Cripto-1基因沉默对人肝癌MHCC-97H细胞侵袭、迁移能力的影响

 目的: 探讨Cripto-1蛋白在人肝癌组织和肝癌细胞系中的表达,研究沉默Cripto-1基因后对肝癌细胞MHCC-97H侵袭和迁移能力的影响及可能的机制。方法: 采用Western blot检测11对肝癌组织及相应的癌旁组织和不同肝癌细胞株中Cripto-1蛋白的表达;收集80例肝癌石蜡标本,免疫组织化学方法检测Cripto-1蛋白的表达情况;Cripto-1 siRNA转染肝癌细胞MHCC-97H细胞后,real-time PCR和Western blot分别检测转染效率;Transwell迁移和侵袭实验分别检测细胞的迁移和侵袭能力;Western blot分别检测基质金属蛋白酶(MMP)-2、MMP-9蛋白的表达。结果: Western blot检测结果显示肝癌组织中Cripto-1蛋白的表达明显高于对应的癌旁组织,Cripto-1蛋白在各肝癌细胞系中均有表达,且表达量高于对照细胞,其中高侵袭性肝癌细胞MHCC-97H表达量最高;免疫组织化学结果显示Cripto-1蛋白在88.7%的肝癌组织中表达;Cripto-1 siRNA转染MHCC-97H细胞后能显著降低Cripto-1 mRNA和蛋白的表达水平,并且能降低其侵袭和迁移能力;Western blot结果表明,Cripto-1基因沉默后能抑制MMP-2和MMP-9蛋白的表达。结论: Cripto-1在肝细胞癌中的高表达可能与肝癌的发生发展密切相关,下调Cripto-1的表达可以抑制肝癌细胞侵袭和迁移能力,其机制可能与下调MMP-2、MMP-9的表达有关。

Effects of Cripto-1 gene silencing on invasion and migration of hepatocellular carcinoma cell line MHCC-97H

AIM: To investigate the expression of Cripto-1 in hepatocellular carcinoma (HCC) and the effects of Cripto-1 gene silencing on the invasion and migration of MHCC-97H cells. METHODS: The protein expression of Cripto-1 in the tumor tissues, paracancerous tissues and different HCC cell lines was detected by Western blot. The hepatocellular carcinoma paraffin-embedded specimens (n=80) were collected. The protein expression of Cripto-1 was detected by immunohistochemical method. The Cripto-1 siRNA or control siRNA was transfected into the MHCC-97H cells. The expression of Cripto-1 at mRNA and protein levels was detected by real-time PCR and Western blot, respectively. Transwell migration and invasion experiments were performed to measure the migration and invasion abilities. Finally, the protein expression of matrix metalloprotein-ase (MMP)-2 and MMP-9 in the MHCC-97H cells transfected with Cripto-1 siRNA was detected by Western blot. RESULTS: The protein expression of Cripto-1 in the tumor tissues was significantly higher than that in the paracancerous tissues. Meanwhile, the protein expression of Cripto-1 in the HCC cell lines (highest in MHCC-97H cells) was higher than that in the control cell line. The results of immunohistochemistry show the positive rate of Cripto-1 in HCC was 88.7%. Compared with control group, the expression of Cripto-1 at mRNA and protein levels was notably down-regulated in the MHCC-97H cells transfected with Cripto-1 siRNA, so was the expression of MMP-2 and MMP-9. In addition, the migration and invasion abilities of MHCC-97H cells transfected with Cripto-1 siRNA were decreased. CONCLUSION: Over-expression of Cripto-1 may be closely associated with the occurrence and development of HCC. Cripto-1 gene silencing decreases the migration and invasion abilities of MHCC-97H cells, which may act via inhibiting the expression of MMP-2 and MMP9.