Deoxypodophyllotoxin Induces Autophagy And Apoptosis Through Inhibit GRP78 In Osteosarcoma Cells

Objective and Significance:Osteosarcoma(OS)is one of the common malignant tumors in children and adolescents.At present,there is a lack of effective OS treatment targets and diagnostic markers,which limits the clinical diagnosis,treatment and prognosis of the disease.Therefore,it is of great significance to find functional prognostic markers of OS.Glucose-regulated protein 78 is a stress partner glucose regulated protein encoded by heat shock protein family A(Hsp70)member 5(HSPA5),which is considered to be a key diagnostic and prognostic biomarker in OS.In this study,we used bioinformatics methods to analyze OS gene expression profile chip,screen and analyze OS prognostic genes and pathways,and explore the pathogenesis of OS at the molecular level.To indicate the direction for the molecular mechanism of deoxypodophyllotoxin(DPT)in the treatment of OS through GRP78,and provide a strong basis for DPT as a potential therapeutic drug for OS.Methods:Based on GEO database,bioinformatics methods were used to screen OS-related differentially expressed genes for biological analysis,focusing on the potential correlation between HSPA5,PSAT1 and ANXA1.The specimens of osteosarcoma and paired adjacent tissues were analyzed by IHC.HSPA5 gene was stably transfected into OS cells for RT-PCR.MTT,Edu and plate clone formation experiments were used to investigate the effect of DPT on the proliferation of OS cells.WB was used to preliminarily explore the molecular mechanism of DPT inhibiting OS.The effect of DPT on apoptosis of OS cells was analyzed by flow cytometry,and the molecular mechanism of DPT promoting apoptosis was explored.The role of GRP78 in the treatment of osteosarcoma by DPT was observed in animal experiments.The molecular mechanism of GRP78 was screened by RNA-seq,and the expression of related proteins was detected by WB and other methods to verify the results of RNA-seq screening.Results:1.Bioinformatics analysis showed that HSPA5 and ANXA1 were enriched in the process of calcium binding and cell adhesion molecule binding,suggesting that they may be closely related at the molecular level.HSPA5 and PSAT1 were highly expressed in osteosarcoma tissues,while ANXA1 was down-regulated.Survival analysis showed that HSPA5 and PSAT1 levels were negatively correlated with OS prognosis.ANXA1 level was positively correlated with OS prognosis.2.In vitro experiments showed that the PSAT1 level in OS cells overexpressing HSPA5 was up-regulated,but had no significant effect on ANXA1.HSPA5 silencing could significantly increase ANXA1 and inhibit the expression of PSAT1.Inhibiting HSPA5 can prevent OS progression by regulating ANXA1 and PSAT1.3.IHC study showed that GRP78 was highly expressed in OS tissues.GRP78 expression was found to be increased in 16(80.0%)of 20 OS tissues,while only 5(25.0%)of paired adjacent normal tissues.GRP78 overexpression was not significantly correlated with age,gender,tumor size,differentiation degree,anatomical location,serum lactate dehydrogenase and alkaline phosphatase levels and distant metastasis,but was closely correlated with clinical stage of OS.4.With the increase of DPT concentration,the expressions of GRP78 and PSAT1 in OS cells were gradually down-regulated and the expression of ANXA1 was up-regulated in a time-dependent manner.DPT promotes apoptosis by up-regulating cPARP and Bax,and down-regulating apoptosis-related genes such as HSPA5,Bcl-2,CCND1 and BIRC5.5.MTT,EdU and plate cloning results showed that DPT significantly inhibited the proliferation of OS cells.DPT induces OS cell apoptosis by stimulating cytochrome C release and Caspase cascade activated mitochondrial signaling pathway,and then affects OS cell growth.6.Flow cytometry showed that DPT can induce apoptosis of OS cells,GRP78 can significantly reduce apoptosis induced by DPT.Knockdown of GRP78 could inhibit the growth of osteosarcoma in mice in coordination with DPT.7.For RNA-seq of siGRP78 HOS cells,it was found that inhibition of GRP78 regulates the expression of autophagy related genes in OS cells.DPT promotes autophagy in OS cells in a dose-dependent manner by inhibiting PI3K/AKT/mTOR signaling pathway.Conclusions:This study shows that DPT promotes OS cell apoptosis by inhibiting the expression of GRP78 and activating the caspase signal cascade.It also promotes OS cell autophagy by inhibiting the PI3K/AKT/mTOR signaling pathway,which plays an important role in OS cell survival.