The Effect Of Epithelial To Mesenchymal Transition In Cyclosporine A-induced Rat Gingival Overgrowth

Cyclosporine A (CsA) is one of the most important immunosuppressive drugs widely used in transplantation and is increasingly being introduced for the treatment of autoimmune diseases such as psoriasis and rheumatoid arthritis. However, use of CsA is limited by one of its unwanted side effects which can lead to gingival overgrowth in oral and which can seriously influence the patients’life with their masticatory function and oral health. It is meaningful to provide science evidence for clinical prevention and treatment by taking further research of the mechanism of CsA-induced gingival overgrowth.Epithelial to mesenchymal transition (EMT) is a process of cellular transdifferentiation during which epithelial cells lose cell-cell contacts and acquire features typical of mesenchymal cells. It is well known that EMT plays an important role in embryonic development but can also occur during tumor progression, tissue repair and fibrosis. Previous research indicated that some evidenced fibrosis had happened in the gingival connective tissues, the epithelial layer apparently enlarged and the elongated rete pegs grown into the connective tissue beneath. Meanwhile, it was found that diminished epithelial E-Cadherin expression and up-regulated expression of fibroblast-specific protein-1 (FSP-1) levels were contained in the drug-induced gingival overgrowth tissues based partly on the histopathology of gingival overgrowth. It can be suggested that EMT could be a biological process that contributes to drug induced gingival overgrowth.The aim of this study is to examine in vivo the expression of EMT markers in CsA-induced rat gingival overgrowth at different time points, including TGF-β1, E-Cadherin, ZEB1, ZEB2, and Snaill. In addition, a group of miRNAs associated with EMT and fibrosis are also investigated in gingivae so as to explore their possible relationship in the process of CsA-induced gingival overgrowth.Material and methods:Part 1:Establish the Rat Model of CsA-induced Gingival OvergrowthThirty-six rats were randomly divided into two groups. The experimental group received CsA therapy subcutaneously in a daily dose of 10mg/kg, and the other group was used as a control. Six rats per group were sacrificed at 20,40 and 60 days, and the gingivae were obtained. After the rats’ mandibles were dissected, fixed,decalcified and HE stained, the width and height of the epithelial layer and the connective tissue, the integrity of basement membrane structures and the epithelial cell phenotypic changes in the gingival tissues were observed by microscope.Part 2:EMT in CsA-induced Gingival Overgrowth in RatsThe expression of TGF-β1, E-Cadherin, ZEB1, ZEB2, and Snaill in rats’ gingival tissues at all periods were examined by quantitative real time PCR (qRT-PCR), western blotting, and immunohistochemistry.Part 3:MicroRNAs in CsA-induced Gingival Overgrowth in RatsA group of microRNAs associated with EMT and fibrosis were detected in gingival tissue by qRT-PCR at day 20,40, and 60.Results:Part 1:We found that after treated with CsA in 40 and 60 days, the width of epithelial layer in rats’ gingival tissues thickened apparently especially in the stratum spinosum and the stratum corneum. The elongated rete pegs extended deep into the underlying connective tissues and the connective tissues enlarged with substantial collagenous fiber and a small amount of vessels.Part 2:We found that the mRNA and protein levels of TGF-β1, ZEB1, and ZEB2 in gingivae were significantly up-regulated after 40 and 60 days of CsA administration. Conversely, the levels of E-Cadherin were significantly down-regulated in overgrowth sample at day 40 and 60. Intense immunohistochemical staining for TGF-β1 were observed in the samples from CsA group at all periods studied. Concomitantly, the densities of E-Cadherin were gradually decreased in the basal layers of epithelium with time.Part 3:We found that three members of miR-200s (miR-200a, miR-200b and miR-200c) were significantly downregulated in CsA-treated rats at 40 and 60 days, while miR-9, miR-23a and miR-155 were significantly upregulated when compared with those of the control group.Conclusions:1. The rat model of CsA-induced gingival overgrowth has been well established by our study. The progression of the gingival overgrowth is associated with the epithelial plasticity and the hyperplasia in gingival epithelial layer and the connective tissue, which demonstrates that EMT may occur during the process of CsA-induced gingival overgrowth in rat model.2.The process of EMT in CsA-induced rat gingival overgrowth is associated with increased expression of TGF-β1, ZEB1, and ZEB2, and decreased expression of E-Cadherin.3. A group of microRNAs is associated with the process of EMT in CsA-induced gingival overgrowth.