| Objective: How human telomerase reverse transcriptase(hTERT)regulate the ferroptosis of gatric cancer cells.Methods: 1)Erastin trialing 5 gastric cancer cells :SNU-1,SGC7901 gastric cancer cells,MGC803,AGS,MKN45 for 24 hours according to concentration of 1 uM and 2.5 uM,5 u,10 uM,20 uM,40 uM to build the ferroposis in gastric caner cells modle.Erastin trial 5 cancer cells for 24 hours,with CCK 8 testing growth inhibition rate,5 strains of gastric cancer cells by q-PCR detection the expression of hTERT and SLC7A11,GPX4 experiment to determine future research object for the SNU-1 and SGC791 these two strains of gastric cancer cells.2.To study whether erastin kill tumor cells by inducing death,with 20 um erastin SNU-1,two strains of SGC7901 gastric cancer cells,then observe the changes of mitochondria in cells by transmission electron microscopy(sem)imaging and other changes of organelles.Respectively with iron inhibitors Fer-1 death,apoptosis inhibitor,Z-VAD-FMK produce most necrosttin necrosis inhibitors-1,autophagy inhibitor 3-MA and erastin processing cells for 24 hours,by CCK-check growth inhibition rate,GSH detection kit to detect the contents of intracellular GSH,MDA kits intracellular lipid peroxidation.3.The study of hTERT iron dies,first through the analysis of the GEO database for gastric cancer,the expression of hTERT and GPX4 related,and erastin stimulate two strains of gastric cancer cells after extracting protein hTRET protein detection.By constructing hTERT stable expression cell lines lenti-hTERT-SNU-1 / lenti-NC-SNU-1;Lenti-hTERT-SGC7901 / lenti-NC-SGC7901.With erastin(20 um)cells after 24 hours,CCK 8 testing growth inhibition rate,Western blot test cells hTERT,the expression of p53,SLC7A11,GPX4,q-PCR to detect the expression of p53.Treated with proteasome inhibitors MG123 and erastin cells,detection of p53 protein in the cell level.4.To study whether hTERT promote p53 protein degradation by MDM2,build the Si-h-MDM2 MDM2 expressed sequence interference,after erstin processing detection level of p53 protein in the cell.5.Use the Graph pad Prism 6.0 data analysis,an analysis of the experimental results,the p value is less than 0.05.Results:1.The growth inhibition of five gastric cancer cells is different when treated with erastin for 24 h according to various concertration,among of these five cells,SNU-1 and SGC7901 gastric cancer cells exploit the highly growth inhibitiong,and q-pCR test shows that the mRNA level of SLC7A11、GPX4 in these two selected celss is low.Erastin processing after 24 hours tem imaging tip cell mitochondria,mitochondrial membrane density increased.Death inhibitors fer-iron erastin + 1 set of growth inhibition rate was lower than those of erastin group,intracellular GSH content is high in the erastin group,the MDA level was lower than those of erastin group.3.Through the analysis of the database,hTERT in gene expression in gastric cancer and GPX4 correlation coefficient is 0.22,p value is less than 0.0001.Erastin treated cells h TERT protein levels rise.4.HTERT stable expression in SNU-1,SGC7901 gastric cancer cells,the expression group SNU-1 cell than the control group after 24 h in erastin processing growth inhibition rate is low.SGC7901 cells express group and control group has no statistically significant difference.HTERT in gastric cancer cells,the expression of SNU-1 cells after erastin processing p53 protein levels,mRNA level without change.SLC7A11 and GPX4 protein level to rise,but there was no statistically significant difference than the control group SGC7901 cells.MG132 inhibitor treatment after gastric cancer cell expression of hTERT SNU-1 the p53 protein levels rise.4.HTERT and MDM2 were located in the nucleus,the interference lenti hTERT-MDM2 expressed in the cell,SNU-1 level of p53 protein in the cell responses.Conclusions: Erastin has induced the ferroptosis in SNU-1 and SGC7901 cells,hTERT has rescued this ferroptosis through promoting wild type p53 protein degradation via the ubiquitin-proteasome pathway and relying on MDM... |
PDF Full Text Request