Mechanistic Study Of Positive Feedback Regulation Between DNA-PKcs And Akt In UV Induced Actinic Keratosis Occurrence

Part1 Tissue level detection of DNA-PKcs and Akt expression levels in AK and SCCObjective:Ultraviolet radiation can induce the occurrence of photo-induced keratosis(AK).This study explored the changes in the expression of DNA-PKcs and Akt in AK induced by ultraviolet radiation.Methods:Collect AK tissue(3 cases),squamous cell carcinoma tissue(3 cases),normal exposure tissue(1 case),and adjacent tissues(2 cases).1.Detect the mRNA expression level of DNA-PKcs and Akt by q-PCR,2.Extract the total protein of nucleus and cytoplasm respectively,WB detect the total protein expression level of DNA-PKcs and Akt,and the phosphorylation level of DNA-PKcs and Akt,3.Co-IP detects whether DNA-PKcs and Akt form a complex.Results:1.q-PCR results:Compared with normal exposed skin and adjacent tissues,the expression levels of DNA-PKcs and Akt in AK and SCC were significantly higher;2.WB results showed:in the nucleus and cytoplasm,AK,SCC has no significant difference in DNA-PKcs,Akt total protein and phosphorylated protein compared with normal exposed skin and adjacent tissues;3.Co-IP:DNA-PKcs are detected in normal exposed skin and adjacent tissues,AK and SCC,Akt forms a complex.Conclusions:1.The expression levels of 1.DNA-PKcs and Akt in tumor cells are increased;2.UV radiation promotes the expression of DNA-PKcs and Akt.Part 2 Cellular level study of the effects of ultraviolet rays on the expression and mutual combination of DNA-PKcs and AktObjective:Early studies at the tissue level found that the expression of DNA-PKcs and Akt was up-regulated in AK and SCC,and further studies on the effect of ultraviolet light on the expression of DNA-PKcs and Akt at the cellular level.Methods:Experiment 1:Culture primary keratinocytes,HaCaT cells,A-431 cells and SCL-1 cells.1.Detect the mRNA expression level of DNA-PKcs and Akt by q-PCR;2.Extract the total protein of nucleus and cytoplasm respectively,detect the total protein expression level of DNA-PKcs and Akt,and the phosphorylation level of DNA-PKcs and Akt by WB;3.Co-IP detects whether DNA-PKcs and Akt form a complex.Experiment 2:Culture primary keratinocytes and divide them into a control group and a UV irradiation group.The UV irradiation group was irradiated with 15mJ/cm2UAB.After irradiation for 2h,4h,24h,1.CCK8 to detect cell viability;2.Plate cloning experiment Detection of cell proliferation;3.Flow cytometric Annexin/PI detection of cell apoptosis;4,Nucleoplasmic separation,extraction of cytoplasm and nuclear proteins,Western Blot detection of DNA-PKcs and Akt total protein,phosphorylated protein expression differences;5.Nucleoplasm separation,extraction of cytoplasm,nuclear protein,Co-IP detection of DNA-PKcs and Akt complex;6.Fluorescence triple labeling for the co-localization of γ-H2AX,DNA-PKcs,and pan-AKT in cells to study DNA-PKcs,Whether Akt is located at the DNA damage,and the number of γ-H2AX focal points.Results:Results of experiment 1:1.q-PCR results:DNA-PKcs and Akt are highly expressed in A-431 cells and SCL-1 cells;2.WB results:p-DNA-PKcs and p-Akt in A-431 cells,SCL-1 cells are highly expressed;3.CO-IP results:DNA-PKcs and Akt complexes were detected in normal cells,A-431 cells,and SCL-1 cells.Results of experiment two:1.CCK8 results:After ultraviolet ray irradiated human keratinocytes,the cell viability increased for 2 hours,and the viability decreased for 24 hours,and the viability gradually decreased with time.2.The results of clone formation experiment:After ultraviolet ray irradiated human keratinocytes,cell proliferation increased for 2h,and proliferation decreased for 24h,and the vitality gradually decreased with time.3.Flow cytometry results:After ultraviolet ray irradiated human keratinocytes,early apoptosis,late apoptosis,and total apoptosis of cells were reduced for 2h,and apoptosis increased at 24h,and apoptosis gradually increased with time.After irradiating human keratinocytes with ultraviolet rays.4.WB results:After ultraviolet radiation keratinocytes,the expression of DNA-PKcs and AKT was up-regulated in the nucleus of cultured 24h,and the expression of p-AKT was up-regulated in the nucleus of 4h.5.CO-IP results:DNA-PKcs and Akt combined with each other in the nucleus and cytoplasm,and the combination of DNA-PKcs and Akt in the cytoplasm increased after UV irradiation.6.Immunofluorescence results:After keratinocytes irradiated by ultraviolet rays,the number of focal points of γ-H2AX increased after 24 hours of culture.Conclusions:1.The expression levels of 1.DNA-PKcs and Akt in tumor cells are increased;2.UV radiation promotes the expression of DNA-PKcs and Akt.Part 3 studies the positive feedback regulation mechanism of DNA-PKCs/Akt at the cellular levelObjective:Previous studies at the tissue level found that the expression of DNA-PKcs and Akt were up-regulated in AK and SCC,and it was also verified at the cell level in vitro that the expression of DNA-PKcs and Akt were up-regulated after ultraviolet radiation on cells,but the correlation between the two has not been shown yet.In this study,we investigated whether the DNA-PKcs and Akt formed positive feedback mechanism in UV-induced AK.MethodS:Primary keratinocytes were cultured in 10 experimental groups.