| BackgroudSkin aging performs as an external manifestation of organism aging. Not only does skin aging bring cosmetic problems, but also connects to a series of age-related diseases, which do harm to people’s physical and intellectual integrity. Owing to its complicated components in the extracellular matrix and the diverse types of cells, dermis plays a dominant role in skin aging. To date, studies about dermis aging have now covered fields involved epigenetics, genomics, proteomics and so on, but its relationship with microRNA(miRNA) is still unclear. miRNA is a short non-coding RNA that can negatively regulate target genes by binding the specific 3’-UTRs sequence, therefore affecting a variety of physiological and pathological processes. Study of age-related miRNA expressed in dermis and its regulation network can help better understand the mechanism of dermis aging, providing innovative views for anti-aging therapies.Object1. To identify the characterization of the miRNA profile in aging dermis, discover the relevant function and signaling pathways and build a regulatory network comprising miRNAs and parts of its target genes related with aging2. To explore the miRNA profile in senescent fibroblasts, compare its coherence and difference with results found in tissues, and discuss its potential regulatory pathway related to cell senescence.MethodIn the first part, human dermis samples from sun-protected body parts were obtained, including 6 for young group and 6 for aged group respectively. miRNA microarray was conducted to discover the differentially expressed miRNA profile between ages. Real-time quantitative PCR was utilized to verify the expression of some miRNAs. Predicted target genes were listed, followed by a GO and pathway enrichment analysis among them. A miRNA-gene-network was then built to acquire the vital regulatory miRNA in this network.In the second part, fibroblasts isolated from dermis were cultured in normal conditions and serum-reduced conditions respectively. After serial passaging of fibroblasts, multiple characteristics of cell senescence were evaluated among early passage and late passage cells, including survival curve, SA-P-GA stain and real time quantitative PCR of senescence-related genes. In the end, assessment of the expression of selected miRNAs was conducted by real time PCR among young and senescent fibroblasts.Results1. The results of miRNA microarray revealed that 40 miRNAs in dermis were differentially expressed with aging.2. The verification of miRNA expression by real time quantitative PCR showed miR-34 family and miR-29 family were significantly highly expressed in the aging dermis, which was in accordance with the results of miRNA microarray.3. With GO and pathway enrichment analysis, predicted target genes of the miRNA profile were found to be mainly involved in processes like cell adhesion, collagen synthesis, positive or negative regulation of transcription, as well as pathways like metabolic pathways, growth factor pathways, ECM-receptor interaction and DNA damage response.4. miRNA-Gene-Network revealed that miR-34 family, miR-29 family and miR-424 may play a dominant role in the regulatory network.5. No matter under normal or nutrition-reduced culture conditions, when compared to early passage cells, fibroblasts in their late passage demonstrated a significantly suppressed proliferation, higher ratio of SA-β-GA positive cells and up-regulation of p16 mRNA, while mRNA of p53 and p21 remained unchanged.6. Real time quantitative PCR of miRNA revealed that, all of the selected 11 miRNAs, i.e. miR-548q, miR-1226*, miR-601, miR-1207-5p, miR-134, miR-34b*, miR-34a, miR-29c*, miR-181a, miR-154, miR-424, showed an up-regulated tendency in senescent fibroblasts under either type of culture conditions. Most miRNAs exhibited similar change to the results of microarray analysis, while a paradoxical result about miR-181a, miR-154 and miR-424 w as found in senescent cells.7. The expression fold of some miRNAs was found to be distinct in the senescent fibroblasts cultured with different nutritional conditions. miR-34 famliy and miR-29 family were most up-regulated miRNAs under normal conditions. However, miR-1226*, miR-601 and miR-1207-5p exhibited a more drastic up-regulation with nutrition-reduced cultural medium; while no overt expression change was seen in miR-29 family.8. Real time quantitative PCR also revealed that some predicted target genes (i.e. E2F3, c-Myc, CREB) of miRNA profile were down-regulated under nutrition-reduced condition.ConclusionThere are differentially expressed miRNAs during dermis aging. The target genes of the differential miRNA profile participate in processes like cell adhesion, collagen synthesis, regulation of transcription, as well as pathways like metabolic pathway, growth factor pathway, ECM-receptor interaction and DNA damage response. The miR-34 family, miR-29 family and miR-424 may play a critical role in affecting dermis aging. A similar miRNA change is observed in senescent fibroblasts in vitro, still paradoxical results of a few miRNAs indicates other mechanisms other than cell senescence may be involved in dermis aging. Additionally, the age-related miRNA profile may interact with p16 pathway to regulate the fibroblasts’senescence. |
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