Transgenic Immortalization of Human Dermal Fibroblasts Mediated Through the MicroRNA/SIRT1 Pathway

Abstract

Background/Aim: Human dermal fibroblasts (HDFs) are widely used as a skin model in cosmetic and pharmaceutical industry due their advantages for the cosmetic industry and medical aspects. Telomeres are key players in controlling cellular aging, in which telomeres and the telomerase enzyme (hTERT) can maintain proliferative capacity and prolong cellular senescence. The primary aim of the study was to elucidate the underlying mechanisms of hTERT/SV40 immortalization of human dermal fibroblasts. Materials and Methods: Transgenic expression of hTERT and SV40 large antigen, as well as co-transfection of both factors was performed and their significance evaluated in terms of HDF immortalization efficiency. Results: The results showed that the immortalized fibroblasts of all conditions can be cultured in over 60 passages and maintain their telomere length. Further, key markers of skin cells, such as COL1A1, KRT18 and ELASTIN, were up-regulated in immortalized cells. In addition, p53 expression was enhanced in all immortalized cells, in accordance with activation of the SIRT1 gene upon transgenic immortalization. The significant role of SIRT1 in fibroblast proliferation was assessed by shRNA-knockdown, and it was found that SIRT1 silencing led to loss of Ki67, a proliferation marker. Moreover, miR-93, a SIRT1-targeted miRNA, also had a significantly reduced expression in the co-transfected immortalized cells, highlighting the linkage of the miRNA and SIRT1 pathway in the immortalization of human dermal fibroblasts. Conclusion: This evidence from this study could benefit the efficient development of human skin cell lines for use in the cosmetic industry in the future.The expansion of somatic cells in vitro for research and medical applications is limited by their short proliferative lifespan (1). Cellular senescence is a state of irreversible arrest of cell proliferation, exhibiting enlarged morphological changes and senescence-associated heterochromatin foci formation. Senescence, originating from the Latin word 'SENAX', refers to a physiological program towards permanent cell-cycle arrest. Cellular senescence can be stimulated via a variety of factors, such as tumour suppressor proteins, oncogene activation, telomere shortening, genomic DNA damage, and activation of the p53/p21 pathway. Oncogene-induced senescence is a cellular response found in pre-malignant tumours. Senescent cells exert a pleotropic effect on development, tissue aging and regeneration, inflammation, wound healing and tumour suppression (2). Currently, cosmeceutical research is developed to correct skin problems, including skin aging, skin inflammation, skin disorders, and acne. In cosmetics, human skin cells are used as a standard model for demonstrating the therapeutic potential of cosmetic products. The use of in vitro tests is a critical step in the development of new products for treating skin problems. The study of human skin cell biology has increased the hope for a successful cell therapy in aesthetic medicine and cosmetics. Various types of ...