The Mechanism Study Of Upregulation Of Aldehyde Dehydrogenase(ALDH2)Activity Inhibited The Proliferation Of Hepatocellular Carcinoma Cells

Background and AbjectiveAccording to the GLOBOCAN 2018 statistics,the incidence and mortality of cancer are still in the rapid growth stage.Liver cancer is one of the leading one both in morbidity or mortality,which reached 841080,781631 respectively.The occurrence of liver cancer related with many factors,including hepatitis B,hepatitis C,alcohol abuse,etc.It is well known that ethanol has been classified as a carcinogen.Acetaldehyde is an extremely dangerous metabolite substances.Among the acetaldehyde metabolism process,aldehyde dehydrogenase 2(ALDH2)plays an important role,which can convert acetaldehyde to acetate to eliminate the toxic effects of acetaldehyde.rs671 is one of the most common SNP in ALDH2.rs671 locus mutation can significantly reduce the activity of ALDH2,thereby weakening its ability to metabolize alcohol.The ALDH2 mutation type include heterozygosis mutation(ALDH2*1/*2)and homozygous mutant type(ALDH2*2/*2),which reduce the enzyme activity to 45%and 5%respectively.According to the existing reports,the relation between ALDH2 gene mutation and hepatocellular carcinoma cells(HCC)remains unclear.Chronic heavy drinking is one of the most important environmental factors that lead to the pathogenesis of HCC.The interaction of ALDH2 gene mutation and environmental risk factors can increase the pathogenesis of HCC greatly.It is reported that the ALDH2 gene mutation rate is higher in East Asian populations.While mechanism underlying of the ALDH2*2 and liver cancer is still unclear.The main purpose of this project is to clarify the significance of genetic polymorphisms of ALDH2 in liver cancer.Moreover,upregulation the activity of ALDH2 inhibits the proliferation of liver cancer cells by repairing ALDH2*2 gene mutations and screening small molecule activators,it provides a certain experimental and theoretical basis for the prevention and treatment of clinical HCC patients.Materials and MethodI.ALDH2 Function study1.Detect ALDH2 protein expression and activity:(1)Use Western blot to detect the expression of ALDH2 in normal mouse tissues.(2)To detect the expression of ALDH2 in liver cancer tissue array by immunohistochemistry.(3)The enzyme activity of ALDH2 and ALDH2*2 in the tissues was detected by the ALDH2 enzyme activity kits.(4)Western blot RT-PCR experiments were used to analyze the expression of ALDH2 protein and mRNA.2.Detect the effects of ALDH2 on the growth of hepatocellular carcinoma cells:(1)Use gene editing tools to knock out and over-express the ALDH2 gene.Western blot was used to detect the infection efficiency.(2)Cell proliferation assay and clone formation assay were used to detect the hepatocellular carcinoma cells growth afterf after over-expression WT or mutant ALDH2.(3)Flow cytometry was used to measured the cell apoptosis after overexpression of WT or mutant ALDH2.3.Validate the role of ALDH2 in alcohol metabolism:(1)Use the ALDH2 enzyme activity kit to analyze the most sensitive concentration of alcohol in liver cancer cells(2)Cell proliferation assay and soft agar clone formation assay were performed to detect the effects of knockout,overexpression of wild-type ALDH2 or ALDH2*2 on alcohol metabolism.4.Investigate the mechanism and signal pathways related with ALDH2:(1)Detect ROS levels and mitochondrial membrane potentials using confocal microscopy.(2)Western blot was used to detect ROS-mediated JNK related apoptotic signal pathways as well as JNK regulated mitochondrial apoptosis signaling pathway and transcription factor STAT3.?.Repair of ALDH2 point mutations1.CRISPR-cas9 technology is used to perform gene knockout first,then HDR template is used to perform human-derived repair.The repair efficiency was calculated based on sequencing data.2.Use the enzyme activity kit to detect the ALDH2 activity in the repaired cells.3.Cell proliferation assay and clone formation assay were used to detect its effect on the proliferation of liver cancer cells.4.Cell proliferation assay and clone formation assay were used to detect the effect of alcohol on it after repair.?.Screening small molecule compounds that can act as ALDH2 activators1.Screening of small molecule of ALDH2 activator:Using computer simulation methods,several candidate compounds were obtained for screening,and finally the compound effect was verified.2.Detect the relationship between activator and ALDH2:(1)Treat cells with activator and detect ALDH2 enzyme activity at different time points.(2)Treat cells with activator and detect ALDH2 protein or mRNA levels by Western blot or RT-PCR.3.The effect of activator on the growth of liver cancer cell:(1)MTT and clone formation assay were used to detect the effect of activator on liver cancer cells.(2)Detect the effect of this compound on the apoptosis and cell cycle by flow cytometry.4.Investigate the mechanism and signal pathways related to the activator and ALDH2:(1)Use confocal microscopy to detect ROS levels and mitochondrial membrane potential.(2)Detect MAPK signal pathway and related apoptosis signal pathways by western blot.Result?.ALDH2 functional study1.ALDH2 is highly expressed in normal liver tissues and low expressed in liver cancer tissues and cells.2.Overexpression of ALDH2 inhibits the proliferation of liver cancer cells and promotes mitochondrial apoptosis pathway.3.Overexpression of ALDH2 promotes apoptosis of liver cancer cells by activating the JNK signaling pathway and down-regulating the transcription level of STAT3 to regulate the production of ROS.?.Repair of ALDH2 point mutationsRepairing ALDH2 mutations inhibits the proliferation of liver cancer cells by restoring its enzyme activity and protein level.?.Screening small molecule compounds that can act as ALDH2 activators1.Butein act as an ALDH2 activator,which can inhibit the proliferation of liver cancer cells.2.Butein can significantly increase the RNA level of ALDH2 as well as the protein level and enzyme activity.3.Butein activated JNK and downregulated STAT3 related signal pathways related to ALDH2.Conclusion1.ALDH2 is low expression in liver cancer tissues and cells.Overexpression of ALDH2 increased ROS production and inhibited the proliferation of liver cancer cells as well as promoted cell apoptosis.2.ALDH2 mutations decreased the enzyme activity and increased the risk of alcohol-related liver cancer.3.Butein,act as an ALDH2 activator,upregulated the expression of ALDH2 and inhibited the HCC cell proliferation.