EphA2通过调控Grb2表达参与肝癌细胞侵袭的机制探讨

The mechanisms of erythropoietin-producing hepatocellular A2 protein participating in the invasion of hepatocellular carcinoma cells via regulating the expression of growth factor receptor bound protein 2

目的:检测促红细胞生成素肝细胞受体A2(EphA2)蛋白在肝细胞肝癌中的表达情况,探讨其参与调控肝癌细胞侵袭的可能内在机制。方法:培养人正常肝细胞HL-7702和肝癌细胞株MHCC97H、Hep3B,通过siRNA干扰法下调肝癌细胞中EphA2、生长因子受体结合蛋白2(Grb2)的表达,分为三组:未处理组(未处理肝癌细胞)、对照组(加入对照siRNA)和siRNA干扰组(加入siRNA干扰EphA2或Grb2)。通过Western blot法检测不同处理前后细胞中EphA2、Grb2蛋白的表达情况,Transwell小室检测细胞侵袭性。多组比较、组间比较分别采用单因素方差分析和LSD-t检验。结果:体外侵袭实验结果显示MHCC97H、Hep3B、HL-7702穿透细胞的数量分别为:(401±3)/10 HPF、(111±4)/10 HPF、(31±3)/10 HPF,人正常肝细胞与人肝癌细胞相比差异具有统计学意义(P <0.05)。Eph A2、Grb2蛋白在人正常肝细胞与人肝癌细胞中的相对表达量相比,差异均有统计学意义(P <0.05)。siRNA法抑制细胞Eph A2表达后,Hep3B和MHCC97H细胞系未处理组、对照组、siRNA干扰组穿透的细胞数量分别为:(111±4)/10 HPF、(108±5)/10 HPF、(53±3)/10 HPF和(405±5)/10 HPF、(399±4)/10 HPF、(155±7)/10 HPF,siRNA干扰组与对照组比较,差异有统计学意义(P<0.001)。检测Eph A2蛋白在Hep3B、MHCC97H细胞系未处理组、对照组、siRNA干扰组中的相对表达量,siRNA干扰组与对照组相比,差异有统计学意义(P <0.05)。siRNA抑制Grb2表达后,Hep3B和MHCC97H细胞系未处理组、对照组、siRNA干扰组穿透的细胞数量分别为:(109±3)/10 HPF、(107±4)/10 HPF、(56±4)/10 HPF和(403±4)/10 HPF、(402±4)/10 HPF、(163±5)/10 HPF,siRNA干扰组与对照组比较,差异有统计学意义(P <0.001)。检测Grb2蛋白在Hep3B和MHCC97H细胞未处理组、对照组、siRNA干扰组组中的相对表达量,siRNA干扰组与对照组比较,差异有统计学意义(P <0.05)。siRNA抑制Eph A2表达后,检测Grb2蛋白在Hep3B、MHCC97H细胞未处理组、对照组、siRNA干扰组中的相对表达量,siRNA干扰组与对照组比较,差异有统计学意义(P <0.05)。结论:Eph A2参与调控肝癌细胞侵袭的内在机制可能是通过调控Grb2蛋白的表达实现的,据此,我们得出Eph A2可作为治疗肝细胞肝癌的新靶点。

Objective:To inquire into the expression and clinical significance of EphA2 protein in the invasion of hepatocellular carcinoma cells,and the possible mechanism of regulating hepatocellular carcinoma cells.Methods:We cultured hepatic cell HL-7702,HCC cell lines MHCC97H and Hep3B.The expression of EphA2 and Grb2 in the MHCC97H and Hep3B was suppressed by siRNA interference,and then were divided into three groups:Untreated group,the control group and the siRNA intervention group.The protein expression of EphA2 and Grb2 was explored by Western blot,the invasion ability of MHCC97H and Hep3B was explored by Transwell chamber.Results:The numbers of HL-7702,Hep3B and MHCC97H cells penetrated the watrigel were(31±3)/10 HPF,(111±4)/10 HPF,(401±3)/10 HPF,respectively.There was significant difference in the number between hepatic cell and HCC cell lines(P<0.05).There was significant difference in the protein expression of EphA2 and Grb2 between hepatic cell and HCC cell lines(P<0.05).Suppress the expression of EphA2,the numbers of Hep3B and MHCC97H cells penetrated the Watrigel in the untreated group,the control group and the siRNA intervention group were(111±4)/10 HPF,(108±5)/10 HPF,(53±3)/10 HPF,and(405±5)/10 HPF,(399±4)/10 HPF,(155±7)/10 HPF.There was significant difference in the number between the control group and the siRNA intervention group(P<0.001).There was significant difference in the protein expression of EphA2 in the MHCC97H and Hep3B cells between the control group and the siRNA intervention group(P<0.05).Suppress the expression of Grb2,the numbers of Hep3B and MHCC97H cells penetrated the watrigel in the untreated group,the control group and the siRNA intervention group were(109±3)/10 HPF,(107±4)/10 HPF,(56±4)/10 HPF and(403±4)/10 HPF,(402±4)/10 HPF,(163±5)/10 HPF.There was significant difference in the number between the control group and the siRNA intervention group(P<0.001).There was significant difference in the protein expression of Grb2 in the MHCC97H and Hep3B cells between the control group and the siRNA intervention group(P<0.05).Suppress the expression of EphA2,there was significant difference in the protein expression of Grb2 in the MHCC97H and Hep3B cells between the control group and the siRNA intervention group(P<0.05).Conclusion:EphA2 may participate in the invasion of HCC cells by regulating the expression of Grb2 protein.EphA2 may be a new target for the treatment of HCC.