Effect And Mechanism Of AJAP1 In Breast Cancer Progression

Objective Breast cancer is a kind of heterogeneous cancer originating from epithelial cells,and it has been the first threat to women's health worldwide for many years.Adherent junction,as one of the ways of connecting epithelial cells(the other two is tight junction?desmosome and hemidesmosome),has also been proved to play a vital role in the progression of cancer.However,there are few reports of adherent junction in breast cancer.Besides,adherent junction associated protein 1(AJAP1),also known as Shrew-1,is a new adhesion junction related protein,which originally was found in epithelial cells.AJAP1 has been proved to play an important role in a variety of tumor progression.However,the role of AJAP1 in breast cancer has not been fully clarified.The purpose of this study is to explore the role of AJAP1 in breast cancer and to explore the mechanism of influencing the progression of breast cancer based on analyzing the results of biological information,above results may improve the understanding of adhesive junctions to people.Methods 1.Firstly,the database was used to analyze the potential research value of AJAP1,and then the clinical data were used to analyze the relationship between AJAP1 and clinicopathological parameters of breast cancer patients,and its effect on the prognosis of breast cancer patients,then the cell lines with overexpressed AJAP1 and knockout AJAP1 expression were conducted.Then MTT,Transwell,wound-healing assay,colony-formation assay and flow cytometry were used to detect the effects of AJAP1 on the migration,proliferation and invasion of breast cancer cells.2.Using STRING software to analyze the protein ?-catenin,which was interacting with AJAP1.And then using immunohistochemical method to analyze the relationship between ?-catenin and clinicopathological parameters of breast cancer patients based on the different localization of ?-catenin.The relationship between ?-catenin and AJAP1 expression was also examined.Co-IP,nuclear-cytoplasmic separation test,ubiquitination test and luciferase assay were used to further clarify the interaction between AJAP1 and ?-catenin and its effect on ?-catenin downstream genes.The recovery experiment of knockout ?-catenin was designed to explore whether AJAP1 exerts its function of inhibiting the migration,invasion and proliferation of breast cancer through ?-catenin both in vivo and in vitro.3.To further explore the upstream molecules of AJAP1 regulating ?-catenin axis,CoIP test,immunofluorescence,nuclear-cytoplasmic separation test,luciferase and construction of mouse model in vivo were used to determine the regulatory effect of EGF/EGFR axis on AJAP1-?-catenin.4.During the process of our experiment,obvious morphological changes were found in the process of constructing AJAP1 and Sh AJAP1 cell lines,and then the effect of AJAP1 on the migration and invasion ability of EMT induced by TGF-?1 were observed by related experiments.5.The targeted micro RNA,which may binding to AJAP1 was predicted by mi Rtarbase and mi RDB database websites,and the specific function of targeted micro RNA in breast cancer was determined by cytological experiments,then the specific binding sites of transcription factors regulating AJAP1 were predicted by Jasper website and verified by Ch IP and other experiments so as to further reveal the specific regulatory mechanism of ZEB1-mi R-3941-AJAP1 in breast cancer migration and invasion induced by TGF-?1.Results: 1.The results of database and immunohistochemistry assay showed that the expression level of AJAP1 was negatively related to histological grade and the number of lymph node metastasis and its high expression also means a good prognosis with breast cancer patients;the results of cellular functional experiments showed that AJAP1 could inhibit the proliferation,migration and invasion of breast cancer and effectively inhibit the cell ratio of G0/G1 phase and increase the cell ratio of S phase and G2 phase.2.The expression level of AJAP1 was related to the different localization of ?-catenin.The experimental results show that AJAP1 and ?-catenin can combine with each other to form a complex,and the knockout of AJAP1 inhibited the ubiquitin degradation of ?-catenin,promoted the entry of ?-catenin from the cytoplasm into the nucleus,and activated the transcriptional activity of ?-catenin and the expression of downstream genes like C-myc and Cyclin D1,and AJAP1 inhibited the proliferation,migration and invasion of breast cancer through ?-catenin.3.EGF could significantly reduce the expression of AJAP1 and promote the nuclear entry activity of ?-catenin.Co-IP confirmed that EGF inhibited the binding efficiency of AJAP1 and ?-catenin complex.Luciferase and other experiments confirmed that EGF inhibited the transcriptional activity of ?-catenin and promoted the expression of downstream genes C-myc and Cyclin D1 of ?-catenin.On the other hand,the expression of AJAP1 was significantly inhibited after EGFR knocked out.The results of recover experiment also confirmed that EGFR/AJAP1 KD group significantly promoted the promoter activity of ?-catenin compared with EGFR KD group,while AJAP1 also counteracted the carcinogenic effect of EGFR in vivo.The above results also proved that EGF/EGFR signal axis weakened the expression of AJAP1 and promoted the nuclear localization of ?-catenin.4.We conducted T47 D cells with knocked down AJAP1 expression and MDA-MB-231 cells with overexpressed AJAP1 expression,the results of RT-q PCR?Western blot and immunofluorescence assay showed that AJAP1 depletion obviously promoted EMT occurrence of breast cancer cells and this process reduced the epithelial marker like E-cad expression but increased the expression of mesenchyme marker like Ncadherin and Vimentin.However,upregulation of AJAP1 obtained the contrary results.Besides,AJAP1 could inhibit TGF-?1 induced EMT?proliferation and invasion ability of breast cancer cell after adding cells with TGF-?1.5.For the sake of explore the potential micro RNA that may targets with AJAP1,we designed different mutants and conducted luciferase assay and the results declared that mi R-3941 can mediate AJAP1 expression.Besides,the KM-plotter database and a series of experiments' results showed that mi R-3941 promoted the proliferation,migration and invasion of breast cancer cells,while Chip and other experiments confirmed that ZEB1 can bind to the AJAP1 promoter to transcriptionally inhibit the expression of AJAP1 while mi R-3941 promoted the expression of ZEB1.Conclusion In breast cells,AJAP1 can regulate the progression of breast cancer through the following two pathways: First,EGF/EGFR negatively feeds back to the regulation of ?-catenin by AJAP1 to promote the entry of ?-catenin into the nucleus and activates the expression of its downstream genes like C-myc and Cyclin D1,which process can mediate the progression of breast cancer;on the other hand,ZEB1 may transcriptionally inhibited the expression of AJAP1 and mi R-3941 increased ZEB1 expression,this process can also mediate the proliferation of breast cells induced by EMT and TGF-?1.