| IntroductionBreast cancer is the most common malignant tumor of women all over the world. The incidence of breast cancer varies greatly around the world, it is greatest in the developed countries and lowest in developing countries. We know that breast cancer severely impair female physical and psychological health. Thus, prevention and treatment of the breast cancer is one of the most popular topics in tumor study.DLC-1 (deleted in liver cancer-1) is a candidate tumor suppressor gene for hepatocellular carcinoma and other cancers,such as breast cancer. etc. The DLC-1 protein contains four major functional domains: a Rho-GAP domain, an N-terminal sterileαmotif (SAM) domain, a serine-rich (SR) region, and a C-terminal steroidogenic acute regulatory protein related lipid-transfer (START) domain. Determination of the DLC-1 cDNA sequence showed that it is the human homologue of rat p122, which has been shown to function as a GTPase activating protein for RhoA, and it may be involved in signal transduction pathways regulating cell proliferation and adhesion. In normal adult tissues the DLC-1 gene is widely expressed. DLC-1 is localized to focal adhesions located at the periphery of cells. Overexpression of DLC-1 in cultured cells resulted in the loss of actin stress fibers and the detachment of cells. This indicates that DLC-1 could regulate cell morphology and adhesion. Transfection of the DLC-1 cDNA caused growth inhibition in HCC cell lines and suppressed tumor formation in breast cancer cell lines in nude mice. These observations strongly suggest that DLC-1 is a tumor suppressor gene.The methylation of some tumor suppressor genes is one of the important mechanisms in tumorigenesis. The methylation state of some genes can be used as a biomarker for tumorigenesis. Aberrant methylation of CpG islands within promoter region usually happened earlier than cell hyperplasia; the global hypomethylation occurs with the cancer genesis and becomes more obvious with cancer development. So changes in DNA methylation patterns can be detected for diagnosis and prognosis evaluation. The promoter region of the DLC1 gene contains a CpG island containing several CpG sites. It can be methylated to promote gene silencing and prevent transcription. Based on the proof of CpG islands having existed in DLC-1 promoter region, research the methylation status of DLC-1 promoter and the relationship with breast cancer. It may help us understand cancer genesis, development, mechanisms of metastasis of breast cancer.Objectives1) To investigate the mechanism of low expression of DLC-1 in human breast cancer cell line.2) To identify whether low expression of gene DLC-1 due to the methylation of DLC-1 promotor.3) To explore the influences of 5-Aza-CdR treatment on the morphology and migration of MCF-7 cells.4) To explore the morphological effects of DLC-1 overexpression on MCF-7 cells.Methods1) Genome DNA of MCF-7 cells was extracted, and treated by bisulfite procedure. Methylation and non- methylation specific primers for DLC-1 were adapted from the literature. The methylation-specific PCP (MSP) was used to examine the methylation status of DLC-1 promoter. MCF-7 cells were treated by different concentrations of DNA methyltransferase inhibitor 5-Aza-CdR. DLC-1 mRNA levels were examined by RT-PCR and Real-time PCR before and after treatment with 5-Aza-CdR.2) MCF-7 cells were treated by different concentrations of 5-Aza-CdR. The expression of DLC-1 mRNA can be examined by RT-PCR before and after treatment with 5'-Aza-CdR. Western Blot were performed to analyze the expression of DLC-1 protein level before and after treatment.3) MCF-7 cells were treated by different concentrations of 5-Aza-CdR. The morphology of cells were detected by inverted microscope. Scratch assay was used to determine the cell migration.4) The DLC-1/pcDNA3.1/Nv5-DESTTM plasmid was transfected into MCF-7 cells. The expression of DLC-1 was determined by polyclonal anti-V5 antibody followed by CY3-conjugated mouse anti-rabbit antibody. The actin stress fibers were stained with FITC-conjugated phalloidin. Immunofluorescence assay was performed to detect the effect of DLC-1 gene on actin organization and cell morphological changes. Results1) Methylation of CpG island of DLC-1 promoter was observed in MCF-7 cells.2) After 5-Aza-CdR treatment, the expression of DLC-1 in MCF-7 cells was significantly increased, which indicated that inhibiting methylation of DLC-1 promoter region could up-regulate the expression of DLC-1.3) MCF-7 cells were treated by 5-Aza-CdR significantly induced cell rounding. Detection of MCF-7 cells migration by the scratch assay. The growth of tumor cells was significantly slower after treated by 5-Aza-CdR.4) Immunofluorescence staining implied that overexpression of DLC-1 can significantly inhibit the capacity of the cytoskeleton's formation. Cell expression of DLC-1 induced cell rounding.Conclusions1) Methylation of DLC-1 promoter might be the main reason of the low expression of DLC-1 in MCF-7 human breast cancer cells.2) The 5-Aza-CdR can inhibit the methylation of DLC-1 promoter.3) MCF-7 cells were treated by 5-Aza-CdR induced cell rounding. The capability of growth and migration of tumor cells was significantly slow after treated by 5-Aza-CdR.4) And the overexpression of DLC1 can inhibit the capacity of the cytoskeleton's formation, and further indicated that it may impact the proliferation and migration of cancer cells. |
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