Establishment Of Cell Sublines With High-and Low-metastatic Potential From Human Breast Cancer Cell Line MCF7 And Their Microarray-based Gene Expression

[Objective] To establish variant cell sublines expressing green fluorescent protein with high and low metastatic potential and to investigate gene expression profile in breast cancer cell sublines with different metastatic potentialities, in order to find new candidate genes related to metastasis of breast cancer to bone.[Methods] MCF7 cells were transfected by GFP gene, two cloned cell sublines(A2, C6) were isolated by limiting dilution technique and electrophoretic technique. They were selected for further research on growth rate, colony formation, cell migration assay, subcutaneous transplantation in nude mice. The gene expression of A2 and C6 cell sublines were investigated by using cDNA microarray analysis. The alterations in ITGA-V and ITGA4 gene expression of these two sublines were confirmed by realtime quantitative RT—PCR.[Results] Two clones expressing Green fluorescent protein with high (A2) and low (C6) metastatic potential were isolated from the parent cell line. Compared with C6, A2 was faster in vitro growth rate (tumor cell doubling time was 25.6 h vs 41.8 h). The clone formation rates were 30.7% for A2 and 14% for C6. Invasion assay in vitro demonstrated that the number of penetrating cells was 136.80±12.36 cells/field for A2 vs. 73.62±9.76/field for C6. After subcutaneous transplantation of A2 and C6, A2 showed a stronger tumorigenicity than C6 after transplantation.There were significant differences in gene expression between high and low metastatic potential cell sublines. Among the target genes, 767 differentially expressed genes were identified in these two cell sublines. Compared to A2, 480 of 767 differentially expressed genes were up-regulated and the other 287 were down-regulated. Realtime quantitative RT—PCR revealed the mean ITGA-V gene copies of A2 cell line were 6574.56 while C6 were 318.84, copies in A2 cell line were significantly higher than those in the C6; the mean ITGA4 gene copies of A2 cell line were 19.98 while C6 were 49.26, copies in A2 cell line were lower than those in the C6.[Conclusion] Two clones expressing Green fluorescent protein with different metastatic potential were established, which could be valuable for further study on the molecular mechanisms and therapeutics of breast carcinoma metastasis to bone. There were different gene expression profiles between high and low metastatic potential cell sublines.These genes may provide important clues for finding genes related to metastasis of breast cancer to bone. The overexpression of integrin aV and loss expression of integrin a4 were related to the invasive behaviour of breast cancer cells which metastasis to bone.