The Expression Of NEK2 In Hepatocellular Carcinoma And Its Effect On The Biological Characteristics

Part one The expression of NEK2 in hepatocellular carcinoma and its relationship with clinical prognosisObjectiveThis study was aimed to observe the expression of NEK2 in hepatocellular carcinoma(HCC)and adjacent liver tissue(ALT),and to preliminary investigate its role in HCC.Methods1.Oncomine database analysis of NEK2 mRNA expression in HCC;2.The relationship between the protein expressions of NEK2,detected by immunohistochemistry(IHC),and the clinicopathological parameters in HCC.ResultsOncomine database showed that the mRNA expression of NEK2 was higher in(?)HCC than that in ALT from three published HCC gene expression studies.IHC was detected in 40 cases of HCC and its ALT,and the expression of NEK2 was mainly located in the cytoplasm and the nucleus.In 40 cases of HCC,23 cases showed NEK2 high expression(57.5%),while only 3 cases(7.5%)in its ALT,which showed that NEK2 expression was higher in HCC than in ALT.NEK2 high expression was positively correlated with tumor size(P<0.01,P=0.002),TNM stage(P<0.05,P=0.028)and tumor thrombus(P<0.05,P=0.039)in HCC patients.Kaplan-Meier method showed that the overall survival(OS)rate(P<0.01)and tumor free survival(TFS)rate(P<0.05)of patients with NEK2 high expression were worse than those with low expression.Univariate and multivariate analysis of Cox showed that NEK2 high expression in HCC was an independent factor influencing the prognosis of patients.Conclusion1.The expression of mRNA and protein NEK2 in HCC was significantly higher than that in the ALT;2.The high expression of NEK2 in HCC was positively correlated with TNM stage,tumor size and tumor thrombus;3.NEK2 high expression is an independent factor influencing the prognosis of HCC.Part two The role of NEK2 in tumor proliferation,apoptosis and cell cycle of hepatocellular carcinomaObjectiveThis study was aimed to investigate the role of NEK2 inhibition in proliferation,apoptosis and cell cycle of hepatocellular carcinoma(HCC).Methods1.Real-time PCR was performed to compare NEK2 mRNA expression in HCC cells and normal hepatocytes;2.NEK2 mRNA and protein expression,after siNEK2 transfection of HCCLM3 and Hep-3B cells,was detected by Real-time PCR and Western blot;3.Cell proliferation was investigated by MTS assay and colony formation assay(CFA)in HCCLM3 and Hep-3B cells,meanwhile cell apoptosis and cell cycle was detected by the flow cytometry,after NEK2 knockdown by siRNA.ResultsThe mRNA levels of NEK2 were higher in the seven HCC cell lines including Hep-G2,Hep-3B,HuH-7,SMMC7721,HCCLM3,Snu387 and Snu475,than that in Chang liver.SiRNA technology was used to knock down NEK2,and its levels were effectively downregulated in HCCLM3 and Hep-3B,verified by Real-time PCR(P<0.001)and Western blotting.MTS assay(P<0.05)and CFA(P<0.01)showed that siNEK2 could inhibit the proliferation of HCC cells.The apoptotic cells of siNEK2 transfected HCCLM3 and Hep-3B were increased(P<0.05),and Western blot assay revealed that silencing NEK2 upregulated expression level of BAD and C-caspase-3,while downregulated BCL-2.Compared with the control group,the proportion of the cells in G2/M was reduced in siNEK2 group(P<0.05),detected by the flow cytometry.The expression of Cyclin B1 and CDK1 was decreased,while P2 7 was increased after NEK2 knockdown.Conclusion1.Chemical synthesis of siNEK2 could effectively downregulate the expression of NEK2 in HCCLM3 and Hep-3B;2.SiNEK2 could inhibit proliferation in HCCLM3 and Hep-3B cells,increase apoptosis and reduce cell mitosis.Part three The role of NEK2 in maintaining self-renewal capacity and drug resistance of cancer stem cells in hepatocellular carcinomaObjectiveThis study was aimed to investigate the role of NEK2 in maintaining self-renewal capacity and drug resistance of cancer stem cells(CSC)in HCC.Methods1.The colony formation assay(CFA)and sphere formation assay(SFA)were used to detect the effect of siNEK2 on the self-renewal ability of HCCLM3 and Hep-3B cells,and CCK-8 was performed to investigate the effect of on the chemotherapeutic drugs resistance of HCC cells for NEK2 inhibition;2.Western blot was performed to detect changes of CSC markers in HCCLM3 and Hep-3B cells after NEK2 deletion;3.Real-time PCR and Western blot was used to detect the expression of β-catenin in HCCLM3 and Hep-3B cells,and its target genes of Wnt/β-catenin signaling pathway.ResultsColony formation assay showed that NEK2 deletion formed less and smaller colonies than their control groups(P<0.01).In the sphere formation assay,HCCLM3(P<0.001)and Hep-3B(P<0.01)cells in which NEK2 expression was knocked down exhibited fewer and smaller spheres.Compared with control cells,NEK2 knockdown cells displayed significantly higher sensitivity to 5-Fu and cisplatin in HCCLM3 and Hep-3B(P<0.05).NEK2 knockdown decreased the levels of CSC genes Bmi-1,Sox2 and Nanog in HCC cells by Western blot.Western blot analysis showed NEK2 knockdown reduced β-catenin protein,meanwhile the expression of its target genes of Wnt/β-catenin signal pathway including CD 133、c-Myc and EpCAM,was also downregulated(P<0.01).Wnt3a,a canonical Wnt ligand,could activate the Wnt/β-catenin signaling pathway.As confirmed,NEK2 knockdown reduced the ability to form tumorspheres,conversely,Wnt3 a addition increased the ability of tumorsphere formation to partially compensate NEK2 inhibition in HCC cells(P<0.01).Conclusion1.NEK2 knockdown reduced CSC properties of HCCLM3 and Hep-3B,including the ability of self-renewal and chemotherapeutic resistance;2.NEK2 was revealed to have an important role in the maintenance of CSC characteristics,maybe by means of Wnt/β-catenin signaling,including self-renewal property and resistance to chemotherapy drugs.