The Role And Mechanism Of Long Non-coding RNA PTTG3P In The Cell Growth And Metastasis Of Pancreatic Cancer

Pancreatic cancer(PC)is a malignant tumor with a very poor prognosis.Its 5-year survival rate is less than 5%and 90%of patients survive for less than one year.The mortality rate of pancreatic cancer ranks sixth of all tumor diseases in China,and the proportion of young patients is increasing per year.Invasion and metastasis are the significant characteristics of PC.The diagnosis of early PC is difficulty with the lack of prior prognostic indicators.Interventions for the early stage or recurrent of the disease are often not timely,and the chemotherapy resistance of PC is prominent.In-depth exploration of the pathogenesis of PC and seeking effective molecular indicators for the diagnosis and prognosis of PC have important practical significance and scientific valueLong non-coding RNA(LncRNA)is a non-coding RNA with a length of more than 200 nucleotides.It plays an important role in tumorigenesis and development.LncRNA regulates various carcinogenic processes involving related signal pathways at transcription,post-transcriptional or epigenetic aspects.Various studies have proved that lncRNAs play a key role in the development of PC.PTTG3P(Pituitary tumor-transforming 3,pseudogene)is a pseudogene with highly similar sequences to the parental genes PTTG1 and PTTG2 As an lncRNA,the expression of PTTG3P is high in a variety of tumor diseases and plays an important carcinogenic effect.PTTG3P can promote the malignant phenotype of tumor cells through a variety of mechanisms.However,its expression,biofunction and clinical significance in PD AC are yet to be determinedThe aim of this study was to reveal the specific role and clinical significance of PTTG3P in PC,and analyze the specific oncogenic molecular pathways and expression regulatory networks of PTTG3P in PC tumor cells.We found that PTTG3P is significantly up-regulated in pancreatic cancer tumor tissue,and the expression level of PTTG3P is closely related to the size of pancreatic cancer tumor and the degree of tumor differentiation.At the same time,the overall survival of patients with high expression of PTTG3P in tumor tissues is significantly shorter.Bioinformatics and cell biology experiments have proved that PTTG3P can promote the growth and metastasis of PC cells through the FoxM1 signaling pathway.As a ceRNA,PTTG3P can competitively bind miR-132/212-3p to regulate the expression of FoxM1,and FoxM1 can transcriptionally activate PTTG3P to form a positive feedback loop that promotes the development of PC Through the above content,we aim to explore the specific role and molecular mechanism of PTTG3P in the occurrence and development of PC,and provide new potential targets for the diagnosis and treatment of itPart 1 Expression of lncRNA PTTG3P in pancreatic cancer patients and its clinical significanceObjective:To analyze the expression level of PTTG3P in pancreatic cancer and its relationship with clinicopathology and prognosisMethods:In situ hybridization staining and real-time PCR were employed to compare the expression of PTTG3P in PC tissues and paired tumor adjacent nomal tissues.The clinical relavanve of PTTG3P in 60 cases of PC tissue with the clinicopathological characteristics and prognosis were analyzed statiscally.Results:The expression of PTTG3P in PC tumor tissues was significantly up-regulated relative to the expression in adjacent tissues.Statistical analysis of PTTG3P expression with clinicopathological characteristics of pancreatic cancer showed that PTTG3P expression was not significantly correlated with gender,age,tumor invasion,lymph node involvement and distant metastasis,while PTTG3P expression was closely related to tumor size and pathological grade.The overall survival time in the high expression of PTTG3P group was significantly shorter than that in the low expression group.Conclusion:The expression of PTTG3P in pancreatic cancer tumor tissue is significantly increased.The expression level of PTTG3P is closely related to the size of pancreatic cancer tumor and the degree of differentiation of tumor cells.The overall survival of patients with high PTTG3P expression is significantly reduced.Part 2 The effects of lncRNA PTTG3P on the biological behavior of PC cellsObjective:To investigate the role of PTTG3P in the proliferation,migration and invasion of PC cellsMethods:Real-time PCR was used to detect the relative expression levels of PTTG3P in normal pancreatic ductal epithelial cells and different PC cell lines.The cell lines with relative high expression of PTTG3P were selected to down-regulationg of PTTG3P,while the cell lines with relative low expression of PTTG3P were selected to up-regulationg of PTTG3P.The effect of PTTG3P on the malignant phenotype of PC cells was observed in vitro by CCK-8,plate clone formation,cell scratch experiment,transwell cell migration and invasion experiment.PC cells were inoculated into subcutaneous tissue or ileocolonic vein in nude mice to observe the effects of PTTG3P on tumor growth and liver metastasis of PC cells.Results:The expression of PTTG3P in PC cells is significantly higher than that in normal pancreatic ductal epithelial cells.CCK-8 growth curve and