Molecular Mechanism Of SCUBE3 Promoting Proliferation Of Hepatoma Cells Through TGFβ Pathway

Background and Purpose:Hepatocellular carcinoma is the fifth most common cancer in the world.The annual death number from hepatocellular carcinoma exceeds 600,000,accounting for one-third of the death number.It is a serious health problem worldwide.The occurrence of hepatocellular carcinoma is a complex process involving many factors,including genetic alterations,abnormal expression of certain proteins,mutations in tumor suppressor factors,and overexpression of oncogenes.It has been determined that the occurrence of hepatocellular carcinoma is associated with many signaling pathways,such as Wnt signaling pathway,MAPK signaling pathway,TGFβ signaling pathway,and NF-κB signaling pathway.Among them,TGF-β signaling is involved in all stages of liver disease progression,from initial liver damage to inflammation,fibrosis,cirrhosis,and tumorigenesis.Studies have shown that the TGF-β signaling pathway plays an important regulatory role in a variety of liver diseases including liver cancer.Recent studies have found that SCUBE3(signal peptide-CUB-EGF-like domain protein 3)can trigger downstream signaling pathways involved in the regulation of various biological processes by binding to the TGF-beta receptor.Studies have shown that SCUBE3 can participate in mouse cardiac hypertrophy and embryo development by regulating TGF-β1-mediated signal transduction.Also.Studies have shown that SCUBE3 is involved in the biological processes of various tumors.For example,SCUBE3 can promote the metastasis and invasion of non-small cell lung cancer by regulating the angiogenesis of lung cancer and promoting mesenchymal transition of epithelium.SCUBE3 can also promote osteosarcoma cells,and is associated with survival prognosis in patients with osteosarcoma.However,the current expression and biological function of SCUBE3 in liver cancer is still unclear.In this study,we constructed a human hepatoma cell line knocked down of SCUBE3,and studied its regulation effect on the proliferation of hepatoma cells by subcutaneous implantation,gene expression profiling,immunoprecipitation,Western Blot,and other means.Methods: The first part:(1)This study first used qRT-PCR and Western blot to detect the expression of SCUBE3 in human hepatoma cell lines Bel7404,Bel7402,HepG2,SMMC7721 and human normal liver cell line LO2.(2)In vitro experiment: The SCUBE3 knockdown Bel7404 cell model was constructed by lentiviral vector transfection technique,and the effect of SCUBE3 on the proliferation of hepatoma cells was detected by MTT assay and EDU assay.The effects on hepatoma cells apoptosis were investigated by Annexin V-APC & PI double staining and Caspase 3/7 activity assay.In addition,the effect of SCUBE3 on the cell cycle of liver cancer was examined by flow cytometry.(3)In vivo experiment: The effect of SCUBE3 on the proliferation of hepatoma cells was investigated by subcutaneous tumor formation in nude mice.The second part:(1)Screening the differentially expressed genes after SCUBE3 knockdown using Affymetrix gene expression profiling chip: To explore the molecular mechanism of SCUBE3 promoting hepatoma cell proliferation,firstly,the differentially expressed genes after SCUBE3 knockdown was screened by Affymetrix gene expression profiling chip.And a series of bioinformatics analyses include mapping scatter plots,volcano plots,hierarchical clustering analysis,and enrichment of diseases and functions to analyze the function of differential genes.(2)Identification of downstream target molecules regulated by SCUBE3: Firstly,genes closely related to the regulatory cell cycle were identified in differentially expressed genes and verified by Western blot to identify downstream target molecules of SCUBE3.The target molecule was down-regulated by lentiviral transfection technology,followed by investigation of the effect of downstream target molecules on proliferation,apoptosis and cell cycle of hepatoma cells.Subsequently,the co-transfection technique was used to simultaneously knock down SCUBE3 and over-regulate the target molecule in the liver cancer cells,and the proliferation,apoptosis and cycle changes of liver cancer cells were detected.(3)To explore the molecular mechanism of SCUBE3 regulating downstream target molecules: Firstly,it was confirmed by immunoprecipitation technique whether SCUBE3 can bind to TGFβRII receptor.In addition,the differentially expressed genes after SCUBE3 knockdown were subjected to pathway enrichment,and key signal pathways were screened and the expression of key molecules was verified by western blot.Results: The first part:(1)The expression of SCUBE3 in liver cancer cells was higher than that of normal human liver cells,and it was the highest in liver cancer cell line BEL7404.(2)In vitro experiments: MTT assay and EDU results showed that SCUBE3 knockdown significantly inhibited the proliferation of hepatoma cells in vitro;apoptosis detection and Caspase3/7 activity assay showed that SCUBE3 knockdown can significantly promote apoptosis of hepatoma cells;The results of the cycle test showed that SCUBE3 knockdown significantly inhibited the transformation of liver cancer cells from G1 to S phase.(3)In vivo experiment: The in vivo imaging results of nude mice showed that the fluorescence value of SCUBE3 knockdown group was significantly lower than that of the control group;the volume growth curve of subcutaneous tumor of nude mice in SCUBE3 knockdown group was significantly lower than that of the control group.The quality of the subcutaneous tumor was significantly lower than that of the control group.The second part:(1)405 up-regulated genes and 489 down-regulated genes had been screened out by using Afymetrix gene expression profile chip.(2)The cell cycle-related proteins in the differential proteins had been screened by the chip,among which,CCNL2,CDK6,CCNE1,CCND1 were significantly different,and the results of Western Blot verification were consistent with the chip results.Since CCNE1 changed most obviously,this study used it as a candidate target for downstream regulation of SCUBE3.According to the molecular Koch’s rule,on the one hand,CCNE1 was knocked down in BEL7404 cells and a series of cell proliferation,apoptosis detection,cell cycle detection and other experiments were carried out.The results showed that knockdown of CCNE1 could inhibit the proliferation of hepatoma cells,promote the liver cancer cells apoptosis and inhibit of G1/S phase transformation of hepatocarcinoma cells,which indicated that knockdown of CCNE1 had the same effect on proliferation of hepatoma cells as knockdown of SCUBE3;on the other hand,SCUBE3 was knocked down while CCNE1 was overexpressed in hepatoma cells,and it was found that overexpression of CCNE1 could reverse the effect of SCUBE3 knockdown on proliferation,apoptosis and cell cycle of hepatocellular carcinoma cells,which suggested that SCUBE3 was likely to promote the proliferation of hepatoma cells by regulating CCNE1 expression.(3)We performed pathway enrichment analysis on the differential genes screened by the expression profile chip,and the results showed that the TGFβ signaling pathway and the PI3K/AKT signaling pathway changed significantly.Since the PI3K/AKT signaling pathway itself is one of the non-canonical signaling pathways of TGFβ,this study used co-immunoprecipitation assay to find that SCUBE3 binded to the TGFβRII receptor.Western blot analysis showed that SCUBE3 knockdown inhibited the PI3K/AKT signaling pathway in hepatoma cells,and inhibited the phosphorylation of GSK3β to inhibit its kinase activity.Conclusions:1.Compared with normal human liver cells,human liver cancer cells highly express SCUBE3.2.SCUBE3 binds to TGFβRII receptor to activate PI3K/AKT pathway,which phosphorylates GSK3β and inhibits its kinase activity,which leads to a decrease in ubiquitination degradation of CCNE1 and leads to the accumulation of CCNE1,which ultimately promotes the proliferation of hepatoma cells.