| Objective: Epigallocatechin-3-gallate(EGCG)is the most abundant ingredient in green tea.Previous studies have reported that EGCG has anti-inflammatory,anti-oxidation and anti-tumor effects,but its specific mechanism is not clear.The purpose of this study is to explore the molecular mechanism of EGCG inhibiting the migration and inflammatory of breast cancer cells,and to provide a scientific basis for the further development of EGCG and its application in the prevention and treatment of breast cancer.Methods:1.The effect of EGCG on the migration of breast cancer cells MDA-MB-231 and MCF-7 in breast cancer cells were treated with different concentrations of EGCG(5 ?M,10 ?M,20 ?M,50 ?M,100 ?M).Then the cell activity was detected by Alarmar blue to determine the optimum concentration and time in 24 h,48 h and 72 h.According to the concentration and time of EGCG determined in the above experiment,breast cancer cells MDA-MB-231 and MCF-7 were treated sequentially,and wound healing and transwell experiments were performed to investigate the effect of EGCG on the migration of breast cancer cells.2.EGCG affected the expression of migration related genes in breast cancer cells After the treatment of breast cancer cells with EGCG,the m RNA and protein respectively expression levels of migration related genes E-cadherin and Vimentin were detected by RT-q PCR and Western blot.The expression of tumor suppressor gene SCUBE2 were detected,and the association between the expression of SCUBE2 and migration related genes was analyzed.3.EGCG inhibited the migration of breast cancer cells by altering the expression of SCUBE2 gene Firstly,In order to determine the correlation between SCUBE2 and the development of breast cancer,the expression of SCEBE2 was detected in breast cancer tissue and adjacent tissues by immunohistochemistry.Then,the sh RNA recombinant lentivirus vector of the SCUBE2 gene was constructed to screen the stable breast cancer cell lines that interfered with SCUBE2,and the expression levels of E-cadherin and Vimentin was detected in SCUBE2-knockdown cells.Finally,EGCG was used to treat SCUBE2-knockdown cells,The effects on cell migration were detected by Wound healing and Transwell experiments.4.EGCG affected the expression of SCUBE2 by affecting its DNA methylation of the promoter region The DNA methylation level in SCUBE2 promoter region was detected in breast cancer tissues and adjacent tissues by using bisulfite treatment and cloning sequencing.After EGCG treatment,the methylation ratio of the SCUBE2 gene promoter was analyzed in breast cancer cells,and the expression of E-cadherin and Vimentin genes was detected by RT-q PCR and Western blot,which suggested that EGCG inhibited the migration of breast cancer cells by affecting the methylation of SCUBE2.5.The effect of EGCG on the release of inflammatory factors in breast cancer cells After EGCG treatment of breast cancer cells,the expression levels of cytokine IL-6 and MCP-1 were detected by the ELISA.In addition,after pretreating the cells using the STAT3 signaling inhibitor S3I-201,the changes of cytokines were detected in EGCG culture medium,and analyzed the possible signaling pathway involved in the inhibition of the cell factor release by EGCG.Result:1.The cell activity assay showed that the activity of breast cancer cells was significantly inhibited when they were treated with 20 ?M EGCG for 24 h(p<0.05).Thus,this condition was used in the subsequent experiments.Wound healing and Transwell experiments showed that EGCG could significantly inhibit the LPS induced migration of breast cancer cells(p<0.01).2.The inhibitory effect of EGCG on breast cancer cells was accompanied by the increased E-cadherin and reduced Vimentin,which were the marker genes of cell migration.In addition,we found that the tumor suppressor gene SCUBE2 was significantly increased.Therefore,we hypothesized that the SCUBE2 gene may be related to the inhibition of EGCG on breast cancer cells.3.The Immunohistochemical assay showed that the expression of SCUBE2 in the breast cancer tissue was significantly lower than that of the adjacent tissues,indicating that the low expression of SCUBE2 could promote the development of the tumor.After interfering SCUBE2,the expression of E-cadherin was reduced and the expression of Vimentin was increased,indicating that SCUBE2 can regulate the expression of the E-cadherin and Vimentin as a upstream gene.After EGCG treating SCUBE2-knockdown stable cell lines,we found that the inhibitory effect of EGCG was weakened,which proved that EGCG could inhibit the migration of breast cancer cells by promoting of the expression of the SCUBE2 gene.4.The degree of DNA methylation of SCUBE2 promoter in breast cancer tissues was significantly higher than that in adjacent tissues(p<0.05),which was consistent with the results of gene expression assay.After EGCG treating breast cancer cells,the changes of methylation of SCUBE2 were detected.The results showed that EGCG could significantly reduce the methylation ratio of SCUBE2(p<0.05).Thus,EGCG regulated the expression level of SCUBE2 by affecting the methylation of the promoter region.5.ELISA assay showed that EGCG treatment significantly inhibited the expression of inflammatory related factors(IL-6 and MCP-1).After pretreatment of cells with STAT3 signaling pathway inhibitor S3I-201 or EGCG,we found that the regulatory role of EGCG on the expression of inflammatory related factors was non-significant compared with that of the S3I-201 treatment group.Therefore,we hypothesized that EGCG may regulate the expression of inflammatory factors related to breast cancer through STAT3 signaling pathway.Conclusion: EGCG regulated its expression by affecting the level of methylation in the SCUBE2 promoter region,and SCUBE2 eventually inhibited the migration of breast cancer cells by regulating the expression of migration related genes(E-cadherin and Vimentin).In addition,EGCG may inhibit the expression of breast cancer related inflammatory factors through STAT3 signaling pathway. |
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