| BACKGROUPThe destruction of the ozone layer has become one of the most important environmental issues, Some research showed that, This may be due to human overuse of chlorofluorocarbons (CFCS) compounds, climate change and other factors. With the destruction of the ozone layer, arrived at the earth’s surface solar ultraviolet will gradually increased, which UVB (ultraviolet radiation B, UVB) increase is particularly prominent. Enhanced ultraviolet radiation will not only harm people’s skin, eyes, damage the human immune system, but also harm the other biological and food crops on the earths. Therefore, the damage of organism by UVB iradiation is also getting more and more attention of researchers.Further more, In recent years, with the development of modern science and technology, the use of UVB in the field of industry and medical treatment also showed an increasing trend, which led to the increasing of UVB occupational exposure. The industry mainly includes the professional personnel engaged in industrial arc welding, carbon arc lamp, mercury lamp, plate making, photography and pilot. In the medical industry, a number of UV treatment instrument lead to increased popularity of the relevant sections of the doctor’s occupational exposure levels. Therefore, the study of ultraviolet radiation on human mechanism causing injury, can also provide important theoretical basis for the prevention and treatment of occupational exposure to U VB damage.Human skin is the most easily and directly affected by the location of UVB radiation. The right amount of UVB irradiation, can promote the body mineral metabolism and vitamin D formation, but if long-term or excessive UVB irradiation, can cause skin erythema, inflammation, skin aging, serious can even cause skin cancer.So which way is the UVB induced radiation damage? Studies have found that 14-3-3σ (SFN or Stratifin) protein are markers of epidermal cell DNA damage and is one of the highly conserved acidic soluble 14-3-3 protein family members, it is mainly present in the epithelial cells, and many intracellular signaling proteins interact to promote DNA damage,then induced cells G2/M phase arrest and the regulation of cell growth, differentiation and proliferation of biological function. In addition, the study shows that some tumor occurrence and development usually exist abnormal expression of 14-3-3 a, such as 14-3-3 a down-regulation in breast cancer, lung cancer,. However, no correlation was found in the expression of skin squamous cell carcinomaIn order to explore the 14-3-3σ in the UVB radiation in human epidermal cell damage is also play their biological functions, and to explore the possibility and the downstream signal molecules; UVB induced skin cancer cells and tissues whether also to have the expression of 14-3-3σ abnormal. In this study, human epidermal cells (HaCaT) were used as the research object, And the stable interfering cell line HaCaTKD was used in the early stage. The intervention factors included UVB at a wavelength of 310nm and a dose of 30mJ/cm2.(The dose of 30mJ/cm2 was the best dose of low dose UVB radiation injury in the early stage of our laboratory.), To explore the 14-3-3 sigma on cell proliferation and cycle effect on the injury induced by UVB. In order to further study the mechanism of radiation damage induced by HaCaT in UVB cells,to explore the possible signaling molecules and 14-3-3 sigma interaction and its mechanism,and 14-3-3 sigma in cutaneous squamous cell carcinoma expression.PURPOSETo explore the mechanism of 14-3-3σ in the proliferation and cell cycle changes of HaCaT cells with 30mJ/cm2 UVB irradiation, and to further explore the downstream signal molecules of 14-3-3σ sigma, and the expression of 14-3-3σ in cutaneous squamous cell carcinoma.Method1. Cell source and cultureHuman epidermal cells HaCaT was purchased from China Center for Type Culture Collection (CCTCC). Human skin squamous cell carcinoma A431 cell line was purchased from Typical culture preservation commission cell bank, Chinese academy of sciences. A431 cells and HaCaT cells were cultured in 10% newborn calf serum and 1 ×105U/L penicillin, 100mg/L streptomycin in DMEM high glucose medium,at 37℃, volume fraction of 5% carbon dioxide incubator in conventional culture, In addition, HaCaTKD cells were added to the final concentration of 100μg/mlGeneticin (G418)2. UVB irradiationWhen