Study On The Role Of PAIP1 On Progress In Pancreatic Cancer

Background:Pancreatic cancer(PC)is one of the most aggressive and mortal cancer.In the clinical presentation of PC,it is often insidious and without specific onset symptoms,which leads to a low excisional rate and poor prognosis.At present,the clinical treatment of PC is mainly based on the use of the anti-metabolic drugs(such as 5-fluorouracil(5-FU),gemcitabine and nab-paclitaxel),and targeted drugs(such as bevacizumab,cetuximb,etc.).Despite remarkable advances in treatment of the disease during the past decade,the survival rate has hardly improved due tohigh invasion of the tumor phenotype and resistance to chemotherapy.Therefore,it is very important to understand the molecular mechanism of PC and search for new effective targets for diagnosis and treatment for improving the prognosis and quality of life of PC patients.Polyadenylate-binding protein-interacting protein 1(PAIP1)is a 479-amino acid protein that encoded by PAIP1,which is located at 5p12.PAIP1 is a mammalian PABP that binds to EIF4A and EIF3 and stimulates translational initiation.PAIP1,as a modulator of translation initiation,it is essential for the control of cell growth,proliferation,and differentiation.PAIP1 also participates mRNA turnover,which stabilizes the c-fos proto-oncogene mRNA by combining with the major protein coding-region determinant of instability(mCRD).Deregulation at this step leads to abnormal gene expression,which in turn alters cell growth and possibly leads to cancer development.Scotto et al found that PAIP1 may be involved in the evolution of cervical cancer by using single nucleotide sequence(SNP)analysis and fluorescence in situ hybridization(FISH).Piao J et al have explained that the over-expression ofPAIPlwas significantly correlated with clinical stage,histological grade and poor prognosis of breast cancer patients,Wang Y et al.have shown that AttenuatedPAIP1 expression inhibition proliferation and EMT-related migration of human lung carcinoma cells by regulating AKT/GSK-3? signaling pathway.These findings indicated that PAIPlmight play pivotal rolesin the development of various types of tumors.However,to date,its accurate expression,function and molecular mechanism in PC remains unclear.Therefore,it is urgent to study the mechanism and the biological function of PAIP1in PC,which identify new molecular targets for the development of effective treatments.Objectives:To investigate the effects of PAIP1 gene on biological behaviors in PC and elucidate the possible molecular mechanism of PAIP1 in PC progression.(1)To analyze the relevance between the PAIP1 expression and the clinicopathological significance in PC patients and provide a novel therapeutic target for PC.(2)To delineate the role of PAIP1 in proliferation,metastasis and angiogenesis in PC progress.(3)To explore the effects of PAIP1 on PC cells behavior in vitro and in vivo,and further identify the mechanism of PAIP1 in regulating the process of PC.Materials and methods:(1)Tissue specimens and database analysis:PAIP1 protein expression in 104 PC patients and 27 normal pancreatic tissues were detected by immunohistochemical method,and analyzes the correlation between PAIP1 protein expression and clinicopathological characteristics of PC patients.TCGA cohort and The Human Protein Atlas cohort were used to suggest the value of evaluation of PAIP1 in prognosis of the PC patients.(2)Experiments in vitro:The protein expression levels of PAIP1 were tested by western blot in PC cell lines,includingSW1990,Miapaca-2,Bxpc-3,Capan-1,and Panc-1.Then,according to the expression level of PAIP1 in PC cell lines,the lentivirus-mediated PAIP1 up-regulating and silencing system were used to modulate PAIP1 expression in PC.3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT assay)and colony formationassay were used to measure the effects of PAIP1 modulation on cell proliferation viability.Wound healing assay,transwell migration and invasion assays were applied to test the ability of PAIP1 on cell migration and invasion.The expressions of epithelia-mesenchymal transition(EMT)markers were detected using western blot and immunofluorescence(IF)staining analysis in PC cells.Western blot assay were used to detect PAIP1 exerts its pro-tumor functions via PI3K/AKT pathway activation.Additionally,the effects of PAIP1 on PC angiogenesis were detected by using vasculogenic mimicry,matrigel tube formation,chorioallantoic membrane(CAM)and western blot assays.