plate clone formation showed that the proliferation ability was significantly enhanced in PTTG3P overexpression cells,while the proliferation ability of PTTG3P down-regulated cells was significantly weakened.The results of cell scratch test and transwell cell migration showed that PTTG3P promotes the migration of pancreatic cancer cells.Transwell invasion experiment further showed that PTTG3P plays an important role in promoting the aggressive phenotype of pancreatic cancer cells.Subcutaneous tumor formation experiments showed that PTTG3P promotes pancreatic cancer tumor growth in vivo.In the liver metastasis model,the number of liver metastases in the up-regulated PTTG3P tumor was significantly greater than that in the control tumor,while the result in the down-regulated PTTG3P group was just the opposite.Conclusion:PTTG3P plays an important role in promoting the proliferation,migration and invasion of PC cells.Part 3 PTTG3P/miR-132/212-3p/FoxM1 regulating the proliferation,migration and invasion of PC cellsObjective:To explore the specific molecular mechanism of PTTG3P in promoting the malignant phenotype of pancreatic cancer cellsMethods:The LinkedOmics database was used to analyze the mRNAs related to PTTG3P,and the correlation between FoxM1 and PTTG3P was verified through the TCGA database and our clinical pathological specimens.The effect of PTTG3P on FoxM1 expression regulation was analyzed by Western-blot and real-time PCR.The role of FoxM1 in promoting cancer by PTTG3P was detected by cytological experiments.Bioinformatics and nuclear plasma separation experiment was used to analyze the location of PTTG3P in pancreatic cancer cells.We predicted whether PTTG3P regulates FoxM1 through the ceRNA mechanism through the Dicer protein intervention experiment and Ago2-RIP experiment.Using bioinformatics and Ago2-RIP experiments to screen out possible miRNAs in the ceRNA mechanism.With the construction of mutant gene plasmids,luciferase reporter experiments and MS2-RIP experiments,the specific binding sites of miRNAs with PTTG3P and FoxM1 were identified respectively.Western blot and real-time PCR verifed the expression regulation relationship between themResults:The oncogene FoxM1,which has a variety of regulatory effects in pancreatic cancer,was related with PTTG3P in the co-expression network.Correlation analysis showed that FoxM1 and PTTG3P are positively relevant in PC.PTTG3P can promote the expression of FoxM1 at both the mRNA level and the protein level in PC cells.CCK-8,cell scratch and transwell invasion experiments showed that down-regulation of FoxM1 can significantly inhibit the promotion of PTTG3P on the proliferation,migration and invasion of PC cells.On the contrary,overexpression of FoxM1 will significantly reduce the tumor suppressor effect caused by down-regulation of PTTG3P.Subcellular localization analysis revealed that PTTG3P is mainly located in the cytoplasm.When Dicer protein was down-regulated in PC cells,the process of PTTG3P up-regulating FoxM1 was prevented.Ago2-RIP showed that PTTG3P may be involved in regulating FoxM1 as a ceRNA.Through miRcode,Targetscan bioinformatics database prediction and Ago2-RIP experimental screening,we found that miR-132/212-3p may mediate the regulatory effect of PTTG3P on FoxM1.Through the double-luciferase reporter experiment and MS2-RIP experiment,we confirmed the target binding sites of miR-132-3p and miR-212-3p with PTTG3P or FoxM1.The inhibition of FoxM1 expression caused by PTTG3P knockdown would be significantly reduced in the presence of miR-132/212-3p inhibitors.At the same time,PTTG3P is closely related to the expression of miR-132/212-3p and FoxM1 in the process of tumor growth.Conclusion:The promoting effect of PTTG3P on the proliferation,migration and invasion of PC cells is at least partially mediated by FoxM1,and PTTG3P can act as a ceRNA to regulate the expression of FoxM1 by competitively binding miR-132/212-3pPart 4 FoxM1 transcriptionally regulates the expression of lncRNA PTTG3P in PC cellsObjective:To explore the upstream regulatory mechanism of abnormal expression of PTTG3P in PC cells and verify the effect of FoxM1 on the expression of PTTG3P,and analyze the specific molecular mechanism.Methods:Bioinformatics were used to analyze the promoter sequence of PTTG3P,and look for its possible transcriptional regulatory factors.The PC cell lines were transfected with FoxM1 overexpression plasmid or siRNA,and real-time PCR was employed to detect the expression changes of PTTG3P.The reporter gene plasmids were constructed according to the position of the potential binding site of FoxM1 on the PTTG3P promoter,and the specific binding site was analyzed by the dual luciferase reporter experiment and ChIP-qPCR experimentResults:Several promising binding sites in the promoter sequence of PTTG3P for FoxM1 protein were found.It suggested that FoxM1 may play a role in transcriptional regulating PTTG3P.FoxM1 promoted the expression of PTTG3P in PC cells.Dual luciferase reporter experiment and ChIP-qPCR experiment showed that FoxM1 can regulate the expression of PTTG3P through transcriptional activation and FoxM1 can target the PTTG3P promoter region TFBS1Conclusion:FoxM1 transcription activates the expression of PTTG3P.