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MHCC97H.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Western blot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Western blot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-t检验.结果 HL-7702、HepG2和MHCC97H中EphA3 mRNA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另别为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EphA3 mRNA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EphA3 mRNA的相对表达量分另别为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);Western blot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(=4.145,P＜0.05);MHCC97H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/10HPF,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).Western blot 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);Western blot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);MHCC97H细胞中VEGF蛋白的相对活力OD值分别为0.98±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.
目的 探討促紅細胞生成素肝細胞受體A3 (EphA3)參與肝癌細胞侵襲的作用機製.方法 培養人肝細胞HL-7702和肝癌細胞株HepG2和MHCC97H.採用siRNA榦擾的方法抑製肝癌細胞中EphA3的錶達.設立未處理組(未處理肝癌細胞),對照組(加入對照siRNA)和siRNA榦擾組(加入siRNA榦擾EphA3).用RT-PCR和Western blot法檢測EphA3的錶達;用Transwell小室檢測不同處理後的HepG2和MHCC97H細胞的侵襲能力;採用Western blot和ELISA法檢測VEGF蛋白錶達和活力的變化情況.多組比較採用單因素方差分析,組間比較採用LSD-t檢驗.結果 HL-7702、HepG2和MHCC97H中EphA3 mRNA的相對錶達量分彆為0.94±0.13、1.76 ±0.16和3.62±0.14; EphA3蛋白的相對錶達量分另彆為0.96 ±0.12、1.59 ±0.11和3.82 ±0.11,非腫瘤細胞與肝癌細胞中EphA3錶達比較,差異有統計學意義(t=2.511,6.437;2.321,6.895,P＜0.05).RT-PCR法檢測HepG2細胞繫中未處理組、對照組、siRNA榦擾組EphA3 mRNA的相對錶達量分彆為0.95 ±0.11、0.96 ±0.12和0.31±0.15,siRNA榦擾組與對照組比較,差異有統計學意義(t=4.051,P＜0.05);MHCC97H細胞中EphA3 mRNA的相對錶達量分另彆為0.97±0.16、0.95 ±0.14和0.40 ±0.11,siRNA榦擾組與對照組比較,差異有統計學意義(t=5.237,P＜0.05);Western blot法檢測3組HepG2細胞中EphA3蛋白的相對錶達量分彆為0.97 ±0.16、0.95 ±0.15和0.32±0.17,siRNA榦擾組與對照組比較,差異有統計學意義(=4.145,P＜0.05);MHCC97H細胞中EphA3蛋白的相對錶達量分彆為0.95 ±0.11、0.96±0.12和0.38±0.17,siRNA榦擾組與對照組比較,差異有統計學意義(t =4.327,P＜0.05).採用體外侵襲實驗,檢測3組穿透的細胞數量,HepG2細胞繫細胞數量分彆為(111±4)箇/10HPF、(109±5)箇/10HPF和(51±3)箇/10HPF,siRNA榦擾組與對照組比較,差異有統計學意義(t =7.582,P＜0.05);MHCC97H細胞繫細胞數量分彆為(402 ±6)箇/10HPF、(397 ±7)箇/10HPF和(152 ±7)箇/10HPF,siRNA榦擾組與對照組比較,差異有統計學意義(t=9.479,P＜0.05).Western blot 法檢測HepG2細胞繫中3組VEGF蛋白的相對錶達量分彆為0.98 ±0.11、0.96 ±0.13和0.57 ±0.11,siRNA榦擾組與對照組比較,差異有統計學意義(t=3.167,P＜0.05);Western blot法檢測MHCC97H細胞繫中3組VEGF蛋白的相對錶達量分彆為0.97 ±0.14、0.98 ±0.12和0.34±0.15,siRNA榦擾組與對照組比較,差異有統計學意義(t =4.278,P＜0.05).ELISA法檢測HepG2細胞繫中3組VEGF蛋白的相對活力OD值分彆為0.96±0.15、0.94 ±0.11和0.47 ±0.13,siRNA榦擾組與對照組比較,差異有統計學意義(t=3.981,P＜0.05);MHCC97H細胞中VEGF蛋白的相對活力OD值分彆為0.98±0.12、0.97 ±0.12和0.38±0.14,siRNA榦擾組與對照組比較,差異有統計學意義(t=4.059,P＜0.05).結論 EphA3可能是通過調控VEGF蛋白錶達和活性來實現肝癌細胞的侵襲,提示EphA3可作為肝癌治療的新靶點.