(3)Experiments in vivo:Different groups ofPC cells were inoculated subcutaneously into the nude mice,and compared the tumor growth in different groups.The model of metastatic tumor in vivo was established by injecting via the tail vein of nude mice,and compared the lung metastasis in different groups.Immunohistochemistry was used to detect the expression levels of PAIP1,Ki67,E-cadherin and Vimentin in collected tumor tissues.Hematoxylin and eosin staining was performed to analyze the metastatic colonies formed in lung tissues.Results:(1)Association between PAIP1 protein and poor prognosis of PC:The IHC results showed that PAIP1 was mainly located in the cytoplasm of PC cells and highly expressed in PC.The positive and strongly positive rate of PAIP1 in PC tissues(71.4%,58.2%)was significantly higher than in normal pancreatic tissues(18.5%,3.7%),and the over-expression of PAI 1 was associated with tumor size,histological grade,clinical stage,lymph node metastasis.Data obtained from TCGA cohort and The Human Protein Atlas cohort showed that patients with high PAIP1 expression had shorter survival time than patients with low PAIP1 expression.(2)PAIP1 significantly promoted the proliferation and colony formation of PC cells:MTT and colony formation assays demonstrated thatPAIP1 promoted PC cells proliferation and clonogenic ability.In vivo experiments furtherproved that the high expression of PAIP1 could promote the subcutaneous tumor formation of nude mice.(3)PAIP1 promoted the migration and invasion of PC cells via EMT process:Wound healing,transwell migration and invasion assays showed that knockdown of PAIP1 markedly inhibitedthe migration and invasion ability of both Bxpc-3 and Capan-1 cells,while over-expression of PAIP1 had the opposite effects.Contrasted with control group,mice inoculated with sh-PAIP1 had fewer lung metastases,while PAIP over-expression group had more.In addition,over-expression of PAIP1 down-regulated the expression levels of epithelial marker(E-cadherin),while up-regulated the expression levels of mesenchymal markers(Vimentin,Slug,Snail)and MMP2.Thus,these results indicated that PAIP1 may promote PC cell migration and invasion via the induction of EMT.(4)PAIP1 promoted angiogenesis in PC.Vasculogenic mimicry and matrigel tube formation assays showed that the ability of tube formation was reduced in PAIP1 knockdown cells,but increased in PAIP 1 over-expression cells.Additionally,CAM assay showed that the blood vessel formation was apparently damaged in the PAEP1-shRNA groups compared with the control groups.And silencing PAIP1 down-regulated the expression level of VEGF was verified via western blot assays.Together,our data indicated that PAIP1 promoted angiogenesis in PC.(5)PAIP1 promoted PC angiogenesis and invasion via the activation of PI3K/AKT pathway.Blocking PI3K/AKT pathway by using PI3K inhibitor LY294002 significantly reduced the proliferation,migration and angiogenesis of PC which induced by PAIP1 over-expression.Also,LY294002 can regulate the EMT process induced by high expression of PAIP1 and down-regulated the expression of VEGF.These results indicated that PAIP1 modulate PC angiogenesis and metastasis though PI3K/AKT pathway.Conclusions:(1)The high expression of PAIP1 was associated with histological grade,clinical stage,LN metastasis,angiogenesis and poor prognosis of PC patients.(2)The elevated PAIP1 expression in PC cells could promote proliferation and the EMT process,whereas the deletion of PAIP1 reversed the malignant progression of PC.(3)The high expression of PAIP1 promoted PC angiogenesis in vitro and in vivo.(4)PAIP1 promoted invasion,angiogenesis and the EMT process via PI3K/AKT pathway in PC.