TET1-CD Inhibits HCC By Activating The Hypernethylated TSGs

Objective:This study aims to explore the demethylation role of the DNA dioxygenase TET1 catalytic domain(TET1-CD)in hepatocellular carcinoma and to explore therapy of hepatocellular carcinoma in vivo and vitro.Methods:1.RT-qPCR and Western Blot were used to detect mRNA and protein expression of TET1 in SMMC 7721 cells and LO2 cells,respectively.2.TET1-CD target fragment was obtained by designing the upstream and downstream primers based on the TET1 gene sequence and used the cDNA of LO2 cells as the template.Then cloned into the eukaryotic expression vector pflag-CMV4 to construct the recombinant expression plasmid pflag-TET1-CD(p-TET1-CD).The mutant recombinant expression plasmid pflag-TET1-mCD(p-TET1-mCD)was obtained by point mutation technique using pflag-TET1-CD as a template(H1672Y,D1674A).3.SMMC 7721 cells were transiently transfected with control,p-TET1-CD and p-TET1-mCD groups respectively.BSP detected the methylation levels of tumor suppressor genes(TSGs)TET1,P16,SOCS1,APC and RASSF1 A and oncogenes c-Myc,Bmi1,EMS1,Kpna2 and c-fos.4.SMMC 7721 cells were transiently transfected with control,p-TET1-CD and p-TET1-mCD groups respectively.RT-qPCR was used to detect the mRNA expressions of TSGs TET1,P16,SOCS1,APC and RASSF1 A and oncogenes c-Myc,Bmi1,EMS1,Kpna2 and c-fos.5.SMMC 7721 cells were transiently transfected with control,p-TET1-CD and p-TET1-mCD groups respectively.The absorbance at OD450 nm was measured after 24 h,48h and 72 h in CCK8 experiment.EDU imaging detected the proliferation ability of three groups.6.p-TET1-mCD groups respectively.Wound Healing,cell migration and invasion assays detected the movement ability of the cells.7.SMMC 7721 cells were transiently transfected with control,p-TET1-CD and p-TET1-mCD groups respectively,and injected into nude mice to establish a subcutaneous tumor model.The tumor growth volume and weight were recorded.Ki-67 expression levels were detected by immunohistochemistry.Results:1.The expression levels of TET1 mRNA and protein in SMMC 7721 cells was lower than that in LO2 cells.2.DNA sequencing confirmed that recombinant expression plasmid pflag-TET1-CD(p-TET1-CD)and pflag-TET1-mCD(p-TET1-mCD)were constructed successfully.3.BSP showed that the methylation levels of TSGs in p-TET1-CD group were significantly lower than that of control and p-TET1-mCD groups.The methylation levels of the oncogenes were slightly lower.4.RT-qPCR revealed that the expression levels of TSGs in p-TET1-CD group were obviously higher than that of control and p-TET1-mCD groups.Only the expression level of c-fos mRNA in the oncogene was significantly increased.5.The absorbance of p-TET1-CD group at OD450 nm was significantly lower than that of control and p-TET1-mCD groups.The number of proliferating cells in p-TET1-CD group was reduced compared with control and p-TET1-mCD groups.6.Compared to control and p-TET1-mCD groups,the scratch area of p-TET1-CD group was greater;the number of migrated and invaded cells were decreased.7.The subcutaneous tumor model of nude mice was successfully established.Tumor volume and weight in p-TET1-CD group were notably determined compared with control and p-TET1-mCD groups;Immunohistochemistry results verified that the protein levels of Ki-67 in p-TET1-CD group was clearly less than that in Control and p-TET1-mCD groups.Conclusion:TET1-CD inhibits HCC by activating the hypermethylated APC,p16,RASSF1 A,SOCS1 and TET1 genes.