Research On Effect Of PcG Proteins In The Process Of The Fibroblast Damage Induced By Ultraviolet A Radiation

Backgrounds: Skin aging is an important sign of human aging.Except the endogenous natural aging, the exogenous environment also can cause human skin aging. The UVA(ultraviolet a, UVA) radiation is the main factor of skin aging exogenous caused by the exogenous environment is also called photoaging. Prolonged UV radiation not only accelerate the aging process of the skin, can also lead to pigmentation spots, telangiectasia, purpura photoaging,even pathological changes precancerous lesions. The current treatment of photoaging include topical tretinoin,Botox injections,soft tissue filling, laser therapy.They can improve the condition of skin,but these treatments is difficult to achieve the underlying aging skin role due to photoaging mechanism is unclear. Pc G(Polycomb group) complex is a modification of chromatin proteins, control the correct pattern of individual development.Pc G is closely related to cell proliferation and tumor differentiation, and participate in the epigenetic regulation of cell and body aging. In the Pc G family complex, multi-bar complex 2(polycomb repressivecomplex2, PRC2) subunit E(z) / EZH2 plays a central role, participate in a variety of molecular mechanisms,such as the formation of chromatin structure,the regulation of gene expression and DNA damage,etc.EZH2 has histone methyltransferase activity, catalyzes histone H3 and H4 N-terminal lysine or arginine residues methylation,so that by adjusting the histone methylation epigenetic regulation of cellular process proliferation,differentiation and tumorigenesis.However,epigenetic studies on photoaging currently rarely reported,we found that our previous skin photoaging process Pc G complex family may be involved in the regulation of the synthesis of hyaluronic acid, thus Pc G complexes may be an important epigenetic molecules in the process of skin photoaging.Purpose: Discussion UVA radiation-induced regulation of Pc G proteins into complex molecular signaling pathway target genes involved in hyaluronic acid synthesis and metabolism of hyaluronic acid and the aging process fiber cell damage,in order to further clarify the apparent skin photoaging process Pc G family genetic regulatory mechanism to provide a solid theoretical basis.Matedals and methods: In this experiment to human skin fibroblasts(human skin fibroblast,HSF) cells as the research object,using the long wave ultraviolet(uv) radiation human skin fibroblasts ultraviolet(uv) radiation induced fibroblast injury model was constructed. 1.To explore the UVA radiation effects on fibroblast phenotype and GSK126 which is EZH2 methyltransferase inhibitors of UVA radiation induced fibroblast phenotype changes. We adopt cell aging beta galactose glucoside enzyme staining to observe different doses of UVA radiation effect on fibroblast of aging and EZH2 GSK126 methyltransferase inhibitors of UVA radiation mediated fibroblasts aging change;Using cell proliferation experiment(cell kit 8- counting,CCK8) tested the different doses of UVA radiation effects on fibroblast proliferation activity and GSK126 of UVA radiation mediated the influence of fibroblast proliferation activity change;The cell apoptosis- Hoechst staining and flow cytometry technique to detect the GSK126 of UVA radiation mediated the influence of the fibroblast apoptosis change;Red blood cell particles exclusion experiments are used to observe different doses of UVA radiation around the fibroblasts hyaluronic acid content and the influence of GSK126 around the UVA radiation mediated fibroblasts hyaluronic acid content change. 2.Discussion related preliminary mechanism of UVA radiation induced aging fibroblasts and GSK126 resistant to fibroblasts aging in the molecular transcriptional level. We use the real-time quantitative polymerase chain reaction polymer(realtime-polymerase chain reaction, rt-pcr) technology to detect different doses of UVA radiation on the Pc G key genes for HA, HA synthesis metabolism related genes, and gene expression related to signal pathway;And tested the GSK126 of UVA radiation induced fibroblast related gene expression changes in aging model. 