OBJECTIVETo observe the biological changes of SMMC 7721 cell line which was stably transfected with HCCR-2 gene and to study the molecular mechanism of HCCR-2 expression in SMMC 7721 cell line.

METHODSSMMC7721 cells were transfected with HCCR-2-pEGFP-N1 and pEGFP-N1 by lipofectmine 2000 and the transfectants were selected and confirmed using Western-blot technique. Cell cycles were tested by flow cytometry. The reproductive activity was detected by MTT assay. SMMC 7721 cells were routinely cultured with treatment of increasing concentrations (0, 50, 100, 200 ng/ml) of EGF for 24 h. Cells were pretreated with the specific inhibitor of PI3K (LY294002) and then cultured with EGF (100 ng/ml) for 24h and the expression of HCCR protein in these cells were measured by Western blot. Another set of SMMC 7721 cells were transfected with DN-Akt-pcDNA3.1 (dominant negative Akt kinase) and pcDNA3.1 by lipofectmine 2000. The transfectants were selected and confirmed using Western-blot technique as before. HCCR-2 and bcl-2 were measured on protein level by Western blot.

RESULTSSMMC 7721 cells were stably transfected with HCCR-2 gene. HCCR-2 gene transfection can increase the proportion of S-phase cells (21.62%+/-1.33% vs 15.76%+/-0.73%, P<0.01) and decrease the cell apoptosis (1.28%+/-0.16% vs 7.72%+/-0.23%, P<0.01). MTT assay showed the growth of HCCR-2 gene transfected cells was faster than that of empty vecter transfected cells. The HCCR-2 protein was up-regulated in a dose-dependent manner in the cells cultured with different concentrations of EGF for 24 h. Treatment of SMMC 7721 cells with specific inhibitor of PI3K (LY294002) suppressed EGF-induced HCCR-2 expression. HCCR-2 protein was down-regulated in dominant negative Akt transfectants.

CONCLUSIONSEGF upregulated HCCR-2 protein expression in SMMC 7721 cell line via PI3K/Akt pathway. The overexpression of HCCR-2 could enhance the division and proliferation of SMMC 7721.