Effects Of Epidermal Permeability Barrier Disruption On UV Induced Pigmentation And Its Mechanisms

Part ?:To verify the effect of TGF?1/Smad signaling pathway on the expression of DNA-PKcs at the cellular level and its mechanismObjective:In order to demonstrate the interaction between the two pathways,this study reversely investigated the effect of TGF-?1/Smad on the level of DNA-PKcs activation at the cellular level.Method:1.The SCL-1 squamous cell carcinoma cell line was selected in this experiment.The upstream factor TGF?RI of TGF-?1/Smad signaling pathway was selected as the target gene,and SCL-1 cells were transfected and expressed from the upstream blocking pathway.This is the experimental group Si-TGF?RI.Transfection of internal reference to normal SCL-1,this is the control group Si-NC.Then,CCK8 cell proliferation assay,EdU assay,and cell scratch assay were performed to detect cell viability,cell proliferation,cell migration ability,etc.of the experimental and control cells,respectively.Real-time quantitative PCR was used to detect the expression of DNA-PKcs gene in SCL-1 squamous carcinoma cells transfected with the target gene TGF?RI.The expression and activation level of DNA-PKcs in SCL-1 squamous carcinoma cells transfected with the target gene TGF?RI were detected by Western Blot.2.SCL-1 cells were transfected into SMD3,a downstream factor of TGF-?1/Smad signaling pathway,and expressed from the downstream blocking pathway.This is the experimental group Si-smad3.Transfect the internal reference to normal SCL-1,which is the control group Si-NC.Then,CCK8 cell proliferation assay,EdU assay,and cell scratch assay were performed to detect cell viability,cell proliferation,cell migration ability,etc.of the experimental and control cells,respectively.The expression of DNA-PKcs gene in SCL-1 squamous carcinoma cells transfected with the target gene Smad3 was detected by real-time fluorescent quantitative PCR.Western blot was used to detect the expression and activation level of DNA-PKcs in SCL-1 transfected with Smad3 in squamous cell carcinoma cells.Results:1.After 24 hours of culture,the SCL-1 cells transfected with si-TGF?RI and si-GAPDH showed that the activity of the cells in the experimental group decreased and the proliferation ability decreased,and the results were statistically significant(P<0.05).After incubation with si-smad3 and si-GAPDH transfected SCL-1 cells for 24 hours,the results of CCK8 showed that the cell viability and proliferation ability of the experimental group were decreased,and the results were statistically significant(P<0.05).2.The results of EDU test of SCL-1 cells transfected with si-TGF?RI and si-GAPDH showed that the proliferation of SCL-1 in the experimental group was decreased(P<0.05),and the results were statistically significant.The EDU test results of Si-smad3 and si-GAPDH transfected SCL-1 cells showed that the proliferation of SCL-1 in the experimental group was decreased(P<0.05),and the results were statistically significant.3.The cell scratch test results of si-TGF?RI and si-GAPDH transfected SCL-1 cells showed that the cell migration ability test group was slightly lower than the control group,but the results were not statistically significant(P>0.05).The cell scratch test results of si-smad3 and si-GAPDH transfected SCL-1 cells showed that the cell migration ability test group was slightly lower than the control group,but the results were not statistically significant(P>0.05).4.The PCR results of si-TGF?RI and si-GAPDH transfected SCL-1 cells showed that the expression of DNA-PKcs gene was increased in the experimental group compared with the control group,and the results were statistically significant(P<0.05).PCR results of si-smad3 and si-GAPDH transfected SCL-1 cells showed that the expression of DNA-PKcs gene was decreased in the experimental group compared with the control group,and the results were statistically significant(P<0.05).5.Western Blot results of si-TGF?RI and si-GAPDH transfected SCL-1 cell proteins showed that the expression of DNA-PKcs and P-DNA-PKcs was increased in the experimental group of SCL-1 cells,and the results of this Western Blot were The PCR expression was the same and statistically significant(P<0.05);The results of Western Blot of Si-smad3 and si-GAPDH transfected SCL-1 cells showed that the expression of DNA-PKcs was slightly decreased in the SCL-1 cells of the experimental group,and the expression of P-DNA-PKcs was not significantly different.There was no statistical significance(P<0.05).Conclusion:1.The main role of TGF?1/Smad signal transduction pathway in squamous cell carcinoma is to promote the proliferation of cancer cells.2.The upstream factor TGF?RI of TGF?1/Smad inhibits the expression and activation of DNA-PKcs.3.The downstream factor Smad3 of TGF?1/Smad had no significant effect on the expression and activation of DNA-PKcs.Part ?:Animal Level Verification of the Effect of DNA-PKcs on the Expression of Two Pathways Under Ultraviolet IrradiationObjective:In order to clarify the interaction between two pathways induced by ultraviolet light(dna-pkcs),this study investigated the effect of dna-pkcs on the expression of key proteins in the two pathways under long-term ultraviolet irradiation at animal level.Method:1.Three C57 female mice(6 weeks old)with knockout DNA-PKcs were used as the experimental group,and 3 normal C57 female mice were selected as the experimental group.Under the same feeding conditions,the mice were sacrificed at a dose of gradually increasing the MED value for 12 weeks,and the back skin of the mice was taken for subsequent protein detection.2.Extracting the back skin of the mouse to extract tissue protein,PIKKs/Akt and TGF?1/Smad-related proteins AKT,P-Akt,S6,P-S6,smad3,P-smad3,and TGF?RI were used to detect pathway expression and activation under the influence of DNA-PKcs under ultraviolet irradiation.Results:1.PIKKs/Akt(DNA-PK/Akt)signaling pathway test results:After 12 weeks of irradiation,Western Blot of the back skin tissue protein of mice knocked out DNA-PKcs showed that the expression of protein AKT,P-Akt,S6 and P-S6 was decreased.The activation state of AKT is lowered,and the activation state of S6 is also lowered.The results were statistically significant(P<0.05).2.TGF?1/Smad signaling pathway detection results:After 12 weeks of irradiation,Western Blot of the dorsal skin tissue protein of mice knocked out DNA-PKcs showed that the expression of Smad3,P-Smad3 and TGF?RI was increased.Smad3 activation state increased.The results were statistically significant(p<0.05).Conclusion:Under long-term ultraviolet irradiation,blocking the expression of dna-pkcs plays an important role in delaying the carcinogenesis and development of squamous cell carcinoma,and can provide a new way of thinking for clinical drug intervention therapy targets.