3.Discussion related preliminary mechanism of UVA radiation induced aging fibroblasts and GSK126 resistant to fibroblasts aging in the molecular translational level. We used sds-page gel electrophoresis to detect the UVA radiation and GSK126 key subunits of Pc G family EZH2, HA synthesis system and histone H3K27me3 protein expression.Results: 1.Different doses of UVA radiation on fibroblast phenotype and influence methyltransferase EZH2 inhibition GSK126 of UVA radiation-induced fibroblast phenotype changes of: β-galactosidase staining showed that compared with the control group 5J/cm2, 10J/cm2 large doses of UVA radiation could induce fibroblast senescence, with a statistically significant result(p<0.05),10J/cm2 UVA radiation after fibroblast senescence highest proportion; CCK8 proliferative activity test results showed that after irradiation fibroblast cell proliferation were decreased compared with the control group, different doses of UVA(2.5J/cm2,5J/cm2,10J/cm2),10J/cm2 UVA radiation dose significantly inhibited fibroblast proliferation activity, the results have statistical significance(p <0.05); erythrocyte exclusion experiments showed that compared with the control group,5J/cm2,10J/cm2 UVA radiation fibroblasts, HSF cells steric exclusion effect on the surrounding red blood cell production reduced after 10J/cm2 UVA radiation HSF cell size exclusion effects on red blood cells disappeared. β-galactosidase staining showed that the control group(10J/cm2) compared to, GSK126 can significantly reduce the proportion of aging UVA radiation induced into fibroblasts, the results statistically different(p<0.05); CCK8 proliferation assay showed compared with the control group(10J/cm2) was added GSK126 of cell proliferation activity increased gradually, the results statistically different(p<0.05); Hoeschst apoptosis staining and flow results show that compared with the control group(10J/cm2),GSK126 could significantly inhibit UVA-induced fibroblast apoptosis, the results have statistical significance(p<0.05); erythrocyte exclusion experiments showed that compared with control group(0J/cm2,5J/cm2,10J/cm2),GSK126 correspondingly enhancing the control fibroblasts row effects on red blood cells outside. 2.Different doses of UVA radiation and EZH2-methyl transferase inhibitor GSK126 fibroblasts of Pc G key genes, signal transduction molecule HA HA synthesis and metabolism-related genes and to participate in related gene expression: PT-PCR results showed that compared with the control group, different doses of UVA radiation fibroblasts Pc G key gene EZH2 and BMI-1, HA system-related genes, HA-related molecules involved in gene expression related signaling pathways are upregulated change appears to 12 h, 24 h declining trend in varying degrees, of which 10J/cm2 UVA radiation group had more genetic changes were statistically significant(P <0.05), 2.5J/cm2,5J/cm2 dose group was statistically significant change in part; Methyltransferase EZH2 inhibitors GSK126 can contribute to UVA radiation induced HAS2,HAS3,CD44,P16,BMI-1 gene increases further raised, lowered UVA radiation-induced increase in MMP-1 gene, the result was statistically significant( P<0.05). 3.Impact on the protein expression of EZH2,HAS1,HAS2, HAS3,H3K27me3 in fibroblasts by UVA radiation and GSK126: Fibroblasts after UVA irradiation, H3K27me3, elevated protein levels HAS3, HAS2, EZH2 protein levels decreased; EZH2 methyltransferase inhibitor GSK126 reduce fibroblasts(normal group and UVA radiation group) H3K27me3, EZH2 protein levels while increasing protein expression of HAS2, HAS3.Conclusions:1.Different doses of UVA radiation fibroblasts may induce varying degrees of fibroblast senescence,decreased cell proliferation,reducing a hyaluronic acid fibroblasts week,10J/cm2 dose radiation group,fibroblast phenotype changed significantly. 2.Different doses of UVA radiation fibroblasts HA anabolic system(HAS1,HAS2,HAS3,CD44,Hyal-1,Hyal-2),HA molecular pathways involved in the regulation of the relevant(EGF/EGFR, Smad2/Smad4,P16,MMP-1) and Pc G protein complex key molecules(EZH2,BMI-1) gene expression was significantly increased at 12 h, 24 h after showing a certain downward trend. 3.We first observed around hyaluronic acid methyltransferase EZH2 inhibitors GSK126 reduces UVA radiation-induced fibroblast senescence extent,improve proliferation UVA radiation damage cell model of aging,reduce the proportion of apoptosis increased change UVA radiation damage induced fibroblast senescence phenotype. 4.At the same time we are also the first study to investigate the expression of histone modifications on HA, we found to inhibit H3K27me3 after school upregulated at the transcriptional level HAS2,HAS3,CD44 and P16 gene, while down-regulated the expression of MMP-1 gene. Upregulated at the protein level of HAS2 and HAS3. These results suggest that: Pc G family can be modified by histone H3K27me3 regulate the expression of HA, in particular HAS2, HAS3 still needs to be confirmed by further experiments Chip- sequence EZH2,H3K27me3 combination with the above key gene promoter region for HA histone modifications Regulation Accumulation scientific basis. We also expect to filter out GSK126 similar small molecules from natural plant single library, by inhibiting H3K27me3 level of skin damage caused by ultraviolet radiation protection,the development of a natural protective skin photoaging agent.