Expression And Roles Of RNF2 In Breast Cancer

Nowadays,cancer is the public security problem which catches global attention and causes serious damage to people’s health.The most common malignant tumor of women is breast cancer.Although many of the tumor biomarkers have been found in breast cancer,the sensitivity and specificity of diagnosis need to be improved.Polycomb group(Pc G)which modifies histones and inhibits gene transcription contains polycomb repressive complex 1 and 2(PRC1 and PRC2,respectively).It plays an important role in maintaining cell identification,regulating cell differentiation,maintaining chromosome structure,pluripotent stem cell differentiation and tumor cell transformation.Ring Finger protein 2(RNF2,Ring1b),as catalytic subunit of PRC1,has E3 ligase activity to catalyze monoubiquitin of H2 A at lysine 119(H2AK119Ub1),promote histones degradation,affect cell proliferation,migration and cell apoptosis through different molecular biological signaling pathways and thus play a vital role in the process of the development of tumor.Objective: To observe the expression of RNF2 protein in breast cancer and breast benign lesions tissues,and analyze the association between RNF2 protein expression and clinicopathological features in breast cancer patients;To explore the effects of RNF2 protein on cell proliferation,migration and invasion of breast cancer and to explore the potential biological mechanisms.Methods:1)Detect the expression of RNF2 protein in 112 breast cancer and 21 breast benign lesions tissues using immunohistochemistry;2)Detect the expression of RNF2 m RNA in non-tumorigenic breast epithelial cells and breast cancer cell lines using QRT-PCR;3)The plasmid to overexpress RNF2 protein was transfected into low RNF2 protein cell line and RNF2-si RNA was transfected into high RNF2 protein cell line.The changes of cell proliferation,colony formation,migration and invasion ability were observed using MTT assay,colony formation assay and Transwell assays after transfection;4)Discuss the mechanism of the influences of RNF2 protein on biology behaviors of breast cancer using immunofluorescence and western blot methods.Results:1)The expression of RNF2 protein was higher in breast cancer tissues than in breast benign lesions tissues confirmed by immunohistochemistry(P<0.05).2)The positive expression of RNF2 protein in breast cancer tissues was closely associated with tumor size,lymph node metastasis and TNM stage(P<0.05),but not obviously related to patient age,tumor grade,and the expression of ER,PR,and HER-2(P>0.05).3)QRT-PCR showed that the expression levels of RNF2 m RNA in 5 breast cancer cell lines were significantly higher than in 2 non-tumorigenic breast epithelial cell lines(P<0.05);4)Vector GFP or plasmid RNF2-GFP prepared by laboratory was transfected into MCF-7 cells for 48 h,while NC(negative control)or RNF2-si RNA was transfected into MDA-MB-231 cells for 48 h,and then cell proliferation ability was detected in d1,d2,d3,d4 by MTT assays.The results showed that cell viability of MCF-7 cells transfected with RNF2-GFP was gradually higher than that in GFP group(P<0.05);cell viability of MDA-MB-231 cells transfected with RNF2-si RNA was graduallylower than that of control group(P<0.05).5)After overexpression of RNF2 protein,the colony formation number of MCF-7cells was more than that in control group(P<0.05);On the contrary,the colony formation number of MDA-MB-231 cells after silencing of RNF2 protein was significantly reduced(P<0.05).6)Overexpression of RNF2 protein in MCF-7 cells significantly increased cell numbers through Transwell chamber while knockdown of RNF2 protein in MDA-MB-231 cells significantly reduced cell numbers through Transwell chamber(P<0.05).7)Immunofluorescence and Western blot assay showed that E-cadherin protein decreased with overexpression of RNF2 protein and increased with knockdown of RNF2 protein.Conclusion:RNF2 protein plays an important role in the development of breast cancer,which may be mediated by down regulation of E-cadherin protein.