Impact Of Norepinephrine On HCC Cells Stemness And Associated Mechanism

BackgroundLiver cancer is the second reason of cancer related death in the world.Some have reported that almost 0.78 million liver cancer would be diagnosis and 0.75 million patients died.As the main histology subtype,HCC occupied 70% to 85%.Because there is no specific symptom so that it is difficult to be discovered,and most patients reached late stage.At present,the first way for therapy HCC remains hepatectomy.But the rate of recurrence and metastasis are high.Therefore,clarification the mechanism of hepatocellular carcinogenesis and recurrence is vital for clinical therapy of HCC.Moreover,the CSCs theory claimed that CSCs is the key of carcinogenesis.Recently,the function of SNS in process of carcinogenesis attracted the attention of scientific community again.Latest investigations have reported that SNS could modulate the characteristics of CSCs to enhance the stemness of melanoma and lung cancer stem cells.and previous study of our group have discovered that the stimulation of SNS associated with hepatocellular carcinogenesis,expression level of HCC stem cell markers CD133 and CD90 were regulated by SNS in the process of DEN inducing HCC.However,whether SNS could modulate characteristics of HCC cells remains to be illustration.ObjectiveWhether stimulation of SNS could modulate CSCs related characteristics of HCC and correlative mechanism.Methods1.Using immunohistochemistry technology to detect the expression level of CD133 in cancer tissue,which was induced by DEN and blocked the activity of SNS in liver.Analysis the relationship of activation of SNS and expression level of CD133.2.Using NE to simulate the activation of SNS to dispose the human HCC cells,Huh7 and PLC.RT-PCR and WB assays were used to tested the impact of NE on expression level of CD133 in vitro.3.Using NE to simulate the stimulation of SNS to dispose the human HCC cells,Huh7 and PLC.Four groups were set,control,NE,NE+PRA and PRA.Cancer cell proliferation,sphere formation and clone formation assays were using to estimate the effect of SNS on proliferation and stemness of Huh7 and PLC HCC cells.4.Establish the activation model of SNS and subcutaneous tumor formation in nude mice for investigation the role of activated SNS in the carcinogenesis process of HCC cell.5.Using NE to simulate the stimulation of SNS to dispose the human HCC cells,Huh7 and PLC.Four groups were set,control,NE,NE+PRA and PRA.RT-PCR and Western blot assays were using to detected the expression level of genes about stemness of CSCs,and survey the signal pathways.Results1.Result of IHC showed that activation of SNS was significantly associated with expression level of CD133 in previous in situ HCC tissues of mice induced by DEN.Lower expression level of CD133 accompanied with weary of SNS.2.Using NE to simulate the activation of SNS to dispose the human HCC cells,Huh7 and PLC,results of RT-PCR and WB assays revealed that NE could promoted the elevation of CD133 in Huh7 and PLC.3.After the disposition of NE,Huh7 and PLC HCC cells showed stronger cell proliferation,sphere formation and clone formation capacity.PRA could block the effect of NE,the capacity of cell sphere formation and clone formation were recovered to the base line,but there wasn't prominent difference between control and NE+PRA,PRA groups.4.Activation of SNS enhanced the subcutaneous tumor formation ability of HCC cell.5.On one hand,NE could elevate the expression level of Sox4,and PRA blocked the impact of NE on Huh7 and PLC HCC cells.On other hand,other genes related to stemness of CSCs were variated irregularly.Accompanied with elevation of Sox4,p-Akt also overexpressed slightly but Akt not.Conclusion1.Activation of SNS significantly associated with the expression level of HCC stem cell marker,CD133.This phenomenon suggested that the probability of SNS regulated stemness of HCC cell.2.NE could promote the expression of CD133 in Huh7 and PLC HCC cells.Simultaneously,stemness characteristics of HCC cells including proliferation,sphere formation and clone formation were enhanced by NE,and activation of SNS promoted subcutaneous tumor formation.But PRA restrained the effect of NE on HCC cells.Expression level of Sox4 and p-Akt were elevated by NE,and PRA blocked the effect.In conclude,activation of SNS may through PI3K/Akt pathway to facilitated Sox4 gene expression to promoted the stemness of HCC cells.