The Effect Of PTBP1 On Growth And Invasion Of Breast Cancer Cells

Objective: Breast cancer is one of the most common malignancies in women.About 120,000 women develop breast cancer every year in the world,and 500,000 women die from breast cancer.Developed countries such as North America and Europe are high-risk areas for breast cancer.According to statistics,one in nine women in the United States will have breast cancer during their lifetime.Asia is a low-risk area for breast cancer,but the incidence of breast cancer is increasing year by year,and there is a tendency to rejuvenate.PTBP1 is a member of heterogeneous nuclear ribonucleoproteins(hnRNPs)family,which can play a variety of functions on many aspects of RNA metabolism.PTBP1 is crucial to the regulation of alternative splicing,initiation of IRES-mediated translation and increase the stability of transcription.The cellular localization of PTBP1 and its interaction with many factors determine the diversity of its functions.It has been reported that PTBP1 is highly expressed in breast cancer tissues compared with normal tissues.PTBP1 is also highly expressed in breast cancer cell lines in cell line assays.PTBP1 can promote the growth and invasion of breast cancer cells,but the specific mechanism is still not clear.The PTEN-AKT signal transduction pathway has a broad and close relationship with tumors.Its imbalance not only promotes tumor development,but also promotes tumor invasion and metastasis,and participates in tumor immune escape.Autophagy also plays an important role in the progression of tumors.Different forms of phosphorylation can occur in about 30% of the body’s proteins,and its phosphorylation sites mainly occur in tyrosine,serine and threonine residues.Abnormal phosphorylation of proteins often leads to abnormalities in cell activities,and even cell damage or canceration.P21-activated kinase(P21-activated kinase,PAKs)is conserved serine / threonine protein kinases,which can be activated by growth factors and other extracellular signals.Through numerous downstream binding protein or kinase substrate,PAKs plays key function in regulating different biological functions,including cytoskeletal reorganization,cell polarity modulation,cell migration and gene transcription regulation,particularly in cell malignant transformation and tumor cell invasion.As a latest discovered and the least researched member of Class II,PAK5 is closely related to tumor progression and development.This study was to investigate the effect and mechanism of PTBP1 on the growth,migration and invasion of breast cancer cells,and to explore how PTBP1 affects the growth of breast cancer cells through PTEN/Akt pathway and autophagy,and the phosphorylation of PTBP1 by PAK5.Provide evidence for the molecular mechanism of development of breast cancer.Methods: Ⅰ.Verifying the expression of PTBP1 in breast cancer cells and breast cancer tissues1.Western blot analyzing PTBP1 protein expression in breast cancer cells.2.Western blot analyzing PTBP1 protein expression in breast cancer tissues.3.Immunohistochemistry analyzing PTBP1 protein expression in breast cancer tissues.Ⅱ.Confiming the important meaning of PTBP1 in breast cancer development1.MTT detecting the effect of PTBP1 on breast cancer cells proliferation.2.Clone formation assay detecting the effect of PTBP1 on breast cancer cells proliferation.3.Transwell detecting the effect of PTBP1 on the migration and invasion capacity of breast cancer cells.4.Nude mice bearing tumor experiment detecting the effect of PTBP1 on breast cancer cells proliferation.Ⅲ.Study on the mechanism of PTBP1 promoting the proliferation of breast cancer cells1.Western blot confirmed the effect of PTBP1 on PTEN-Akt signaling pathway.2.Western blot verified the effect of PTBP1 on autophagy.3.Western blot analying the relationship between PAK5 and PTBP1 protein expression.Ⅳ.Verification of interaction between PAK5 and PTBP1.1.TNT combined with GST pulldown test confirmed that PAK5 and PTBP1 were directly combined in vitro.2.Co immunoprecipitation experiments confirmed the in vivo interaction between PAK5 and PTBP1.3.Overexpressed PAK5 in breast cancer cells,Western Blot detecting the effect of PAK5 on the expression of PTBP1 protein.4.In vitro kinase assay was used to detect the effect of PAK5 on the phosphorylation of PTBP1.Results: Ⅰ.Verifying the expression of PTBP1 in breast cancer cells and breast cancer tissues1.PTBP1 is highly expressed in breast cancer cell lines.2.PTBP1 is highly expressed in breast cancer tissues.Ⅱ.Confiming the important meaning of PTBP1 in breast cancer development1.PTBP1 could promote the proliferation of breast cancer cells.2.PTBP1 promotes the migration and invasion of breast cancer cells.3.PTBP1 could promote the proliferation of breast cancer cells in nude mice.Ⅲ.Study on the mechanism of PTBP1 promoting the proliferation of breast cancer cells1.PTBP1 could active p-Akt signaling pathway through downregulating PTEN.2.PTBP1 could inhibit cell autophagy.3.PTBP1 and p-Akt expression in breast cancer tissues present strongly correlated.Ⅳ.Verification of interaction between PAK5 and PTBP1.1.PAK5 and PTBP1 can directly combine in vitro.2.PAK5 and PTBP1 can interact in vivo.3.PTBP1 is a new physiological substrate for PAK5.4.PAK5 can downregulate PTBP1 expression Conclusion:1.PTBP1 is highly expressed in breast cancer cell lines and breast cancer tissues.2.PTBP1 promotes the growth of breast cancer cells through PTEN/Akt pathway and autophagy.3.PTBP1 is a new physiological substrate for PAK5.