Analysis of the Role of Rab5A in Metastasis of Hepatoma Cells Using RNA Interference

• Main Authors: Ming-Lin Tsai, 蔡明霖
• Other Authors: Chin-Wen Chi
• Format: Others
• Language: zh-TW
• Published: 2006
• Online Access: http://ndltd.ncl.edu.tw/handle/48774252081373763706

碩士 === 國立陽明大學 === 藥理學研究所 === 94 === Hepatoma is the leading cause of malignant tumors in Taiwan. It has been observed clinically that some of the patients with small hepatocellular carcinoma (HCC) progressed very rapidly than others. In order to analyze the changes in the expression of genes in these patients, microarray had been used and it was found that Rab5A was among several genes with increased expression as compared to non-tumorous liver tissue. Small GTPases of the Rab family are important modulators of vesicle formation. It has been reported that Rab5A is an early rate limiting control factor of receptor internalization and related with actin remodeling. In this study, I have examined and compared the expression of Rab5A in 16 paired hepatoma and adjacent liver tissue samples from patients with or without recurrence. Quantitative PCR revealed that the Rab5A mRNA level was higher in tumors from patients with recurrence. Rab5A was found to be differentially expressed in three human hepatoma cell lines examined by RT-PCR and Western blot analyses. Immunostaining of Rab5A in hepatoma cells revealed that it is mainly found in the cytoplasm. Moreover, either transiently transfect Rab5A siRNA or stably knockdown Rab5A in HA22T/VGH cells significantly decreased the expression of Rab5A at 48 hours. Trypan blue exclusion and MTT assay were used for analysis of the cell growth of hepatoma cells with knockdown Rab5A expression and flowcytometry was used to analyze the cell cycle. Reduced expression of Rab5A decreased the cell proliferation but had no effect on cell cycle progression of Rab5A knockdown HA22T/VGH cells. HA22T/VGH cells with transient or stable knock down Rab5A showed reduced invasion and migration ability using invasion and motility assays. In addition, the MMP-9 production decreased in Rab5A knockdown clones. These results together suggest that Rab5A may be involved in human hepatoma cells metastasis.