Effects Of GRP78 On The Biological Behavior Of SiHa Cells And Its Mediated Mechanism Of Paclitaxel-induced Pyroptosis In SiHa Cells

Objective:The expression of glucose-regulated protein 78（GRP78）is significantly increased in a variety of malignant tumors and is involved in tumor proliferation,invasion,migration and drug resistance,and its expression level is correlated with the prognosis of patients.The purpose of this study was to investigate the effects of GRP78 protein on the biological behavior of cervical cancer SiHa cells and the effects of GRP78 small interfering RNA combined with paclitaxel treatment on drug sensitivity of SiHa cells and the mechanism of action.Thus provide a new theoretical basis for clinical diagnosis and treatment of cervical cancer.Methods:1.Cell Culture:Establishment of SiHa cells culture model in vitro.2.Transfection:GRP78-siRNA was transfected into SiHa cells mediated by liposome to ensure successful transfection,and the siRNA with the highest silencing efficiency was selected.3.qRT-PCR:It is used to determine the mRNA expression levels of GRP78,Caspase-1,GSDMD and IL-1β.4.Western Blot:It is used to detect the expression levels of GRP78,P-AKT^ser473,P-GSK3β^ser9,AKT,GSK3β,Cyclin D1,CDK4,CHOP,Bcl-2,E-cadherin,N-cadherin,Vimentin,Caspase-1,GSDMD and IL-1β.5.Plate Clone Formation Assay:It is used to detect the proliferation ability of SiHa cells.6.Scratch Test And Transwell Test:It is used to detect the changes in migration and invasion ability of SiHa cells.7.Flow Cytometry:It is used to detect the effect of transfection on the apoptosis rate of SiHa cells.8.MTT:It is used to measure the half inhibitory concentration（IC50）of paclitaxel on SiHa cells.9.Statistical Methods:Graph Pad Prism（Version5）software was used for data analysis and statistics.Experimental results were expressed as the mean±standard deviation.Independent sample t test was used for comparison between the two groups of data,P<0.05 means the difference is statistically significant.Results:1.Effects of siRNA transfection at different sites on the expression of GRP78The expression levels of GRP78 protein and GRP78 mRNA in SiHa cells transfected with GRP78-siRNA were significantly decreased,and the silencing effect of GRP78-siRNA1501 interference chain was the most significant.2.Effect of GRP78 gene silencing on proliferation of SiHa cellsSilencing GRP78 gene resulted in a significant decrease in the number of SiHa cell clones（P<0.001）.To further clarify the mechanism of action,the expression levels of AKT and GSK3βwere detected,the results showed that the expression levels of P-AKT^ser473and P-GSK3β^ser9were significantly decreased,while the total AKT and GSK3βremained basically unchanged.In addition,the downstream molecules cyclin D1 and CDK4 proteins were also significantly reduced（P<0.001）.3.Effects of GRP78 gene silencing on the migration and invasion of SiHa cellsSilencing GRP78 gene significantly reduced the mobility of SiHa cells at12h,24h and 48h detection points（P<0.01）.Transwell experiment showed that the number of transmembrane of SiHa cells was significantly reduced（P<0.001）.In order to further explore the mechanism of action,the expression levels of EMT-related proteins were further detected,and the results showed that E-cadherin expression was significantly increased,while the N-cadherin and Vimentin expressions were significantly decreased（P<0.001）.4.Effects of GRP78 gene silencing on apoptosis of SiHa cellsSilencing GRP78 gene resulted in CHOP protein increased significantly,while Bcl-2 protein decreased significantly（P<0.01）.The apoptosis rate of SiHa cells was significantly increased（P<0.001）.5.Effects of GRP78 gene silencing combined with paclitaxel treatment on drug sensitivity of SiHa cellsGRP78 small interfering RNA combined with paclitaxel treatment,gene level results showed that the expression levels of caspase-1 mRNA,GSDMD mRNA and IL-1βmRNA were significantly increased with the addition of paclitaxe,when GRP78small interfering RNA was combined with paclitaxel,the expression levels of caspase-1 mRNA,GSDMD mRNA and IL-1βmRNA were increased more obviously,the differences were statistically significant（P<0.001）;Protein level results showed that the general trends of caspase-1,GSDMD and IL-1βprotein expression were consistent with gene level.Conclusions:1.GRP78 protein can promote the proliferation of SiHa cells,it may function through activation of the AKT/GSK3β/Cyclin D1 pathway.2.GRP78 protein can promote the invasion and migration of SiHa cells,which may be related to the promotion of EMT process.3.Silencing GRP78 gene significantly increased the apoptosis rate of SiHa cells and the expression of CHOP protein.4.Paclitaxel can induce pyroptosis in SiHa cells and decrease the expression of GRP78 protein can promote the paclitaxel-induced pyroptosis process,thereby increasing the sensitivity of SiHa cells to paclitaxel.