| Background&Aims:Hepatocellular carcinoma (HCC) is one of the most aggressive malignant tumors highly prevalent in the world. Studies demonstrated that even if early surgical resection for HCC,the5-year recurrence rate may achieve50%.Therefore, it is very urgent to elucidate the mechanisms of invasion and metastasis,and search for new targets in the treatment of HCC. Cathepsin S(Cat S) is one of the members of the cathepsins family that can be found in a great variety of cell types. As a cysteine protease,Cat S can degrade extracellular matrix (ECM) elements such as laminin,fibronectin,elastin and collagens. Cat S has been demonstrated to be involved in multiple types of cancers such as lung cancer, prostate cancer, gastric cancer and astrocytoma.Moreover,recent experimental studies confirmed that Cat S play a key role in tumourigenesis, angiogenesis,invasion and metastasis.Our previous work indicated that Cat S is aberrantly overexpressed in the endothelial cells of HCC and the expression level is evidently correlated with portal vein embolus, extrahepatic metastasis and the differentiation degree. However, very little is known about the function of Cat S in the invasion and metastasis of HCC. Thus, the objectives of this study was to confirm the expression of Cat S in human HCC cell lines and illustrate the function of Cat S in the invasion and metastasis of HCC in vitro.Methods:1. Real-time quantitative PCR and western blot were performed to examine Cat S expression in HCC cell lines with different metastatic potential.2. Three pairs of Cat S gene targeted siRNA and a pair of negative control siRNA were transfected into MHCC97-H cells respectively. Real-time quantitative PCR and Western blot were performed to evaluate the interfering effects of three candidate sequences and a missense sequence to down-regulate Cat S in MHCC97-H cells.3. After transfection with siRNA, the MTT assay were performed to examined the effect of Cat S silencing on proliferation of MHCC97-H cells. The Annexin V-FITC/PI double stain assay was used to quantitatively analyze the apoptosis of MHCC97-H cells. The transwell assay were used to determine the impact of knockdown of Cat S on MHCC97-H cells invasion.Results:1. Cat S mRNA and protein levels in HCC lines (MHCC97-L and MHCC97-H) were higher than those in normal hepatic cell line L02. Among the three cell lines analyzed, MHCC97-H cells have the highest Cat S expression.2. The Cat S expression was significantly inhibited transfected with siRNA-1011compared with siRNA-NC and blank control.The quantification analysis showed that the siRNA reduced Cat S mRNA levels by48%(siRNA-1011),26%(siRNA-770) and22%(siRNA-669) compared to the blank control (P＜0.05).3. Cell proliferation was significantly inhibited at48h and72h after transfection with siRNA-1011. After transfection with siRNA-1011for48h,the apoptosis rate of MHCC97-H cells was42.2%±9.9%,significantly higher than in those for the siRNA-NC(10.7%±7.6%) and blank control(9.8%±7.1%).The number of invade through the matrigel MHCC97-H cells were significantly decreased after transfect with siRNA-1011compared with that in siRNA-NC infected cells and blank control.Conclusions:1. Both Cat S mRNA and protein were expressed at a very high level in human HCC cells with with highly metastatic potential.2. The expression level of Cat S gene was inhibited by RNA interference.3. Cat S has an important role in HCC cells proliferation and invasion and might be a potential target for HCC therapy. |
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