| Background and Aims:Hepatocellular carcinoma is one of the most common malignant tumors in China.In recent years,the incidence rate is increasing and the age of onset becomes earlier and earlier.Notably,due to the high invasion rate,fast recurrence after surgery and high mortality of liver cancers,the life expectancy is less than 10 months without treatment.In recent years,the clinical research of liver cancer has made great progress,however,how liver cancer occurs and the underlying molecular mechanisms are largely unknown.It is found that activation of β-adrenergic receptor(β-AR)is able to promote tumor angiogenesis,migration,epithelial transformation and inflammation and some other malignant biological behaviors through regulating various signaling pathways associated with these malignant biological behaviors.To this end,this project mainly studies the role of β-AR on the proliferation and migration of HepG2 cells and the underlying mechanism were investigated.Methods:1.The expression of β1-AR and β2-AR genes in HepG2 cells: Extracted the total RNA from liver cancer cells(HepG2)and normal liver cells(LO2),and then measured the m RNA expression level of β1-AR and β2-AR by quantitative real time PCR(q RT-PCR)or reverse transcription-PCR(RT-PCR).2.The effect of NE on HepG2 cell proliferation: The proliferation of cells was measured by MTT assay,after HepG2 cells and LO2 cells were stimulated with different concentrations of NE at 0,1,2,5,10,20 and 30μM respectively for 48 h.Then the two cells were treated with different concentrations o f NE at 0,5,10 and 20μM for 24 h,48h and 72 h,respectively and then the proliferation of HepG2 cells were measured by MTT assay.3.The effect of N E on cell migration and cell morphology: Cell wound healing assay and EMT assay were performed to detect the cell migration and cell morphology.4.The mechanisms of promoting cell proliferation: 1)The effects of NE on HepG2 cell proliferation by PRO,a specific inhibitor of β-AR,and U0126,an ERK1/2 specific inhibitor: HepG2 cells pretreated with the above inhib itors for 30 min,and stimulated with NE for 48 h,then measured by MTT.2)si RNA that targeting C REB was used to knockdown the expression of C REB in HepG2 cells and then stimulated with NE for 48 h,the proliferation of cells was measured by MTT assay.3)The effects of various inhibitors on NE-induced downstream signal pathway: HepG2 cells treated with 10 μM NE for 0,2,5,10,20,30,60 and 120 min respectively.The phosphorylation of ERK1/2、CREB、AKT and PDK1 were determined by western blot,and observed the effects of various inhibitors on the phosphorylation of NE-mediated downstream target protein.Results:1.The results of q RT PCR and RT-PCR indicated that the m RNA expression level of β1-AR and β2-AR in HepG2 were much higher than that in LO2 cells.2.NE significantly promoted the proliferation of HepG2 cells in a dose dependent manner.The optimum concentration of N E was 10 μM,and the optical time treatment was 48 h.3.NE had no significant effect on cell migration and cell morphology.4.Mechanism analysis: 1)PRO,U0126 and si-CREB can all inhibited the migration of HepG2 cells.2)10 μM of NE can significantly increase of phosphorylation of ERK1/2,CREB,AKT and PDK1.3)NE-induced phosphorylation of ERK1/2 and CREB can be inhibited by PRO and U0126.Conclusion:1.The m RNA expression level of β1-AR and β2-AR in hepatocellular carcinoma cell HepG2 are much higher than normal liver LO2 cells.2.NE can promote the proliferation of HepG2 cells,but has no effect on cellmigration and cell morphology.3.NE promotes the proliferation of HepG2 cells might via the ERK1/2 /CREB pathway and PI3K/PKD1/AKT pathway. |
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