Circular RNA CircSP3 Promotes The Proliferation,Migration And Invasion Of Hepatocellular Carcinoma By Regulating CDK4 Expression As A Sponge Of MiR-198

Objective:To explore the expression and role of circSP3 in hepatocellular carcinoma and the regulation of CDK4 by circSP3 as a miR-198 sponge Mechanisms that promote the proliferation,migration,and invasion of hepatocellular carcinoma.Methods:The expression level of circSP3 in cancer tissues and normal liver tissues of patients with hepatocellular carcinoma was detected by real-time fluorescent quantitative PCR(qRT-PCR).In addition,four kinds of hepatoma cells(Hep3B,Huh7,Bel-7402,SMMC-7721)and expression levels in normal hepatocytes(HL-77O2)were detected.Hep-3B and Huh-7cells were infected with the plasmid and lentivirus,and then hepatocellular carcinoma cell lines stably overexpressing and knocking down circSP3 were screened.The effects of circSP3 on cell proliferation,migration,and invasion were tested by CCK-8 experiments,Transwell migrationexperiments,and Transwell invasion experiments.Respectively,Nude tumor formation experiments were used to determine the effect of circSP3 on cell proliferation in vivo.Next,the relationship between circSP3,miR-198 and CDK4 was analyzed using bioinformatics analysis,qRT-PCR,dual luciferase assay,and Western blot.Rescue experiments verified that circSP3 promotes the proliferation,migration,and invasion of hepatocellular carcinoma by regulating CDK4 expression as a miR-198 sponge.RESULTS:The qRT-PCR results showed that compared with adjacent liver tissues and normal liver cells,circSP3 in liver cancer tissues(P<0.001)and in four kinds of hepatoma cells Hep3 B,Huh7,Bel-7402,SMMC-7721(P<0.05,P < 0.001,P < 0.001,and P < 0.001)significantly increased expression.Chi-square test results showed that the expression of circSP3 was highly correlated with tumor size(P < 0.001)and TNM stage(P <0.01).After plasmid transfection,the expression level of circSP3 in the cells of the circSP3 group was significantly enhanced(P<0.001),and the expression level of circSP3 in the cells of the sicircSP3-2 group was significantly decreased(P<0.001).After lentivirus infection,the cells of the LV3-circSP3 group were significantly increased.The expression level of circSP3 was significantly reduced(P < 0.01).The results of CCK-8proliferation experiments showed that the cell proliferation activity of thecircSP3 group was significantly enhanced compared with the NC group,and was most obvious at 72 hours(P < 0.05);on the contrary,the cell proliferation activity of the si-circSP3-2 group was significantly weakened compared with the si-NC group.48 hours(P<0.05)and 72 hours(P<0.01)were more obvious.The results of the Transwell migration and invasion experiments showed that the number of cells migrated(P < 0.01)and invaded(P < 0.01)in the circSP3 group was significantly increased compared with the cells in the NC group;on the contrary,compared with the si-NC group,The number of cell migration(P<0.01)and invasion(P< 0.01)in the si-circSP3 group was significantly reduced.Nude tumor formation experiments showed that compared with the LV3-NC group,tumor growth in the LV3-circSP3 group was slower and tumor volume(P< 0.05,P < 0.01)and weight(P < 0.05,P < 0.001)were significantly reduced.small.Subsequently,the levels of miR-198 in normal liver cells and hepatocellular carcinoma cells were detected by qRT-PCR.It was found that the expression of miR198 in liver cancer cells(P<0.001,P<0.001,P<0.001 and P<0.001)was significantly lower than that of normal liver tissue and normal liver cells.Correlation analysis revealed a negative correlation between miR-198 and circSP3 expression levels(r=-0.2524).qRT-PCR results showed that miR-198 expression was low in circSP3 group cells(P < 0.001),and miR-198 expression was increased insi-circSP3 group cells(P < 0.001);circSP3 expression was reduced in miR-198 mimic group cells(P < 0.001).the expression of circSP3 was increased in mi-198 inhibitor group cells(P<0.001).The results of double luciferase assay showed that miR198 mimic could reduce the fluorescence activity of cells in circSP3-wt group(P < 0.01,P < 0.001),while miR-198 contorl had no effect on the fluorescence activity of cells in circSP3-wt group.qRT-PCR and Westornblot results showed that miR-198 mimic reversed the promotion of CDK4 overexpression of circSP3(P < 0.01 and P < 0.001),and miR-198 inhibitor reversed the inhibitory effect of knockdown circSP3 on CDK4(P<0.01 and P<0.001).Rescue experiments showed that miR-198 mimic reversed the promotion of Hep3 B cell proliferation,migration,and invasion by overexpression of circSP3(P< 0.01),and miR-198 inhibitor reversed the inhibition of Huh-7 cell proliferation,migration,and invasion by knocking down circSP3(P<0.05).Conclusion:The expression of circSP3 in hepatocellular carcinoma and hepatocellular carcinoma tissues was increased.The expression of circSP3 is related to tumor size and TNM stage.CircSP3 can promote proliferation,migration and invasion.The expression levels of circSP3 and miR-198 are inversely related.CircSP3 acts as a sponge for miR-198 and then regulates the downstream target gene CDK4.CircSP3 may be an important biomarker and therapeutic target for hepatocellular carcinoma,whichdeserves further study.