| Background and ObjectiveRadiation includes ionizing radiation and non-ionizing radiation.Ultraviolet(UV)is a non-ionizing radiation with a wavelength of 100-400 um.According to thedifferent wavelengths of ultraviolet light,it can be divided into short-wave ultraviolet UVC(200-280 nm),medium wave UVB(280-320 nm)and long-wave UVA(320-400 nm).And in the atmosphere through the wavelength of less than 290 un UV will be absorbed by the ozone in the atmosphere,to reach the ground is mainly UVA and a small amount of UVB.Radiation-induced bystander effects(Bystander effects,BE)refers to the reaction of the bystander cells such as cell death,gene mutations,chromosomal instability,and the like,which are directly affected by radiation.A bystander cell refers to a cell adjacent to an irradiated cell and also a cell culture with a supernatant of irradiated cells.In addition to direct damage to irradiated cells,ultaviolet radiation can also cause indirect damage to adjacent cells by ultraviolet irradiation(UV-BE).These bystander effects include oxidative stress,gene mutation,death,inflammatory response,immunosuppression,and even tumor lesions.In order to eliminate the effects of UV radiation and maintain a healthy chromosome,cells produce a range of protective mechanisms,including DNA repair,cell cycle arrest,and apoptosis.In the bystander effect,the DNA damage response of the irradiated cells is connected through the intercellular gap,and the extracellular soluble factor is inter-cellularly communicated with the irradiated cells.Recent studies have shown that small molecule RNAs(miRNAs)play an important role in the signaling pathways of irradiated cells and bystander cells.In the past,people think that miRNAs only play a role in the cell,but with the deepening of the study,the researchers found that miRNAs will be secreted into the cell gap and play a function.A large number of data show that the extracellular miRNA is very stable,miRNA has been able to exist in the cell gap,because it is wrapped in extracellular vesicles,extracellular vesicles(EVs)by the phospholipid bilayer closed to different sizes of particles(20～2000 nm),in vivo and in-vitro almost all types of cells will release EVs into the extracellular media.So far,according to different formation mechanisms and physiological characteristics are divided into three different forms of EVs:exosomes,cell membrane particles,and apoptotic bodies.Exosomes produced in the nucleus carrying more bubbles,cell membrane particles and apoptotic bodies derived from the cytoplasmic membrane release of microcapsules(Micro-vesicles,MVs).The miRNAs encapsulated in EVs are called"secretory miRNAs," which protect "secretory miRNAs,from degradation by various enzymes and are transported to the corresponding receptor cells and regulate the cells of the recipient cells features.So,in ultraviolet radiation,"Bystander effect"in the cell damage is mediated by the miRNA or by what kind of miRNA-mediated it?The aim of this study was to screen out secretory miRNAs that may play an important role in the acute and long-wave ultraviolet radiation "bystander effect" and to predict its possible mechanisms.Methods(1)The establishment and verification of the "spectator effect" model of ultraviolet radiationUVA20 J/cm2 and UVB 60mJ/cm2 were used to irradiate human skin fibroblasts(HSF)with different doses of extracellular vesicles The cells were treated with UVA and UVB,and the supernatant was extracted at 24 h after irradiation.The cells were divided into control group(Ctrl group),UVA group(UVA group),UVB group(BE-group),bystander cell UVA group(BE-UVA group),bystander cell UVB group(BE-UVB group),observed under inverted microscope directly under the radiation cells and Bystander cell morphology,cell count CCK-8 method was measured directly by irradiated cells and bystander cell proliferation rate,flow cytometry was measured directly by irradiated cells and bystander cell apoptosis rate,DCFH-DA kit was detected directly by radiation Cells and bystander cells of ROS.(2)The miRNA microarray screening of differentially expressed secretory miRNAs and bioinformatics analysis.The human skin fibroblasts were irradiated with ultraviolet light in the above dose and incubated with the extracellular vesicle culture medium for 24 hours.The supernatants of the irradiated cells were collected and analyzed by miRNA microarray.The logarithmic intensity and>5 were used to screen up-regulated miRNAs in down-regulated miRNAs and down-regulated miRNAs.QRT-PCR was used to validate the up-regulated miRNAs in UVA vs Ctr group and UVB vs Ctr group.Target genes were predicted by targetScan,microRNAorg and PITA database.The target genes of miRNAs were analyzed by GO and pathogenic enrichment.(3)qRT-PCR method to further verify the differential expression of secretory miRNA.The differentially expressed miRNAs were verified by qRT-PCR(real-time quantitative PCR).The expression of miRNAs in irradiated cells,directly irradiated cells and bystander cells were measured.Screening out the secretory miRNAs that may be associated with the UV"e;bystander effect"e;and examining the expression of the precursor RNA(pre-miRNA)in bystander cells.Results(1)Ultraviolet radiation after human skin fibroblasts can induce UV "bystander effect" occursThe supernatants induced by UVB 60 J/cm2 and UVB 60mJ/cm2 radiation were incubated with the bystander cells for 24 hours.The cells were observed directly under the microscope and irradiated by irradiated cells and bystander cells directly by irradiated cells and bystander cells CCK-8 results suggest that the proliferation rate of radiation cells and bystander cells were slowed down.Flow cytometry results showed that the cells were directly affected by radiation cells and bystander cells.The ROS results suggest that the fluorescence intensity of reactive oxygen species in radiation cells and bystander cells is higher than that in non-radiation group.(2)miRNA chips were screened for differentially expressed miRNAs.The results showed that the standard was fold change>5.0,and 54 in the UVA vs Ctr group,and the results were found in the supernatant induced by ultraviolet radiation.MiRNA expression was up-regulated and 11 miRNAs were down-regulated.50 miRNAs were up-regulated in the UVB vs Ctr group and down to 4 miRNAs.Among them,the number of miRNAs in the up-regulated miRNAs was 19,which were hsa-miR-8071,hsa-miR-769-5p in the UVA vs Ctr group with Fold change>= 5.0 and UVB vs Ctr group Fold change>Hsa-miR-758-3p,hsa-miR-7515,hsa-miR-6856-5p,hsa-miR-6837-5p,hsa-miR-6769a-5p,hsa-miR-6743-5p,hsa-561,hsa-miR-4694-3p,hsa-miR-4655-3p;,hsa-miR-4514,hsa-miR-4513,hsa-miR-4422.hsa-miR-432-5p,hsa-miR-Hsa-miR-3163,hsa-miR-22-5p,hsa-miR-1299.Biomechanical analysis of differentially expressed secretory miRNAs was carried out to predict target gene function.UVA group and UVB group were significantly enriched in the metabolic pathway.The gene was mainly enriched in energy metabolism,transcription regulation,cell proliferation,transmembrane signal transduction and these genes are mainly involved in the Rapl signaling pathway,focal adhesion kinase(FAK)-related signaling pathway,MAPK signal pathway and other important metabolic pathways.(3)Differential expression of secreted miRNAs for further validation.The expression of hsa-miR-4655-3p and hsa-miR-769-5p in the irradiated cells and bystander cells were significantly higher than those in the pre-miR-4655-3p,pre-miR-769-5p showed low expression.ConclusionAcute long-wave ultraviolet radiation can induce the expression of secretory miRNAs in human skin fibroblasts,and hsa-miR-4655-3p and hsa-miR-769-5p are significantly up-regulated by acute long-wave ultraviolet radiation.Most likely to participate in the regulation of UV "bystander effect",worthy of further study. |
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