the logarithm of the long-term HaCaT cells to reach the 80%～90% culture dish area, with PBS wash cells 2 times. at each time point,3 dishes were set. According to the need of the experiment, the dishes were randomly divided into the control group and the UVB group, Then add a certain amount of PBS (including 10cm culture dish PBS 5mL,6cm dish PBS 1.9mL,96 96 well plate plus 28 μL PBS), Control group were simulated (In addition to the non irradiated UVB, the other treatment processes are consistent with the treatment group), the UVB group is placed in the box of the UV light source, which is located 40 cm from the distance light source, UVB irradiation 3 min, the cumulative radiation dose of 30m J/cm2. After ultraviolet radiation, all the fresh DMEM high glucose medium were replaced, and the total protein and RNA of HaCaT cells were collected at 3,6,12,18,24 and 30h time points, respectively. UVB treated the cells at that time as a control group (Oh).3. Crystal violet staining3×105 HaCaT cells and HaCaTKD were seeded in 3.5 cm dish. When HaCaT cells grow to 70%-80%, the two kinds of cells were staining with crystal violet after treated with 0,10,20,30,40,50,60 and 80 mJ/cm2 irradiation 48 h.5 fields of view were randomly photographed by microscope with ×200. Calculate the average number of cells, In the absence of irradiation group (0 mJ/cm2) as control.The experiment was repeated 3 times.4. Cell proliferation was detected by CCK-8 assay8×103 HaCaT and HaCaTKD cells were seeded in a 96 well plate, Cell suspension with 100μl per holes. Incubate 24 h after UVB irradiation，Irradiation after 0,12,24,48 and 72 h, respectively,10μl CCK-8 solution was added to each hole.continue to culture for 2 h, measuring 450 nm absorbance OD value.5. Cell cycle was detected by PI stainingIn a 6cm dish, HaCaT and HaCaTKD cells were seeded with 1×106 cells. Cells were collected after inoculation 0,6,12,18, and 24 h,then the cell cycle was detected by PI staining. HaCaT and HaCaTKD cells were seeded with 1×106 cells. When HaCaT cells grow to 70%～80%, two groups of cells were treated with 30 mJ/cm2UVB irradiation. Cells were collected at 0,6,12,18, and 24 h after irradiation,then the cell cycle was detected by PI staining. PI single staining cell cycle was detected by kit. The experiment was repeated 3 times.6. western blotHaCaT cells and 1×106 cells were inoculated with the same density, and the cells were treated with UVB irradiation when HaCaT cells grew to 70%-80%. The cells were collected at 0,6,12,18 and 24 h after irradiation, and the total protein of the cells was extracted. Add 20μg protein per hole,5% stacking gel and 10% separation gel by electrophoresis, and transferred to a polyvinylidene fluorine ethylene (polyvinylidene, PVDF) membrane,5% nonfat milk closed for 1 h at room temperature, primary antibody were incubated overnight at 4℃.After TBST rinse,the secondary antibody incubate for 1 hour. ECL luminescence kit for detection of luminous intensity. The experiment was repeated 3 times7. q-PCRTotal RNA was extracted, its concentration and purity were measured, The RNA reverse transcription was cDNA, Primer was first tested by PCR. And then PCR was amplified with cDNA. Fluorescence quantitative PCR was carried out using cDNA as raw material. The configuration of the reaction system were added to the q-PCR board, measured CT value. The relative mRNA content is calculated according to the formula.8. immunohistochemicalThe paraffin section of dewaxing.3% H2O2 incubation 5～10 minutes at the room temperature.5～10% normal goat serum blocking, incubation at room temperature for 10 minutes. Add appropriate dilution proportion of primary antibody. 37℃ incubation 1-2 hours or 4℃ for the night. Add appropriate dilution proportion of two resistance (1% BSA-PBS dilute). Add appropriate dilution proportion SABC. Chromogenic agent (DAB or AEC). Tap water rinse fully, after dyeing, dehydration, transparent (if necessary), sealing film, microscopy.9. Statistical analysisThe results expressed in x±S. Analysis by SPSS 20.0 software. Comparing multiple experimental group and control group mean using analysis of variance Dunnett t test. Two factor analysis of multiple time point data using factorial analysis of variance. The comparison of two sample mean using independent sample t test. Inspection level of alpha=0.05.Results1.The expression of mRNA