Effects Of Overexpression Of UCHL1-AS1 And Combined Treatment With Rapamycin On Proliferation Of Hepatocellular Carcinoma Cells And Bioinformatic Analysis Of Related Genes

OBJECTIVE: Human hepatocellular carcinoma cell line Bel-7404 was transfected with lentiviral to up-regulatethe expression of UCHL1-AS1.Investigating the effect of overexpression UCHL1-AS1 on the proliferation and migration of human hepatoma cell line Bel-7404.And in vestigating the effect of UCHL1-AS1 combined treatment with rapamycin on the proliferation of hepatoma cell line.Finally using the bioinformatics to analyze the possible mechanism of combination therapy.METHODS: 1.Lentiviral vector was transfected into human hepatoma cell line Bel-7404 to over expression UCHL1-AS1 and to establish stable transgenic plants.Fluorescence microscopy and fluorescence quantitative qRT-PCR were used to detect the transfection effect.2.The proliferation ability of hepatocarcinoma cells overexpressing UCHL1-AS1 was detected by MTT assay,and the cell migration ability was detected by scratch test.3.Seting groups for the following experiments,such as control group(blank control group),NC group(negative control group),UCHL1-AS1 group(experimental group),rapamycin +control group,rapamycin +NC group,rapamycin +UCHL1-AS1 group.Different concentrations of rapamycin(0 nmol/L,10 nmol/L,20 nmol/L,40 nmol/L,80 nmol/L,100 nmol/L,200nmol/L,500nmol/L,1000 nmol/L)treatment group alone.After 48 h treatment,MTT assay was used to investgate the effect of rapamycin combined with overexpression of UCHL1-AS1 on inhibiting the proliferation of Bel-7404 cells.4.The correlation between the expression of UCHL1-AS1 and UCHL1 in 369 HCC patients and the correlation between the gene target of mTORC1 downstream of mTOR pathway were detected by data mining in TCGA database.RESULTS: 1.The expression of UCHL1-AS1 in the experimental group was significantly higher than that in the control group and the NC group(P<0.05).The difference was statistically significant,the experimental group cells can be used in subsequent experiments.2.The results of MTT assay showed that there was no significant difference in the growth rate between UCHL1-AS1 group and control group and NC group(P> 0.05).3.The scratch test results showed that the cell migration ability of UCHL1-AS1 group was significantly inhibited.The difference between control group and NC group was statistically significant(P <0.05).4.UCHL1-AS1 combined treatment with rapamycin after48 h,when the rapamycin drug concentration was 200nmol/L and 500nmol/L,it has obvious difference between rapamycin +UCHL1-AS1 group and rapamycin+ NC group and rapamycin + control group,P < 0.01,statistically significant.5.There was a positive correlation between the expression of UCHL1 and EIF4EBP1,ATG13 and PTPA(P <0.05).There was also a positive correlation between UCHL1-AS1 and UCHL1 expression level(P <0.05).CONCLUSION: 1.Overexpression of UCHL1-AS1 had no significant effect on cell proliferation of Bel-7404 hepatoma cells,but could inhibit cellmigration.2.Overexpression of UCHL1-AS1 combined with rapamycin significantly inhibited the proliferation of human hepatocellular carcinoma cell line Bel-7404,and the effect was stronger than that of UCHL1-AS1 and rapamycin alone.3.The mechanism of overexpression of UCHL1-AS1 and rapamycin may be related to EIF4EBP1,ATG13 and PTPA.