| Background:Protein4.1is a member of the protein4.1super family which includes the4.1R,4.1G,4.1N and4.1B homologous proteins encoded by different genes. Protein4.1molecule family members have been found that they all contain three conserved domains through which interact with other proteins:FERM (Four.1protein, Ezrin, Radixin, Moesin), SABD (Spectrin and Actin-binding Domain) and CTD (C-Terminal Domain). In addition to the conservative domain, these members include three distinct domains U1, U2, and U3. The N terminal amino acid compositions in these unique domains that have unidentified function are distinctly different in every members of protein4.1super family, which may be different structural basis represent different4.1molecular functions.Previous studies suggested that protein4.1R have some important functions in maintaining the mechanical stability of red cells and promoting the interaction of spectrin-actin with some transmembrane proteins during this process. Besides, more studies have shown that the members of proteins4.1family have some relations with cell proliferation and motality. Latest studies found that the members of protein4.1family can be regarded as a negative regulator of tumor growth. For example, the loss of4.1B was occurred in lung cancer, breast cancer, prostate cancer and meningeoma, etc.4.1R and4.1G have been found in the progress of brain tumor occurrence or development.4.1B was reported to have connection with the metastasis of sarcoma and prostate cancer. Breast cancer can be regarded as one of the most common malignant tumors in women. Though great progresses have been made in the diagnosis and therapy of breast cancer, the distant metastasis still remains a main problem. The5-year survival rate in non-metastatic breast cancer cases was reported up to98%, but can dramatically decreased to27%when internal organs and brain transfer occurs. Thus, it is important to identify new bio-markers of metastasis and study the mechanisms in breast cancer.Our previous study has suggested that4.1N protein may have roles in breast cancer metastasis. Base on this finding, further study on the function of4.1N in breast cancer metastasis will be discussed in this study.Objective:To study the effects of4.1N on the ability of cell migration and invasion in MCF-7breast cancer cells.Methods:RNAi plasmid vector pEZsiRNA6.1-4.1N was constructed and verified by PCR and sequencing, followed by transfection into MCF-7cells observed by fluorescence. Western blot was used to test the protein level of4.1N before and after the cell thasfection of MCF-7. The changes of biological characteristics of MCF-7cells before and after transfection were assessed by cell proliferation assay, cell adhesion assay, wound healing assay, cell migration assay and cell invasion assay to identify the function of4.1N in metastasis.Results:1. The RNAi plasmid vector pEZsiRNA6.1-4.1N was successfully constructed and and verified by PCR and sequencing. Western blot showed that the4.1N protein level was decreased in MCF-7cells after transfection.2. Biological characteristics study of transfected cells showed that the cell proliferation was significantly enhanced in pENTRsh4.1N-human group and pEZsiRNA6.1-4.1N group compared to negative control group and non-transfected group (P<0.01). The negative results were observed as to the adhesion of cells to matrix Fibronectin (P<0.01). The cell migration (P<0.01) and cell invasion (P<0.01) were also significantly enhanced in pENTRsh4.1N-human group and pEZsiRNA6.1-4.1N group.Conclusions:Protein4.1N can inhibite the cell proliferation, migration and invasion in MCF-7cell line, which suggested a potential role to be a negative regulator of breast cancer metastasis and a molecular marker for metastasis and prognosis in breast cancer. |
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