Study On The Mechanism Of Calreticulin Exposure Induced By Thrombospondin-1 In Human Mucoepidermoid Carcinoma MC-3 Cell Line By PERK/eIF2α Signaling Pathway

Objective:To study the immunogenic death of human mucoepidermoid carcinoma cell line MC-3 induced by thrombospondin-1,and to further explore whether thrombospondin can induce calreticulin exposure in human mucoepidermoid carcinoma cell line MC-3 by PERK/e IF2αsignaling pathway.Methods:1.Mucoepidermoid carcinoma MC-3 cell line was selected as the research object,CCK-8 was used to screen the optimal seeding density of MC-3 cells,and the growth curve of MC-3 cells was drawn.At the same time,the optimal concentration and time of TSP-1 were detected;2.Flow cytometry Annexin V/PI double staining method was used to detect the apoptosis of cells in different experimental groups.The morphology of MC-3 cell line was observed by morphological changes of cells in control group,TSP-1-treated group,PTX-treated group and TSP-1+PTX-treated group were observed under microscope;3.The cells were divided into control group,PTX-treated group,TSP-1-treated group,TSP-1+ISRIB-treated group,TSP-1+PTX-treated group,and the changes of CRT expression in each group were detected by flow cytometry;4.The expression of PERK,e IF2αand CRT in MC-3 cell line was detected by immunofluorescence detection,Western blot,QPCR and other experimental methods;5.The above experiments were repeated three times independently.The experimental results were analyzed by Graph Pad prism 8.0 statistical software,and the statistical results were expressed by mean±standard deviation.The comparison between multiple groups was performed by one-way ANOVA of multi-sample mean,and the comparison between the two groups was performed by independent sample t test;P<0.05 means that the difference is statistically significant.Results:MC-3 cell line can reach the growth plateau on the sixth day according to the inoculation density of 2×10~4/m1,which meets the experimental requirements;Furthermore,CCK-8 experiment was used to detect the cytotoxicity of TSP-1.The results showed that0.1μmol/L was the best concentration and 72 hours was the best detection point.The results of flow cytometry,immunofluorescence staining,Western blot,and QPCR experiments all suggested that TSP-1 could induce the exposure of calreticulin CRT in MC-3 cell lines.The difference was statistically significant compared with the control group(P<0.05),but compared with the positive control group,the difference was not statistically significant(P<0.05).The experimental results also showed that after TSP-1acted on MC-3 cell lines for 72 hours,the simultaneous up-regulation of CRT was accompanied by the up-regulation of PERK and e IF2α(P<0.05);TSP-1 combined with paclitaxel can significantly enhance the above experimental process,and the up-regulation of PERK,e IF2αand CRT is more obvious than that of single drug(P<0.05);After 72hours of ISRIB inhibitor treatment,the results showed that the expression of PERK,e IF2αand CRT were down-regulated,and the difference was statistically significant(P<0.05).Conclusion:TSP-l protein can induce immunogenic cell death in mucoepidermoid carcinoma MC-3 cell line,which is accompanied by exposure of CRT,a damage-related molecular model,and may be related to the activation of PERK/e IF2αsignaling pathway.