The Role Of SATB1 In The Proliferation And Metastasis Of Melanoma

Cutaneous malignant melanoma, a highly malignant tumor of the melanocytic cells in the skin, a variety of genes were found abnormal expressed, but the real molecular mechanism about its occurrence and development was still unknown. SATB1, a matrix attachment regions binding protein, recently, was found to play an important role in the progression of many kinds of tumors, such as breast cancer, however, there was no report about the correlation between SATB1 and melanoma could be found until now. Therefore, we analyzed in this study (ⅰ) the expression of SATB1 in human melanoma, (ⅱ) the functional role of SATB1 in the proliferation and invasion of melanoma cells in vitro and in vivo.Part 1:The expression of SATB1 in human melanoma cell lines and tissueBy Semi-quantity RT-PCR, compared to melanocytic cells from two healthy donors and breast cancer cell line MDA-MB-231, the expression of SATB1 mRNA in melanoma cell lines A375, M14 and MV3 was investigated. As a result, similar to MDA-MB-231, SATB1 mRNA was overexpressed in the three melanoma cell lines, especially A375, but was negative in normal melanocytic cells.On the level of protein, by western blot, compared to normal melanocytic cells, the expression of SATB1 in the three melanoma cell lines was investigated; compared to 6 case of benign melanocytic nevi, the expression of SATB1 in tissue from 8 patients with melanoma was investigated too. As a result, SATB1 was overexpresed in the all melanoma cell lines and tissue from patients with melanoma, normal melanocytic cells were negative, though benign melanocytic nevi were positive also (0.53±0.28), the level was lower than in tissue from patients with melanoma(1.10±0.32)(P<0.05).The overexpression of SATB1 mRNA and protein showed it may play a role in the development of melanoma as a promoter. Part 2:The effects of knockdown SATBl on melanoma cell line A375To investigate whether SATBl was required for the proliferation and invasion of melanoma, a specific short hairpin RNAs (shRNA) was expressed in melanoma cell line A375 which wide type overexpressed SATBl. When it was confirmed that the expression of SATBl mRNA and protein was remarkably reduced (23.21%and 34.57% of the parental cells, respectively), a series of experiments were put into practice.To investigate the effects of the reduced SATBl on the proliferation of A375 cells, proliferative activities were measured by cell counting and the cell cycle was analyzed by flow cytometry in vitro; by constructing the subcutaneous transplanted melanoma model in athymism mouse, the proliferation was analyzed in vivo. As a result, the number of SATBl-depleted A375 cells was obviously less than of control cells at 24h, 48h and 72h, and the difference increased with time. In vivo, the incubation period of transplanted melanoma model of SATBl-depleted A375 cells (8.17±2.86 days), was longer than that of control cells (4.83±1.17 and 5.33±0.82 days, respectively) (P< 0.05); the tumor volume was smaller at regular intervals than that of control cells, and consequently, the weight of tumors of SATBl-depleted A375 cells (0.65±0.36 g) was smaller than that of control cells (1.44±0.75 g and 1.52±0.74 g, respectively) (P< 0.05). Both in vitro and in vivo study suggested, overexpression of SATB1 may be a promoter to the proliferation of melanoma, and knockdown SATB1 may weaken its proliferation. Though the flow cytometry experiment showed that the G1 phase and apoptosis cells were increased, and the G2 and S phase cells were decreased, these were not statistically significant, the real mechanism needs further research.Furthermore, the effects of SATB1 knockdown on the invasive capacity of melanoma were as investigated. In vitro, a reconstructed basement membrane, as a barrier to discriminate invasive cells from non-invasive cells, served to evaluate the invasive capacity of SATB1 knockdown melanoma cells; and then, by injecting SATBl-depleted A375 cells and control cells into the lateral tail vein of athymic mice, the effect of SATBl depletion on metastasis was evaluated in vivo. Consequently, Compared with the control groups(31.92±6.93 and 35.83±7.46, respectively), the number of SATBl-depleted A375 cells (3.92±2.71) passed through the reconstructed basement membrane was significantly decreased (P<0.05); and the area proportion of tumors developed in athymic mice lungs was markedly reduced from the SATB1 knockdown cells(0.08±0.05), compared with the control groups (0.37±0.22 and 0.40±0.12, respectively)(P<0.05), showed tumours developed in athymic mice lungs was markedly reduced in the SATB1 knockdown cells. So, both in vitro and in vivo, overexpression of SATB1 in A375 cell may contribute to metastasis and knockdown SATB1 may weaken its capacity of metastasis.ConclusionThis study showed when overexpression of SATB1 was depleted, the capacity of proliferation and metastasis was weakened in melanoma, suggested SATB1 may be a promoter to these malignant behaviors, and may be useful as a therapeutic target for melanoma.