and protein of 14-3-3σ in HaCaT induced by UVB0、6、12、18、24h after 30mJ/cm2 UVB irradiation, the expression of mRNA and protein levels of 14-3-3σ, compared with Oh group, increased gradually over time.2.14-3-3σ influence the proliferation of HaCaTAfter UVB irradiation, the cell density of HaCaT and HaCaTKD decreased in a dose-dependent manner at 48 h. The difference was statistically significant between HaCaT cells and HaCaTKD cells without irradiation (P<0.05). Compared with the non irradiated group, the normal HaCaT cells were significantly inhibited after 20 mJ/cm2 irradiation. (P<0.05). HaCaTKD cells were significantly inhibited after irradiation at 10 mJ/cm2 (P<0.05)Factorial analysis of variance,HaCaT group and HaCaTKD group at different time points of OD value, the interaction between group and time. The proliferation of HaCaTKD cells was significantly lower than that of HaCaT cells(P<0.05). At the same time point compared to the two groups, except 48 and 72 h time points in HaCaT group was higher than HaCaTKD group(P<0.05), there was no significant difference in other time points(P>0.05). Fixed groups at different time points, the differences were statistically significant.By factorial analysis of variance, UVB+HaCaT group and UVB+HaCaTKD group at different time points of OD value,the interaction between group and time. The proliferation of HaCaTKD cells was significantly lower than that of HaCaT cells(P<0.05). At the same time point compared to the two groups, In addition to the 0 h time point, the other time points had significant difference(P<0.05). Fixed groups at different time points, the differences were statistically significant (P< 0.05).3. 14-3-3σ influence HaCaT cell cycle.HaCaT cells in G2/M phase cells was increased at 6 h,12h peak,18 h decreased significantly, and the 3 time points, the differences were statistically significant compared with the 0 h(P<0.05). And HaCaTKD cell group, the observation time point compared with 0 h group, there were no statistically significant difference (P> 0.05).HaCaT cells in G2/M phase cells was increased at 6～18 h. The content of G2/M compared with 0 h group after HaCaT cells were treat with 6,12,18h (P> 0.05). the differences were statistically significant. In HaCaTKD cells, G2/M short period increased after irradiation at 6 h, then decreased gradually, no significant G2/M phase arrest.4. The expression 14-3-3σ and downstream cyclin proteins After UVB irradiationIn normal HaCaT cells, Irradiation after 6,12,18,24h,14-3-3σ expression was significantly increased compare with the Oh, (P<0.05),Cdc2 down-regulate,24h no rebound; Cdc25c、CyclinB1 down-regulate (P<0.05), Cdc25c have a slight rebound in 24 h, CyclinB1 have a slight rebound in 18、24h. In HaCaTKD cells,irradiation after 24h,14-3-3σ expression increased significantly (P< 0.05) compare with the Oh, Cdc25c,CyclinBl down-regulate,there was no significant change in the expression of Cdc2 at all time points (P> 0.05).5.After UVB irradiation,14-3-3σ synergy with the TPD52L1Sequencing analysis showed that after UVB irradiation, the transcription levels of 14-3-3 sigma first and gradually increased, and then decreased, and the transcription level of TPD52L1 then showed the opposite trend. Furder,Q-PCR method to verify the expression level of mRNA of 14-3-3σ and TPD52L1,it is found that the mRNA expression of 14-3-3σ and TPD52L1 trend coincides with sequencing results.14-3-3σ expression level gradually increased to reach the maximum after 18h began to decline, while TPD52L1 expression level gradually increased to 24h reaches a peak and then gradually decline.14-3-3σ is basically consistent with the trend of protein levels TPD52L16.The expressed of 14-3-3σ in skin squamous cell carcinoma and squamous cell carcinoma tissue samplesThe expression of 14-3-3σ in squamous cell carcinoma cells A431 is lower than normal skin cells. The expression of 14-3-3σ in skin squamous cell carcinoma was lower than that in adjacent normal tissues.Conclusion1. UVB irradiation can cause HaCaT cell growth inhibition, cycle arrest and induces apoptosis in a dose-dependent, and its mechanism may be 14-3-3σ regulate cycle related proteins Cdc2, Cdc25c, CyclinBl,and collaborative TPD52L12. The expression of 14-3-3σ in squamous cell carcinoma cells and squamous tissuse is lower than HaCaT... |
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