Mechanisms of erythropoietin-producing hepatocellular A3 participating in the invasion of hepatocellular carcinoma cells via regulating vascular endothelial growth factor

Abstract

Objective To investigate the mechanisms of erythropoietin-producing hepatocellular A3 (EphA3) in the invasion of hepatocellular carcinoma (HCC) cells.Methods Hepatic cell HL-7702 and HCC cell and HCC cell lines HepG2 and MHCC97H were cultured.The expression of EphA3 in the HepG2 and MHCC97H cells was suppressed by siRNA interference,and then were divided into the untreated group,the control group and the siRNA intervention group.The expression of EphA3 was detected by RT-PCR and Western blot.The invasion ability of HepG2 and MHCC97H was detected by Transwell chamber.The protein expression of VEGF and activity of vascular endothelial growth factor (VEGF) were detected by western blot and ELISA.All data were analyzed using the analysis of variance or LSD-t test.Results The relative mRNA expressions of EphA3 in HL-7702,HepG2,and MHCC97H cells were 0.94 ±0.13,1.76 ±0.16 and 3.62 ±0.14,respectively,and the protein expressions of EphA3 in the 3 cells were 0.96 ±0.12,1.59 ±0.11 and 3.82 ±0.11.There was significant difference in the EphA3 expression between HL-7702 cells and HepG2,MHCC97H cells (t =2.511,6.437 ; 2.321,6.895,P ＜ 0.05).The relative mRNA expressions of EphA3 in the HepG2 cells in the untreated group,the control group and the siRNA intervention group were 0.95 ±0.11,0.96 ±0.12 and 0.31 ±0.15,respectively.There was significant difference in the mRNA expression of EphA3 in the HepG2 cells between the siRNA intervention group and the control group (t =4.051,P ＜ 0.05).The relative mRNA expressions of EphA3 in the MHCC97H cells in the untreated group,the control group and the siRNA intervention group were 0.97 ± 0.16,0.95 ± 0.14 and 0.40 ± 0.11,respectively.There was significant difference in the mRNA expression of EphA3 in the MHCC97H cells between the siRNA interference group and the control group (t =5.237,P ＜0.05).The relative protein expressions of EphA3 in the HepG2 cells in the untreated group,the control group and the siRNA intervention group were 0.97 ± 0.16,0.95 ± 0.15 and 0.32 ± 0.17,respectively.There was significant difference in the protein expression of EphA3 in the HepG2 cells between the siRNA interference group and the control group (t =4.145,P ＜ 0.05).The relative protein expressions of EphA3 in the MHCC97H cells in the untreated group,the control group and the siRNA intervention group were 0.95 ± 0.11,0.96 ± 0.12 and 0.38 ±0.17,respectively.There was significant difference in the protein expressions of EphA3 in the MHCC97H cells between the siRNA interference group and the control group (t =4.327,P ＜ 0.05).The numbers of HepG2 cells penetrated the Watrigel in the untreated group,the control group and the siRNA intervention group were (111 ±4)/10HPF,(109 ±5)/10HPF and (51 ±3)/10HPF,respectively.There was significant difference in the number of HepG2 cells between the siRNA interference group and the control group (t =7.582,P ＜ 0.05).The numbers of MHCC97H cells penetrated the Watrigel in the untreated group,the control group and the siRNA intervention group were (402 ± 6)/10HPF,(397 ± 7)/10HPF and (152 ± 7)/10HPF,respectively.There was significant difference in the number of MHCC97H cells between the siRNA interference group and the control group (t =9.479,P ＜ 0.05).The relative protein expressions of VEGF in the HepG2 cells in the untreated group,the control group and the siRNA intervention group were 0.98 ± 0.11,0.96 ± 0.13 and 0.57 ± 0.11,respectively.There was significant difference in the protein expression of VEGF of the HepG2 cells between the siRNA interference group and the control group (t =3.167,P ＜ 0.05).The relative protein expression of VEGF in the MHCC97H cells in the untreated group,the control group and the siRNA intervention group were 0.97 ±0.14,0.98 ±0.12 and 0.34 ± 0.15,respectively.There was significant difference in the protein expression of VEGF of the MHCC97H cells between the siRNA interference group and the control group (t =4.278,P ＜ 0.05).The relative activities of VEGF proteins of HepG2 cells in the untreated group,the control group and the siRNA intervention group were 0.96 ±0.15,0.94 ±0.11 and 0.47 ±0.13,respectively.There was significant difference in the activity of VEGF protein in the HepG2 cells between the siRNA interference group and the control group (t =3.981,P ＜ 0.05).The relative activities of VEGF proteins in MHCC97H cells in the untreated group,the control group and the siRNA intervention group were 0.98 ±0.12,0.97 ±0.12 and 0.38 ±0.14,respectively.There was significant difference in the activity of VEGF protein in the MHCC97H cells between the siRNA interference group and the control group (t =4.059,P ＜ 0.05).Conclusions EphA3 plays an important role in the invasion of HCC cells via regulating the protein expression and activity of VEGF.EphA3 might be a new target for the treatment of HCC. Key words: Liver neoplasms; Erythropoietin-producing hepatocellular A3; Vascular endothelial growth factor; Invasion