①Primary keratinocytes ②Primary keratinocytes+15mJ/cm2 UVB irradiation③ Primary keratinocytes+si-NC④Primary keratinocytes+si-NC+15mJ/cm2 UVB irradiation⑤Primary keratinocytes+si-Akt ⑥Primary keratinocytes+si-Akt+15mJ/cm2 UVB ⑦primary keratinocytes+si-DNA-PK⑧primary keratinocytes+si-DNA-PKcs+15mJ/cm2 UV⑨primary keratinocytes+si-Akt+DNA-PK⑩primary keratinocytes+si-DNA-PKcs+si-Akt+15mJ/cm2 UVB.48h after transfection,15mJ/cm2UVB irradiation,2h,4h,24h after irradiation culture,1.CCK8 detection of cell viability;2.Cell proliferation was detected by plate cloning assay;3.Cell apoptosis was detected by flow Annexin/PI.4.Nuclear plasma separation,cytoplasm and nuclear proteins were extracted,and the differences in total protein and phosphorylated protein expression between DNA-PKcs and Akt were detected by Western Blot.5.Nuclear plasma separation,cytoplasm and nuclear proteins were extracted,and DNA-PKcs and Akt complex were detected by Co-IP.6,The co-localization of γ-H2AX,DNA-PKcs and Pan-Akt in cells was carried out with fluorescence three markers to study the negative location of DNA-PKcs and Akt in DNA damage sites and the number of γ-H2AX focal points.Results:1.CCK8 results:after UV irradiation,compared with the non-irradiation group,it promoted cell viability for 2h,inhibited DNA-PKcs and Akt for 24h.after uv irradiation,cell viability increased for 2h,and began to decrease with time,and showed inhibition for 24h.2.After UV irradiation,compared with the non-irradiated group,2h clones increased,24h clones decreased,and DNA-PKcs and Akt were inhibited.After UV irradiation,2h cell clones increased,with the increase of time,clone proliferation began to decrease,and 24h clone proliferation decreased obviously.3.Flow cytometry results:After UV irradiation,compared with non-irradiation group,early apoptosis,late apoptosis and total apoptosis decreased at 2h,increased at 24h,and inhibited DNA-PKcs and Akt.After UV irradiation,early apoptosis,late apoptosis and total apoptosis decreased at 2h,but increased with time,which showed early apoptosis,late apoptosis and total apoptosis at 24h.4.WB results:Compared with the non-irradiated group,the expressions of DNA-PKcs,Akt,p-DNA-PKcs and p-Akt were up-regulated in the nucleus and cytoplasm,which inhibited DNA-PKcs and Akt.5.CO-IP results:After the interference of DNA-PKcs and Akt,the binding between DNA-PKcs and Akt decreased,and after UV irradiation,the binding between DNA-PKcs and Akt increased significantly,especially in cytoplasm.6.Fluorescent immune results:DNA-PKcs and Akt exist in nucleus and cytoplasm,while DNA-PKcs is mainly located in nucleus and Akt is mainly located in cytoplasm.After UV irradiation,DNA damage of cells caused DNA-PKcs to shift from nucleus to cytoplasm,phosphorylated Akt in cytoplasm,and caused Akt to shift to nucleus.When interfering with DNA-PKcs and Akt alone,the focus number of γ-H2AX increased with the extension of UV irradiation time,while when interfering with the expression of DNA-PKcs and Akt,the trend of increasing focus number of γ-H2AX with the extension of UV irradiation time slowed down.Conclusions:1.DNA-PKcs and Akt may play important roles in tumor formation;2.The positive feedback regulation of DNA-PKCs and Akt does not seem to play an important role in the UV-induced Ak.Part4 The positive feedback regulation mechanism of DNA-PKcs/Akt was studied at the mouse levelObjective:In this study,we investigated the positive feedback effect of DNA-PKcs/Akt in UV-induced AK at the animal level.Methods:Five C57B/6 mice(18-22g)were selected as the control group,and five PRKDC-/-mice with gene knockout DNA-PKcs were selected as the experimental group.Both groups were treated with ultraviolet radiation at the same time.The ultraviolet radiation was treated with a mixed UVA and UVB suberythema dose(90%MED),which was increased to 1 MED in the third week.After that,the MED was increased by 12.5%every week.At the 8th week,the irradiation dose was increased to UVB325mJ/cm2 and UVA4875mJ/cm2.After that,the irradiation dose was maintained for 20 weeks.Some skin tissues were isolated from epidermal tissue for WB detection of relative expression levels of Akt,DNA-PKcs,Akt Ser473,Akt Thr308,Bax,Bcl-2,p53,p53 serine-15,SP1,c-fos and c-myc.Part of skin tissue(including true epidermis)was removed for HE staining and histopathological identification.Immunofluorescence confocal staining was used to detect and count γ-H2AX focus(confocal),Ki-67(immunofluorescence)was used to detect cell viability,and Tunel staining was used to detect cell apoptosis.Results:1.The experimental group grew new organisms at 12 weeks,the control group grew new organisms at 18 weeks,and the experimental group grew new organisms earlier;2.After 20 weeks of irradiation,the back tissue protein WB of mice with DNA-PKCs knockout showed decreased expression of Akt S473,Aktt308,Bcl2,Bcl2/Bax and c-fos,and the results were statistically significant(p<0.05);The expression of p53 serine-15 was upregulated,and the results were statistically significant(p<0.05);2.Compared with the control group,the number of γ-H2AX decreased,the cell viability decreased and the apoptosis increased in the experimental group,and the results were statistically significant(p<0.05).Conclusions:1.The positive regulation of DNA-PKcs on Akt was verified at the animal level;2.DNA-PKcs can promote cell proliferation,inhibit apoptosis,and play a role in promoting cancer.