The Role Of Ovol2 In Regulating The Stemness Of Breast Cancer Cells

Objective:To analyze the expression characteristics of OVOL2 in breast cancer cell lines;to study the effects of OVOL2 on breast cancer cell surface marker CD44 and to explore the regulatory mechanism of OVOL2 on breast cancer cell stemness.To detect the effects of overexpression of OVOL2 on breast cancer cell proliferation,migration and invasion.The correlation between OVOL2 and clinical prognosis of breast cancer was analyzed by studying the survival time of breast cancer patients without recurrence.Methods:1.Western blotting was used to detect the expression of OVOL2 protein in different breast cancer cells;overexpression and knockdown of OVOL2 in different breast cancer cells were used to detect the expression of CD44 protein.2.Flow cytometry was used to detect the effects of overexpression of OVOL2 on the percentage of breast cancer cells labeled with CD44⁺/CD24^-/low.3.The effects of OVOL2 on proliferation,invasion and migration of breast cancer cells were studied by cell culture,scratch test,Transwell test and breast microsphere culture.4.The binding sites of OVOL2 on CD44 promoter were detected by chromatin immunoprecipitation assay to study the structural basis of OVOL2 regulating stemness.5.The survival data of Kaplan-Meier Plotter database were analyzed to study the correlation between OVOL2 and clinical prognosis of breast cancer.6.By using SPSS19.0 software to statistical data,P value<0.05 is defined as having differences,statistically significance.Results:1.Western blotting results showed that the expression level of OVOL2 was different in different breast cancer cells.2.OVOL2 significantly inhibited the percentage of breast cancer cells labeled with CD44⁺/CD24^-/low;OVOL2 significantly inhibited the expression of CD44 on the surface of MDA-MB-231 cells,and the expression of CD44 in MDA-MB-231 cells was up-regulated after knocking down OVOL2.3.The results of cell culture showed that overexpression of OVOL2 significantly inhibited mammospheres formation in MDA-MB-231 cells;overexpression of OVOL2 significantly inhibited invasion and migration of breast cancer cells.4.Survival analysis of Kaplan-Meier Plotter database showed that OVOL2 was closely related to breast cancer recurrence and supported the function of OVOL2 in inhibiting breast cancer cell stemness.5.Chromatin immunoprecipitation?CHIP?assay revealed the most likely binding site of OVOL2 on CD44 promoter.Conclusions:OVOL2 may inhibit the phenotype of breast cancer stem cells by down-regulating the expression of CD44 on the surface of breast cancer cells,and the most likely binding site of OVOL2 was found on the CD44 promoter.It will lay a foundation for the further study of molecular mechanism and provide theoretical help for the development of targeted drugs.