Mechanism Of Proliferation Regulation By TROAP In Human Normal Hepatocyte HL-7702 And Hepatocellar Carcinoma HepG2 Cells

TROAP is a cytoplasmic protein,which plays an important role in embryo implantation and microtubule regulation and is one of the important cytoplasmic proteins required for spindle assembly and cell invasion.It is highly expressed in a variety of cancer cells,and the role of TROAP in gastric cancer,breast cancer and other cancers has been reported in previous studies,but there are few studies on its role in liver cancer and related diseases.In this paper,the online bioinformatics analysis website GEPIA2 was used for statistical analysis of TCGA and GTEX tumor databases.We found that the expression of TROAP gene was higher in various cancers than in adjacent tissues,and the difference was significant.The survival rate of HCC patients with low expression was higher（P<0.05）.In the High-throughput sequencing,we found that the expression of TROAP increased during 24-72 h after partial hepatectomy,which was in the proliferative stage of liver regeneration,suggesting that TROAP may play an important regulatory role in the proliferation of liver cells.The above information suggests that TROAP has an important function in the liver.Therefore,in this study,human normal cell HL-7702 and hepatoma cell HepG2 were used as the main experimental materials to study the role of TROAP in the regulation of cell proliferation and its specific mechanism.In this study,siRNA knockdown gene was transfected to down-regulate the expression of TROAP,and the viability of cells was detected by CCK-8 method.The results showed that 48 h after transfection,the viability of cells in the experimental group was significantly decreased compared with the control group（P<0.05）.Ed U kit was used to detect cell proliferation,and the results showed that knocking down the expression of TROAP inhibited the proliferation of HL-7702 and HepG2 cells.Cell migration ability was detected by scratch assay and Transwell assay,and the results showed that the downregulation of TROAP expression slowed down the invasion and metastasis of HepG2 cells.Meanwhile,in HL-7702 cells,the contents of ALT,AST and GSH decreased in the experimental group.Flow cytometry and fluorescence staining were used to detect the cell cycle and apoptosis.It was found that the downregulation of Troap expression in both groups could significantly block the process of cell cycle（P<0.01）and promote cell apoptosis.Immunofluorescence,quantitative real-time PCR and Western blot results showed that the expression levels of p-AKT,P65,BCL-2,CASEPASE3 and other genes and proteins in PI3K/AKT,NF-κB,mitochondrial apoptosis pathway and other signaling pathways were significantly changed in both cells（P<0.01）.These results suggest that TROAP may regulate the proliferation of HL-7702 and HepG2 cells through PI3 K,NF-κB,AKT,mitochondrial apoptosis pathway and other related signaling pathways.This study is expected to provide a new target for the diagnosis and treatment of liver-related diseases.