Ubiquitin-conjugating Enzyme UBE2K Regulates C-Myc To Promote Malignant Progression Of Hepatocellular Carcinoma

Object:Hepatocellular carcinoma(HCC)is a type of digestive system cancers that poses a serious threat to human health and life worldwide because of its high morbidity and mortality.Most patients with HCC are diagnosed at an advanced stage.Treatment options for HCC include ablative therapy,liver resection,and liver transplantation,etc.In order to discover more effective treatments and improve the prognosis of HCC patients,it is crucial to explore the molecular mechanisms of HCC progression.The E2 s have been reported to be involved in the progression of HCC,including the regulation of proliferation,migration,invasion,apoptosis,and drug resistance of HCC cells.The ubiquitin-conjugating enzyme UBE2 K,also known as Huntington protein-interacting protein-2(HIP2),regulates the proliferation,migration,and invasion of gastric cancer cells.c-Myc as a transcription factor is dysregulated in the expression of many tumors.While the study and molecular mechanism of UBE2 K in HCC have not been reported,therefore,the aim of this study was to investigate the expression of UBE2 K in HCC,the effect of UBE2 K on proliferation,migration,and stemness of HCC cells,the molecular mechanism of UBE2 K regulation of HCC cells through c-Myc and its effect on HCC tumor growth.Methods:The expression of UBE2 K in HCC,the relationship between UBE2 K expression and clinical prognosis of HCC patients,the correlation between UBE2 K and hepatocellular tumor stage,the correlation between UBE2 K and c-Myc expression were analyzed bioinformatically.Cell biology assay was performed to detect the effects of UBE2 K expression on the proliferation,migration and stemness characteristics of HCC cells.m RNA expression of UBE2 K and c-Myc was detected by RT-q PCR.Expression of UBE2 K,c-Myc and stemness protein was detected by Western blot.Subcutaneous tumorigenesis assay was performed to detect the effect of UBE2 K expression on the growth of HCC tumors in mice.Results:Bioinformatics analysis obtained that the m RNA expression levels of UBE2 K were higher in human HCC tissues than in non-tumor liver tissues,and HCC patients with high UBE2 K expression had a poorer prognosis.Protein and m RNA expression levels of UBE2 K were higher in HCC cells than in normal liver cells.Stably transfected human HCC cells Huh7 and Hep3 B and mouse HCC cells Hepa1-6were infected with lentivirus of UBE2 K and screened with Blasticidin S.The m RNA and protein expression levels of UBE2 K were elevated in human HCC cells in the UBE2 K overexpression group compared to the UBE2 K overexpression control human HCC cells.The m RNA and protein expression levels of UBE2 K were increased in the UBE2 K knockdown group compared with the UBE2 K knockdown control group.Compared with the UBE2 K overexpression control human HCC cells,the proliferation,migration,and stemness expression ability of tumor cells in the UBE2 K overexpression group were elevated.In contrast,the proliferation,migration,and stemness expression of tumor cells were decreased in the UBE2 K knockdown group compared with the UBE2 K knockdown control human HCC cells.The expression of stemness proteins CD133,KLF4,NANOG,and Bmi1 was elevated in the UBE2 K overexpression group of human HCC cells compared to the UBE2 K overexpression control group of human HCC cells.In contrast,the expression of stemness proteins CD133,KLF4,NANOG,and Bmi1 was decreased in the UBE2 K knockdown group compared with the UBE2 K knockdown control human HCC cells.UBE2K could regulate the m RNA and protein expression levels of c-Myc,and c-Myc was positively correlated with the expression of UBE2 K.Transient transfection of c-Myc plasmid in UBE2 K knockdown human HCC cells showed significantly higher proliferation and migration ability compared to UBE2 K knockdown human HCC cells.Transient transfection of small interfering RNA targeting c-Myc in UBE2 K overexpressed human HCC cells resulted in significantly lower proliferation and migration capacity compared to UBE2 K overexpressed human HCC cells.Successful construction of an experimental model of subcutaneous tumor formation in HCC mice.Compared with the UBE2 K overexpression control group,the tumors in the UBE2 K overexpression group showed better growth ability and greater tumor weight.In contrast,the tumors in the UBE2 K knockdown group had poorer growth ability and smaller tumor weight compared with the UBE2 K knockdown control group.Conclusion:UBE2K expression is upregulated in HCC and is a potentially good biomarker for clinical HCC patients.The expression of UBE2 K promoted the proliferation,migration,and tumor stemness ability of HCC cells.UBE2 K regulates the expression of stemness proteins in HCC cells.UBE2 K can positively regulate the expression of c-Myc,a oncogene.c-Myc can reverse the ability of UBE2 K on the proliferation and migration of HCC cells.UBE2 K promotes the malignant progression of HCC by regulating the downstream molecule c-Myc.UBE2 K expression promotes the growth of HCC tumors and the progression of HCC in mice.