EphA3通过调控VEGF参与肝癌细胞侵袭的机制

目的探讨促红细胞生成素肝细胞受体A3（EphA3）参与肝癌细胞侵袭的作用机制。方法培养人肝细胞HL-7702和肝癌细胞株HepG2和NHCC97H。采用siRNA干扰的方法抑制肝癌细胞中EphA3的表达。设立未处理组（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EphA3）。用RT-PCR和Westernblot法检测EphA3的表达；用Transwell小室检测不同处理后的HepG2和MHCC97H细胞的侵袭能力；采用Westernblot和ELISA法检测VEGF蛋白表达和活力的变化情况。多组比较采用单因素方差分析，组间比较采用LSD—f检验。结果HL-7702、HepG2和MHCC97H中EphA3mRNA的相对表达量分别为0．94±0．13、1．76±0．16和3．62±0．14；EphA3蛋白的相对表达量分别为0．96±0．12、1．59±0．11和3．82±0．11，非肿瘤细胞与肝癌细胞中EphA3表达比较，差异有统计学意义（t=2．511，6．437；2．321，6．895，P〈0．05）。RT—PCR法检测HepG2细胞系中未处理组、对照组、siRNA干扰组EphA3mRNA的相对表达量分别为0．95±0．11、0．96±0．12和0．31±0．15，siRNA干扰组与对照组比较，差异有统计学意义（t=4．051，P〈0．05）；MHCC97H细胞中EphA3mRNA的相对表达量分别为0．97±0．16、0．95±0．14和0．40±0．11，siRNA干扰组与对照组比较，差异有统计学意义（t=5．237，P〈0．05）；Westernblot法检测3组HepG2细胞中EphA3蛋白的相对表达量分别为0．97±0．16、0．95±0．15和0．32±0．17，siRNA干扰组与对照组比较，差异有统计学意义（t=4．145，P〈0．05）；MHCC97I-I细胞中EphA3蛋白的相对表达量分别为0．95±0．11、0．96±0．12和0．38±0．17，siRNA干扰组与对照组比较，差异有统计学意义（t=4．327，P〈0．05）。采用体外侵袭实验，检测3组穿透的细胞数量，HepG2细胞系细胞数量分别为（111±4）个／10HPF、（109±5）个／10HPF和（51±3）个／IOHPF，siRNA干扰组与对照组比较，差异有统计学意义（t=7．582，P〈0．05）；MHCC97H细胞系细胞数量分别为（402±6）个／10HPF、（397±7）个／10HPF和（152±7）个／10HPF，siRNA干扰组与对照组比较，差异有统计学意义（t=9．479，P〈0．05）。Westernblot法检测HepG2细胞系中3组VEGF蛋白的相对表达量分别为0．98±0．11、0．96±0．13和0．57±0．11，siRNA干扰组与对照组比较，差异有统计学意义（t=3．167，P〈0．05）；Westernblot法检测MHCC97H细胞系中3组VEGF蛋白的相对表达量分别为0．97±0．14、0．98±0．12和0．34±0．15，siRNA干扰组与对照组比较，差异有统计学意义（t=4．278，P〈0．05）。ELISA法检测HepG2细胞系中3组VEGF蛋白的相对活力OD值分别为0．96±0．15、0．94±0．11和0．47±0．13，siRNA干扰组与对照组比较，差异有统计学意义（t=3．981，P〈0．05）；NHCC97H细胞中VEGF蛋白的相对活力OD值分别为n98±0．12、0．97±0．12和0．38±0．14，siRNA干扰组与对照组比较，差异有统计学意义（t=4．059，P〈0．05）。结论EphA3可能是通过调控VEGF蛋白表达和活性来实现肝癌细胞的侵袭，提示EphA3可作为肝癌治疗的新靶点。

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

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 ceils in the untreated group, the control group and the siRNA intervention group were O. 97 ± O. 16, O. 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 O. 95 ± O. 11, O. 96 ± O. 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 〈 O. 05）. The numbers of HepG2 ceils 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 O. 98 ±0.11, 0.96 ±0.13 and 0.57 ±0.11, respectively. There was sig- nificant 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 O. 97 ± O. 14, O. 98 ± O. 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 O. 96 ± O. 15, O. 94 ± O. 11 and O. 47 ± O. 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 ± O. 12 and 0.38 ± 0.14, respectively. There was significant difference in the activity of ~EGF protein in the MHCC97H cells between the siRNA interference group and the control group （ t = 4. 059, P 〈 O. 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.