목적 탐토촉홍세포생성소간세포수체A3 (EphA3)삼여간암세포침습적작용궤제.방법 배양인간세포HL-7702화간암세포주HepG2화MHCC97H.채용siRNA간우적방법억제간암세포중EphA3적표체.설립미처리조(미처리간암세포),대조조(가입대조siRNA)화siRNA간우조(가입siRNA간우EphA3).용RT-PCR화Western blot법검측EphA3적표체;용Transwell소실검측불동처리후적HepG2화MHCC97H세포적침습능력;채용Western blot화ELISA법검측VEGF단백표체화활력적변화정황.다조비교채용단인소방차분석,조간비교채용LSD-t검험.결과 HL-7702、HepG2화MHCC97H중EphA3 mRNA적상대표체량분별위0.94±0.13、1.76 ±0.16화3.62±0.14; EphA3단백적상대표체량분령별위0.96 ±0.12、1.59 ±0.11화3.82 ±0.11,비종류세포여간암세포중EphA3표체비교,차이유통계학의의(t=2.511,6.437;2.321,6.895,P＜0.05).RT-PCR법검측HepG2세포계중미처리조、대조조、siRNA간우조EphA3 mRNA적상대표체량분별위0.95 ±0.11、0.96 ±0.12화0.31±0.15,siRNA간우조여대조조비교,차이유통계학의의(t=4.051,P＜0.05);MHCC97H세포중EphA3 mRNA적상대표체량분령별위0.97±0.16、0.95 ±0.14화0.40 ±0.11,siRNA간우조여대조조비교,차이유통계학의의(t=5.237,P＜0.05);Western blot법검측3조HepG2세포중EphA3단백적상대표체량분별위0.97 ±0.16、0.95 ±0.15화0.32±0.17,siRNA간우조여대조조비교,차이유통계학의의(=4.145,P＜0.05);MHCC97H세포중EphA3단백적상대표체량분별위0.95 ±0.11、0.96±0.12화0.38±0.17,siRNA간우조여대조조비교,차이유통계학의의(t =4.327,P＜0.05).채용체외침습실험,검측3조천투적세포수량,HepG2세포계세포수량분별위(111±4)개/10HPF、(109±5)개/10HPF화(51±3)개/10HPF,siRNA간우조여대조조비교,차이유통계학의의(t =7.582,P＜0.05);MHCC97H세포계세포수량분별위(402 ±6)개/10HPF、(397 ±7)개/10HPF화(152 ±7)개/10HPF,siRNA간우조여대조조비교,차이유통계학의의(t=9.479,P＜0.05).Western blot 법검측HepG2세포계중3조VEGF단백적상대표체량분별위0.98 ±0.11、0.96 ±0.13화0.57 ±0.11,siRNA간우조여대조조비교,차이유통계학의의(t=3.167,P＜0.05);Western blot법검측MHCC97H세포계중3조VEGF단백적상대표체량분별위0.97 ±0.14、0.98 ±0.12화0.34±0.15,siRNA간우조여대조조비교,차이유통계학의의(t =4.278,P＜0.05).ELISA법검측HepG2세포계중3조VEGF단백적상대활력OD치분별위0.96±0.15、0.94 ±0.11화0.47 ±0.13,siRNA간우조여대조조비교,차이유통계학의의(t=3.981,P＜0.05);MHCC97H세포중VEGF단백적상대활력OD치분별위0.98±0.12、0.97 ±0.12화0.38±0.14,siRNA간우조여대조조비교,차이유통계학의의(t=4.059,P＜0.05).결론 EphA3가능시통과조공VEGF단백표체화활성래실현간암세포적침습,제시EphA3가작위간암치료적신파점.
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.
