The Expression Of BAG-1 In Cutaneous Melanoma And Its Effects On Biological Behavior Of Melanoma Cells In Vitro

Malignant melanoma is one of the most malignant tumors. Cutaneous malignant melanoma accounts for 80% mortality of all cutaneous tumours, and the incidence of malignant melanoma is rising rapidly. As a multifunctional protein, BAG-1 has many important biological activities in tumor genesis, development and therapy response. Interestingly, there's rare report about the expression of BAG-1 in cutaneous malignant melanoma tissues and its relationship with clinicopathologic characteristics, and the exact effects of BAG-1 on malignant melanoma is still unclear.ObjectiveDetect the expression of BAG-1 protein in cutaneous malignant melanoma and explore its correlation with clinicopathological characteristics. Further investigate the effect of BAG-1 gene inhibition on cell proliferation, cell cycle, cell apoptosis and cellular response to docetaxel in mouse melanoma cell line B16F10.Materials and methodsPart 1: Immunohistochemistry was done to examine the expression of BAG-1, p53, and Bcl-2 in paraffin-embedded tissue blocks containing 32 cutaneous malignant melanoma, 20 pigemented nevi and 10 nomal skin tissues. The correlations between BAG-1 and clinicopathological characteristics were analyzed. The expression of BAG-1 in melanoma cell lines M14 and B16F10 was also analyzed.Part 2: The recombinant plasmids of BAG-1, pRNAT-U6.1/Neo-BAG-1-1, -2, -3, were constructed and transfected into melanoma cell line B16F10, the positive clone was selected by G418 method. RT-PCR and western blot were performed to detect the expression of BAG-1 mRNA and protein in transfected, negative control and untransfected cells. The most effective shRNA plasmid was selected to next step.Part 3: Flow cytometry was used to analyze cell cycle, MTT assay and clone formation assay to confirm cell proliferation, TUNEL assay to determine cell apoptosis. After using MTT assay to determine proliferation inhibition rate, docetaxel was added to cells including untransfected, transfected negative control and most effective shRNA plasmid in 10 umol/l. Flow cytometry was done to analyze cell cycle, TUNEL assay to determine cell apoptosis. ResultsPart 1: The positive rate of BAG-1 expression was significantly higher in malignant melanoma than pigmented nevi and normal skin tissues (78% vs. 15%, 0%, P<0.001) . Statistically significant association was observed between BAG-1 expression and Clark level (r=0.47, P<0.05). Expression of BAG-1 was not correlated with p53 and Bcl-2 (r=0.05, 0.11, P>0.05) . The positive expression of BAG-1 was confirmed in melanoma cell lines M14 and B16F10.Part 2: The eukaryotic expression plasmids-pRNAT-U6.1/Neo-BAG-1-1, -2, -3 were constructed and verified by restriction enzyme digestion and DNA sequencing. The transfection rates in B16F10 cells was 20～30%. Comparing with the untransfected and negative control cells, the expressions of BAG-1 mRNA and protein in shRNA plasmid transfected cells were significantly inhibited by BAG-1 shRNA (P<0.05) . The most effective shRNA plasmid was pRNAT-U6.1 /Neo-BAG-1 -3, its inhibition rates were (77±4) % and (62±2) %, respectively.Part 3: Statistically significant differences were found in cell cycle, cell proliferation and cell apoptosis among transfected, negative control and untranfected groups (P<0.05) . Cells transfected with pRNAT-U6.1/Neo-BAG-1-3 exhibited higher percentge of S-phase cell, lower proliferation rapid, less cell clones and more apoptotic cells.24 h after docetaxel added, cells exhibited higher G2-phase percentage. Statistically significant difference was not found among groups; 48 h later, G2-phase percentage in cells transfected with pRNAT-U6.1/Neo-BAG-1-3 was higher than negative control and untranfected cells (P<0.05). About cell apoptosis, the number of apoptotic cells was higher in cells transfected with pRNAT-U6.1/Neo-BAG-1-3 than negative control and untranfected cells both 24 h later and 48 h later (P<0.05) .ConclusionsBAG-1 protein is highly expressed in cutaneous malignant melanoma, and its expression is positively corrected with Clark level. The shRNA plasmid pRNAT-U6.1/Neo-BAG-1-3 can predomiantly and specifically inhibit the expression of BAG-1 gene in mouse melanoma cell line B16F10. BAG-1 can affect the cell cycle, cell proliferation, cell apoptosis and cellular response to docetaxel of mouse melanoma cell line B16F10. After the expression of BAG-1 gene was inhibited, B16F10 cell exhibited S-phase stasis, lower proliferation ability, more apoptotic cells, and more sensitive to docetaxel induced G2-phase stasis and apoptosis.