| Metastasis and recurrence is the main obstacle for the living of the patients with hepatocellular carcinoma (HCC). To study the molecular target about invasion and metastasis of hepatoma carcinoma cell is significance for preventing the recurrence of HCC and improving the therapeutic effect after surgical resection. RhoC is a member of ras gene superfamily. It participates in signal transduction in cells and reconstruction of cytoskeleton. Existed studies revealed that the over-expression of RhoC was closely correlated with the metastasis of malignancy tumor,which can lead to a role like"switch"gene. While, it is still unclear that if we can inhibit the invasion and metastasis of HCC by down-regulating the expression of RhoC. We assumed that it would be valuable for preventing metastasis and recurrence of HCC after surgical resection with the gene therapy aimed to RhoC.Objective: Constructing a RhoC-siRNA expression vector and establishing a RhoC knock down model of hepatoma carcinoma cell by stably transfection of the vector, on this basis, the roles of RhoC-siRNA on the invasive and metastasis ability were studied, which can provide a new target and experiment data for gene therapy on HCC.Methods: (1) Designing the RhoC specific siRNA expression vector on web with bioinformatics technology and constructing the pSilencer2.1-RhoC-siRNA in vitro. (2) After separately transfection with pSilencer2.1-RhoC-siRNA and pSilencer2.1-HK, and selection with G418, two stable transfection cell lines were built, which were renamed with SK-Hep1/RhoC-siRNA cell and SK-Hep1/HK cell separately. (3) The role of RhoC-siRNA on the expression of RhoC protein in SK-Hep1 cell was verificated by the methods of Western-Blot. (4) Cell morphous was observed by microscopy and by stained with HE, the growth curve was made, the cell adhesion was measured with MTT assay, the cell migration was surveyed by scratch method, the cell invasion in vitro was determined with Boyden chamber, and the production of VEGF was determined with ELISA kit. (5) The SK-Hep1 cell, the SK-Hep1/HK cell and the SK-Hep1/RhoC-siRNA cell were transplanted subcutaneously in nude mice separately, and the growth and metastasis of the three groups of cell was oberved in vivo.Results: (1) Rho-siRNA expression vector was confirmed to be successfully constructed with the assay of enzyme digestion and sequencing. (2) The expression of RhoC protein decreased by 60% in SK-Hep1/RhoC-siRNA cell compared with that in SK-Hep1 cell and in SK-Hep1/HK cell. (3) There were no obvious differences in cell morphous and in cell growing manner among SK-Hep1 cell, SK-Hep1/HK cell and SK-Hep1/RhoC-siRNA cell. (4) Cell proliferation of SK-Hep1/RhoC-siRNA cell and SK-Hep1/HK cell fell behind to that of SK-Hep1 cell (p<0.05), especially in the fourth and fifth day, while the cell growth curve has no significant difference between SK-Hep1/HK cell and SK-Hep1/RhoC-siRNA cell (p>0.05). (5) The adhesion rate of SK-Hep1 on Matrigel at 20min, 40min and 60min after seeding was (38.1±10.37)%,(68.4±11.52)%,(85.1±8.38)%, which was (34.3±9.56)%,(62.5±12.31)%,(80.6±9.43)% in SK-Hep1/HK cell, and was (31.2±7.10)%,(57.2±8.08)%,(71.5±4.19)% in SK-Hep1/RhoC-siRNA cell. There was significant difference only at 60min after seeding between SK-Hep1/RhoC-siRNA cell and the other 2 groups (p<0.05). (6) The scratch wound had almost been filled with SK-Hep1 cell or SK-Hep1/HK cell at 48h after scratching, while the wound was still apparent in the SK-Hep1/RhoC-siRNA cell, which meant that RhoC-siRNA could inhibit the migration of SK-Hep1 cells. (7) The number of cells that penetrated polycarbonate coated with Matrigel when incubation for 16h was only 67±18.5 in SK-Hep1/RhoC-siRNA cell, which was much lesser than SK-Hep1 cell (146±26.8) and SK-Hep1/HK cell (129±19.0) (p<0.05). (8) After incubation for 24h, the VEGF production of SK-Hep1/RhoC-siRNA cells was 91.2±13.7pg/105 cells, which was much lower than that of SK-Hep1 cell (141.7±24.1pg/105 cells) and SK-Hep1/HK cell (134.6±16.1pg/105 cells) (p<0.01). (9) The tumor was formed at the fifth day after planting SK-Hep1 cell or SK-Hep1/HK cell subcutaneously in nude mice, which grew more quick than that of SK-Hep1/RhoC-siRNA cell, while the latter formed tumor at the eighth day after planting, and the size of tumor was much smaller than the other two groups (p<0.05).Conclusions: (1) Rho-siRNA expression vector was successfully constructed, after stably transfected in SK-Hep1 cells, it could effectively inhibit the expression of RhoC, and the inhibition ratio was about 60%. (2) Rho-siRNA didn't influence the cell morphous, the growing manner, and the proliferation activity of SK-Hep1 cell, while it could significantly inhibit the ability of adhesion to extracellular matrix, the ability of migration to scratch wound, and the ability of invasion in vitro, and it could decrease the production of VEGF, at the same time, the growth of the hepatoma carcinoma cell transfected with RhoC-siRNA expression vector in nude mice was inhibited. (3) RhoC-siRNA could inhibit the invasive and metastasis potential of SK-Hep1 cells, and the gene intervention targeted on RhoC may partly reverse the malignant phenotype of hepatoma carcinoma cells. There is clinical prospect of RhoC-siRNA in the prevention of metastasis and recurrence of HCC. |
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