| Background and Aims: Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in China. Metastasis and recurrence are the most principal factors for their prognosis of the patients with the tumor. KAI1 gene, isolated from human metastatic prostate tumor in 1995, was regarded as a metastasis suppressor gene for prostate cancer. Further studies showed decreased KAI1 expression was observed not only in human prostate cancer but also in several common solid epithelial tumors. The aim of present study is to explore the effects of KAI1 gene on the growth and invasion of HCC cell line MHCC97-H.Methods: KAI1 sense and antisense eukaryotic expression plasmids were constructed using subclone technique and transfected into MHCC97-H cells respectively by DOTAP liposome transfection system to directly interfere in the expression of KAI1 gene. PCR was used to confirm the transfection, and Western blot to understand if the recombinants could function in cells. Several items about cell growth and invasion were measured.Results: (1) Two recombinants from subclone construction were digested and resulted in two fragments respectively as 0.75kb and 7.72kb, 2.25kb and 6.22kb. These results were consistent with expected recombinant plasmids. (2) PCR showed a specific fragment with length 790bp was amplified from MHCC97-H-S cells (transfected with sense KAI1), MHCC97-H-AS (transfected with antisense KAI1) and MHCC97-H-pCI (transfected with vector pCI-neo), but none from MHCC97-H. The results exhibited that the recombinants and the vector pCI-neo had integrated into the chromosome of MHCC97-H cells respectively. Western blot analysis showed enhanced expression of KAI1 protein in MHCC97-H-S, but decreased expression in MHCC97-H-AS, and no obvious difference in MHCC97-H-pCI in contrast with MHCC97-H. (3) No significant differences in growth curve and cell cycles were observed among MHCC97-H-S, MHCC97-H-AS, and MHCC97-H. (4) Under observation with electrical microscope, the amount of mitochondrions was decreased in MHCC97-H-S cells, but increased in MHCC97-H-AS cells. Furthermore, increased amount of rough endoplasmic reticulum, Golgi apparatus andexpanded endoplasmic reticulum was also noted in MHCC97-H-AS cells. (5) KAI1 immunoreaction was exhibited in the cell cytoplasm. Stronger staining was present in MHCC97-H-S whereas weaker staining in MHCC97-H-AS compared with MHCC97-H. (6)Plate cloning test showed there existed no statistical difference between MHCC97-H-S(36.25±3.30) and MHCC97-H (38.50±1.91)(P>0.05). However, MHCC97-H-AS (132.50±12.87)showed higher clone formation (P<0.01). (7) Boyden Chamber test revealed that the cells penetrating the artificial basement membrane in MHCC97-H-S(59.67±3.51) were fewer than that in MHCC97-H(92.67±1.53) (P<0.01), however, more penetrating cells in MHCC97-H-AS (188.00±4.51)were noted as compared with that in MHCC97-H (P<0.01). Conclusion: Sense and antisense KAI1 eukaryotic expression plasmids were successfully constructed and transfected into MHCC97-H respectively. Sense KAI1 gene could upregulate the expression of KAI1 protein in MHCC97-H cells, however, antisense KAI1 gene could downregulate the expression of KAI1 protein. KAI1 gene had no obvious effects on cell cycles and growth of MHCC97-H, but could alter amount and morphology of some organells. Enhanced KAI1 expression could decrease the invasive ability of MHCC97-H. These results suggest that it may be an effective route to upregulate KAI1 expression for inhibiting the metastasis of HCC.
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