Effect Of ASCT2 On Malignant Behavior Of Hepatocellular Carcinoma Cell And Its Mechanism

Backgrounds Hepatocellular carcinoma(HCC)is the most prevalent liver cancer and the fifth most deadly cancer worldwide.Liver cirrhosis caused by chronic hepatitis B,alcoholism,or hepatitis C infection is a major risk factor for HCC,followed by nonalcoholic steatohepatitis.HCC is a multifactorial disease caused by a variety of risk factors,including hepatitis B virus or hepatitis C virus infection,aflatoxin B1,alcohol overdose,smoking,and host factors such as cirrhosis,and nonalcoholic fatty liver disease.The development of HCC requires unique overlapping risk factors,complex interactions of pre-cancerous genomic alterations.Compared with other human malignant solid tumors,HCC has a very high invasion and metastasis ability.There are even opinions that malignant tumors are actually a metabolic disease.Higher glutamine metabolism is a prominent feature of malignant tumors.Because of the high consumption of tumor cells,more glutamine is required,and extracellular glutamine needs to be transported into the cell by means of a specific amino acid transporter on the cell membrane.Therefore,tumor cells may be transported through high expression of amino acids.The vector was used to test the high uptake of glutamine.ASCT2,also known as SLC1A5(Solute Carrier Family 1 Member 5),is a neutral amino acid transporter that is dependent on sodium ions on the cell membrane and mediates the transport of glutamine into cells.Although glutamine is a non-essential amino acid to cells,the lack of glutamine affects the biology of cells.The demand for glutamine in tumor cells is significantly higher than that of normal cells.Glutamine is produced by the production of large amounts of ATP and synthetic proteins to meet the high metabolic requirements of tumor cells.By inhibiting ASCT2 protein to reduce glutamine intake,the malignant biology of inhibiting tumor cells has been effectively demonstrated in gastric cancer,breast cancer,and acute leukemia,so ASCT2 will be the target of new tumor therapy.Research contents 1.The expressions of ASCT2 m RNA and protein in 5 liver cancer cell lines and normal liver cell lines were detected.The expression of ASCT2 in hepatocellular carcinoma and normal liver tissues was found in the oncomine database and statistical analysis was performed.2.SMMC-7721 cell line and BEL-7404 cell line were selected.The ASCT2 was knocked down by a lentiviral system,and the transfection efficiency was detected by microscopic observation of fluorescence.The knockout effect was detected by Western blot,and a stable cell line was established by screening with puromycin.3.Whether the knockdown of ASCT2 affects the proliferation of hepatoma cells by plate cloning and MTT assay;whether the effect of ASCT2 knockdown on the migration and invasion of hepatoma cells is detected by migration and invasion experiments.4.The expression changes of AKT/m TOR signaling pathway after ASCT2 knockdown was detected by Western Blot.Research results 1.The expression of ASCT2 m RNA and protein in 5 liver cancer cell lines was higher than that in normal liver cell lines.And through the data analysis in the oncomine database,the expression of ASCT2 in hepatocellular carcinoma tissue is higher than that in normal liver tissue in clinical tissue samples.2.After observation by fluorescence inversion microscope,SMMC-7721 cells and BEL-7404 cells were fluorescent after lentivirus transfection and puromycin screening.The expression of ASCT2 protein in the SMMC-7721-inhibit group and the BEL-7404-inhibit group was significantly lower than that in the NC group by Western blot.3.The results of colony formation and MTT assay showed that the knockdown of ASCT2 significantly inhibited the proliferation of SMMC-7721 cells and BEL-7404 cells.The results of migration and invasion experiments indicated that the knockdown of ASCT2 significantly inhibited the migration and invasion of SMMC-7721 cells and BEL-7404 cells.4.In terms of mechanism,the expression of p-AKT and m TOR was significantly decreased by Western blot after ASCT2 knockdown.It indicates that the expression changes of ASCT2 affect the intracellular AKT/ m TOR signaling pathway.Conclusion 1.In hepatocellular carcinoma cell lines and liver cancer tissues the expression of ASCT2 was significantly higher than that of normal liver cells and liver tissues.2.Inhibition of expression of ASCT2 inhibits the malignant behavior of hepatocellular carcinoma cells by affecting the intracellular AKT/m TOR signaling pathway.