined Chandan Sen's lab in the Department of Surgery at the Wexner Medical Center at The Ohio State University in July 2011 as a postdoctoral researcher. Ghatak is currently focused on studying the involvement of microRNAs in cutaneous wound healing.

What is the issue or problem addressed in your research?

In the United States, human skin wounds represent a major public health problem affecting 6.5 million patients. Following injury, one of the major goals of the healing process is to rapidly re-establish the barrier function of the skin. This is achieved by restoration of an intact epidermal barrier through wound re-epithelialization. Keratinocytes populate the wound bed below the scab by migration, which is supported by keratinocyte proliferation in the surrounding epidermis both at and adjacent to the wound edge. Defects in one or multiple keratinocyte function(s) related to re-epithelialization can have pleiotropic effects, ranging from excessive scarring to the formation of chronic ulcers.
The significance of miRNAs in this process remains unknown. We hypothesized that keratinocytes specific conditional ablation of Dicer, one of the key RNase III responsible for miRNAs maturation, will result in depletion of mature miRNAs in the skin epidermis and compromise wound re-epithelialization.

What methodology did you use in your research?

Keratinocytes specific Dicer ablated mouse was generated by crossing Dicerflox/flox mouse with another transgenic mouse having Cre recombinase protein fused to estrogen-receptor ligand binding domain under keratin 14 promoter. Murine excisional wound model was induced by 8 × 16 mm full-thickness excision on the dorsal skin, equidistant from the midline and adjacent to the four limbs. Expression of miRNAs was performed by Luminex microarray. Expression of mRNAs and proteins were validated by quantitative real time PCR and Western blot respectively. Localization of proteins of interest was validated by immunohistochemistry. The miRNA expression profiling of wound-edge tissue during cutaneous wound healing of wild-type mice revealed a global up regulation of miRNAs on day 14 post wounding.  Re-epithelization was associated with more than 2.5 fold elevated expression of Dicer protein (n=6; p<0.001). Dicer ablation attenuated wound re-epithelization by 25% (n=3; p<0.05) on day 14 post wounding. Epithelial cell proliferation at the wound-edge tissue was also attenuated as evident from ki67 immunohistochemistry (n=3; p< 0.01). Real time expression of the cyclin/CDK inhibitor p21waf1/CIP1 from laser captured keratinocytes revealed more than 2.5 fold elevated mRNA expression in Dicer ablated skin epidermis compared to normal epidermis (n=6; p<0.001). Increased expression of p21waf1/CIP1 in Dicer ablated wound edge tissue was also confirmed by western blot and immunohistochemistry, suggesting a role of miRNAs in the suppression of p21waf1/CIP1.

What are the purpose/rationale and implications of your research?

The discovery of miRNAs and their significance in biology represents a major breakthrough in molecular biology. We now know that miRNA represent a key mechanism in post transcriptional gene silencing (PTGS). PTGS by miRNA results in sequence-specific inhibition of gene expression; this can occur at levels as different as chromatin modification and silencing, translational repression and mRNA degradation. Given the recognized role of injury sensitive coding genes in cutaneous wound repair, it becomes critically important to elucidate miRNA dependent mechanisms that regulate wound-induced gene function. Elucidating the basic mechanisms of miRNA biology in wound healing will enable prudent assessment of the risk-benefit ratio of potential miRNA-based therapeutics. Thus it lends itself to clinical therapeutics and is of extraordinary translational and commercial value. Once firm knowledge establishing the significance of specific miRNAs in cutaneous wound outcomes is developed, topical delivery (e.g. topical cream) of miRNA mimetics and antagomiRNAs may be considered as first attempts to favorably regulate the expression of sets of target coding genes.