INVOLUCRIN EXPRESSION IN THREE-DIMENTIONAL CELL CULTURE AND PROGNOSTIC PARAMETERS OF CANINE CUTANEOUS SQUAMOUS CELL CARCINOMA (SCC) IN CANINE PATIENTS AND IN ULTRAVIOLET RADIATION INDUCED CULTURED CELLS

Abstract

The objectives of this study are 1) to demonstrate the involucrin (INV) expression and prognostic parameters in normal skin and cutaneous squamous cell carcinoma (CSCC), 2) to develop the artificial epidermis in dog and 3) to study the effect of INV expression on pathogenesis of ultraviolet radiation induced CSCC in dog.The first experiment is to determine the protein expression by Immunofluorescence technic (IFC) and mRNA expression of INV,Cytokeratin 10 (CK10), Ki67 and p53 normal skin(n=6) and CSCC (n=6) in dog.The protein expression pattern was demonstrated that INV and CK10 were positive in intra-cytoplasmic pattern in nucleated epidermis. While, intra-nuclear pattern was positive with Ki67 but was negative for p53. The levels of the mRNA expression of INV, CK10, Ki67, and p53 in the normal skin and CSCC in dog are in correspondence with IFC that INV of normal skin revealed more than CSCC, on the contraryKi67 in normal skin was less than CSCC with statistically difference (p<0.05). The papilloma virus was negative in all samples. Though, p53 has not shown any statistically difference (p>0.05), and the mRNA expression in CSCC tends to be more than in normal skin.The second experiment is the cultured commercial canine keratinocyte cell line (CPEK, C CELLnTEC Advanced Cell Systems, Switzerland) was successfully conducted by producing the 3 dimensional (3D) epidermal skin appearance under air-liquid interface technique. The 3D cell culture showed the epidermal characteristic similar to the epidermis on 14 days cultured. For IFC, the INV, CK10 revealed positive result while ki67 and p53 showed negative result. The mRNA expression, there were no statistical difference of INV, CK10 between 3D cultured CPEK and normal skin (p>0.05). The p53 and ki67 were negative in 3D cell cultured. In the same cultured condition, the primary cell culture both normal skin and CSCC could not be obtained good yields. The third experiment was designed siRNA for INV by online program ((https://us.bioneer.com/sirna/custom-sirna-ex.aspx), the 3 sets; INV-1, INV-2 and INV-3 could be obtained. The treatment of cultured CPEK with INV-1 siRNA for INV knockdown showed the best result among 3 sets in the reduction of mRNA levels. From the titration, 300 pmol demonstrated the highly effective with minimum cytoxicity to cultured cells. The mRNA expression of INV started to decrease with statistical difference (p<0.05) at 0 h after 5 h transfection and slightly increasing at 48 h. The cytologic appearance of cultured CPEK were initially observed after 300 mJ/cm2 of UVB irradiation exposure, at 6 h. The cell showed a number of dead and sloughing cells in the siRNA transfected with UVB irradiation group in comparison to control group. The mRNA expression of wild type p53 of cultured cell was increased after 6 h exposure of 300 mJ/cm2 of UVB and gradually increased its expression when increasing exposure time respectively. After the cells were harvested at 24 h, the results of p53 mRNA expression were statistically difference increased in cultured cell both; with and without siRNA transfection under UVB irradiation groups more than without UVB irradiation groups (p<0.05). There was no statistically difference of INV, CK10, and Ki67 expression between with and without UVB irradiation groups. The obtained results demonstrated that the protein expression by IFC and the level of mRNA expression of INV are difference between normal skin and SCC in dog. The 3D cultured CPEK could be developed as an artificial epidermis in dog. Finally, this is the first report on the design of siRNA transfected in the cultured CPEKfor INV’s knockdown. It is suggested that INV doesn’t effect on the induced in cultured keratinocyte by the UVB irradiation at 300 mJ/cm2, which is the major cause of CSCC in dog.