Mechanisms Of SFRP1 Regulation By 5-aza-2’-deoxycytidine And Effects On The Epithelial-mesenchymal Transition Of Colorectal Cancer Cells

Objectives: 1. In order to study(5-aza-d C) impact on SFRP1 expression in the colorectal cancer(CRC) cell line SW480. 2. To observe the effects of cell proliferation, migration, invasion with the application of 5-aza-d C in SW480 cells. 3. Analysis the relationship between Wnt pathway antagonist SFRP1 and epithelial mesenchymal transition(epithelial-mesenchymal transition, EMT) transcription factor Slug and to explore the related mechanism.Methods: 1. Immunohistochemical(IHC) analysis was used to detect the expression of SFRP1 and Slug in 85 archival surgical specimens of human CRC and 40 adjacent “normal” tissues. 2. Using the methylation inhibitor 5-aza-d C to intervene the methylation status of SW480 cells. Cells were treated with different concentrations of 5-aza-d C different concentrations for 24 h, 48 h, 72 h in CRC cells. Next, cell activity was detected with CCK8(Cell Counting Kit-8), then chosen the smaller concentration effect on cell proliferation of subsequent experiments. 3. Using Transwell migration assay and Wound healing assay detect migration of SW480 cells caused by 5-aza-d C treated; Transwell invasion assay was used to detect the cell invasiveness. 4. Using Western blot, RT-PCR to detect the expression of SFRP1, β-catenin, Slug and E-cadherin with 5-aza-d C was treated in SW480 cellsResults: 1. Analysis SFRP1 and Slug expression in colorectal cancer by Immunohistochemistry Immunohistochemistry showed: SFRP1 protein positive expression rate in colorectal cancer group(21/85, 24.7%) lower than those in adjacent "normal" tissues(27/40, 67.5%), Slug protein positive expression rate in colorectal cancer group(34/85, 40%) higher than those in adjacent "normal" tissues(3/40, 7.5%). There was no relationship between SFRP1 and Slug expression and gender, age, diameter of tumor, location(p>0.05). The expression rates of Slug were significantly associated with the differentiation, lymph node metastasis and TNM stages(p<0.05). SFRP1 expression significantly correlated with the depth of invasion, lymph node metastasis and TNM stages(p<0.05). The expression of SFRP1 had a negative correlation with Slug expression(r=-0.245, P=0.024). 2. Effect of 5-aza-d C on colorectal cancer SW480 cells proliferation, migration and invasion CCK-8 results showed that, 5-aza-d C has significant inhibitory effect on the proliferation of colorectal cancer SW480 cells, and with increasing of concentration and incubation time of, the more significant inhibition. Selection of 5-aza-d C concentrations(10 μmol/L) less impact on the ability of cell proliferation with Transwell and Wound healing assay experiments, the results show that 5-aza-d C can reduce invasion and migration ability of cells(p<0.05). 3. Effect of 5-aza-d C on the expression of SFPR1 in colorectal cancer cell Select 10 μmol/L concentration of 5-aza-d C treated cells, 48 h later, using Western blot, RT-PCR to detect the expression of SFRP1 found SFRP1 expression was significantly increased 4. To observe the influence of SFRP1 on the expression of canonical Wnt pathway key factor β-catenin and EMT transcription factor Slug Select 10 μmol/L concentration of 5-aza-d C to restore SFPR1 expression after 48 h, using the Western blot and RT-PCR to detect the expression of β-catenin, Slug and E-cadherin, the results showed that the expression of β-catenin and Slug decreased invarying degrees, increased expression of E-cadherin.Conclusion: 1. 5-aza-d C may restore the expression of SFRP1 gene with inhibiting SFRP1 gene promoter methylation. 2. Relevant concentrations of 5-aza-d C may inhibit proliferation, migration and invasion of colon cancer cells. 3. SPFR1 may reduce Slug expression through canonical Wnt/β-catenin signaling pathway, thereby inhibiting the process of EMT in colorectal cancer cells.