Study On The Antiproliferation Of Cardamonin By Suppression Of The Expression Of Raptor On HepG2 Cells

Aim1 To clarify the correlation between the expression of RPTOR（regulatory associated protein of mTOR）gene and the occurrence and prognosis of hepatocellular carcinoma.2 To investigate the mechanism of cardamonin（CAR）down-regulating the expression of Raptor protein in HepG2 cells and affecting its proliferation and apoptosis.Methods1 Bioinformatics analysis1.1 Download the TCGA database（the Cancer Genome Atlas）,find the transcriptional expression level of RPTOR gene in HCC,analyze the clinical information data of liver Cancer patients in the database,and discuss the relationship between RPTOR gene and clinicopathological characteristics and staging of liver Cancer patients;1.2 To analyze the mutation and co-expression of RPTOR gene in HCC;1.3 Kaplan-Meier method was used to analyze the relationship between the expression of RPTOR gene and the prognostic indicators such as Overall survival（OS）and disease-free survival（DFS）of hepatocellular carcinoma,so as to identify RPTOR gene as an independent prognostic indicator of HCC.2 Cell-level validation CAR targets the mTOR signaling pathway to inhibit the proliferation and apoptosis of HCC HepG2 cells2.1 cell cultureHuman HepG2 cell lines were cultured at 37℃and 5%CO₂ condition of incubator,and cultured with glucose DMEM,10%fetal bovine serum,100 g·ml^-1streptomycin and 100 U·ml^-1 penicillin medium.When the cells were in good condition and grew adherent to 90%,trypsin was added for digestive passage.2.2 Groups and drugs interventionHuman HepG2 cell lines were divided into blank control group,CAR high and low concentration group（10,40μM）and positive control group:rapamycin（0.1μM）and AZD8055（0.1μM）.2.3 Determination of cell proliferation and protein expression2.3.1 CCK-8 assay were used to detect the effects of CAR（10,40μM）at different concentrations on the proliferation of HepG2 cells;2.3.2 Ed U staining assay the effects of CAR（10,40μM）at different concentrations on the proliferation of HepG2 cells;2.3.3 Flow cytometry was used to detect the effects of CAR（10,40μM）at different concentrations on apoptosis of HepG2 cells;2.3.4 Western Blotting was used to examine the presentation of apoptotic protein cleaved caspase 3,mTORC1 signaling pathway key proteins mTOR,p-mTOR、S6K1、p-S6K1 and mTORC1 specific binding protein Raptor.Results1 RPTOR gene is highly expressed in HCC and indicates poor prognosis.1.1 The expression level of RPTOR gene in HCC tissues was significantly higher than that in normal tissues（P<0.05）.There was no statistical difference in the expression of RPTOR in HCC patients with different race,gender,age,weight and tumor stage;1.2 RPTOR gene was mutated in 5%of patients with HCC;1.3 The overall survival rate and disease-free survival rate of HCC patients with high expression of RPTOR gene were significantly lower than those with low expression of RPTOR（P<0.05）;1.4 The co-expression of RPTOR gene and protein network analysis suggested that RPTOR was involved in cell proliferation,differentiation,apoptosis and glycolipid metabolism;2 CAR decreased the expression of Raptor protein In HepG2 cells;3 CAR inhibited the proliferation of HepG2 cells in a dose-time dependent manner;4 CAR dose-dependent induction of apoptosis in HepG2 cells increased the expression of apoptotic related protein cleaved caspase3;5 CAR inhibites the phosphorylation of mTOR and S6K1 protein in a concentration-dependent manner,but not affect the total protein expression.Conclusion1 RPTOR gene is highly expressed in patients with hepatocellular carcinoma and affects the overall survival rate and disease-free survival rate;2 CAR inhibits the proliferation of HepG2 cells and promotes their apoptosis,and its mechanism may be related to suppression of the expression of Raptor protein and then result in inhibition of mTORC1 activity.