Clinical Significance Of KLF8 Expression In Breast Cancer And Its Effect On Proliferation,migration And Invasion Of Breast Cancer Cells

ObjectiveTo investigate the expression of Krüppel like factor 8(KLF8)in breast cancer tissues and cell lines,and to explore its relationship with clinicopathological features and prognosis of breast cancer patients.The effect of KLF8 expression on the proliferation,migration and invasion of breast cancer cells was verified by cell function experiments.Methods(1)The differential expression of KLF8 in breast cancer was analyzed by Oncomine database,and the relationship between KLF8 m RNA expression and prognosis of breast cancer patients were analyzed by Kaplan Meier plotter database.(2)The expression of KLF8 in 16 cases of breast cancer tissues and their adjacent normal tissues were detected by q RT-PCR and Western blot.(3)The expression levels of normal breast epithelial cell lines MCF-10 A and 7 breast cancer cell lines MDA-MB-231,MCF-12 A,HS-578 T,MCF-7,BT-474,MDA-MB-453 and ZR-75-30 were measured.The expression and localization of KLF8 in MCF-7 and HS-578 T breast cancer cell lines were detected by Immunofluorescence.(4)The expression of KLF8 in 139 cases of breast cancer tissue microarray were detected by immunohistochemistry,and the relationship between KLF8 expression and clinicopathological characteristics and prognosis of breast cancer patients were analyzed.(5)We built KLF8 overexpression breast cancer cell lines: HS-578T—pc DNA 3.1—KLF8,MCF-7—pc DNA 3.1-KLF8,and negative control group :HS –578T —pc DNA 3.1—Vector,MCF-7-pc DNA 3.1 – Vector.Western blot were used to detect the KLF8 transfection efficiency.The effects of KLF8 overexpression on the proliferation,migration and invasion ability of breast cancer cells were investigated by WST-1 assay,Western blot and Transwell invasion assay.Results(1)Finak microarray data in Oncomine database showed that the expression of KLF8 in breast cancer were significantly higher than that in normal breast tissues(P< 0.01).Kaplan Meier plotter database showed that RFS(Relapse Free Survival)、OS(Overall Surival)、DMFS(Distant Metastasis-Free Surval)、PPS(Post Progression Survival)in patients with KLF8 m RNA high expression group were significantly worse than those in low expression group(P<0.05)。(2)The results of q RT-PCR showed that the expression of KLF8 m RNA in 12 cases of breast cancer tissues were significantly higher than that of adjacent normal tissues(P=0.002),Western blot results also showed that the KLF8 expression in breast cancer tissues were significantly higher than that in adjacent normal tissues(P<0.001).(3)The expression of KLF8 in 7 breast cancer cell lines were significantly higher than that in normal breast epithelial cell lines.Immunofluorescence results showed that KLF8 was mainly expressed in cytoplasm in MCF-7 and HS-578 T breast cancer cell lines.(4)The results of tissue microarray immunohistochemical staining were evaluated by H-score,with 75 points as the cut-off point.After excluding 4 cases of exfoliation,there were 63 cases in the high expression group and 72 cases in the low expression group.KLF8 protein expression was correlated with ER(Estrogen Receptor,χ 2=5.183,P=0.023)and PR(Progesterone Receptor,χ 2=8.150,P=0.004)expression.Kaplan-Meier analysis showed that KLF8 protein expression(P=0.002),TNM stage(P=0.003),ER expression(P=0.009),and PR expression(P=0.039)were significantly associated with prognosis.Multivariate Cox regression analysis suggested that TNM stage(P=0.003)and KLF8 expression level(P=0.004)were independent prognostic factors of breast cancer.(5)After transfection of KLF8 in 2 breast cancer cell lines,the successful transfection were confirmed by Western blot,WST-1 assey results indicate the proliferation ability in KLF8 high expression group were significantly higher than the control group,the difference were statistically significant(P<0.005).Then we detected the EMT related markers by western blot,the results showed that the expression of epithelial marker,E-cadherin,were decreased in KLF8 overexpression group,but the expression of mesenchymal markers,Vimentin and,N-cadherin,were significantly increased.Transwell invasion assay showed that the invasion ability of KLF8 overexpression group was significantly higher than the control group,the differences were statistically significant(P<0.005).Conclusions(1)The expression level of KLF8 in breast cancer tissues were significantly higher in public databases and clinical tissues;The expression of KLF8 protein were increased in breast cancer cell lines,and were mainly expressed in cytoplasm.(2)KLF8 protein expression were correlated with ER and PR expression in breast cancer patients;TNM stage,ER,PR and KLF8 expression were related to the prognosis of breast cancer patients.TNM stage and KLF8 expression can be used as independent prognostic factors of breast cancer.(3)KLF8 promoted the proliferation,migration and invasion of breast cancer cells.