| BackgroundPancreatic cancer is a kind of lethal cancer with high malignancy.It is a rapidly progressive disease.Until now,the major therapy for pancreatic cancer is radical surgery,but a majority of patients have loss the chance for surgery at the time of the first diagnosis.It is challenging to realize the disease,for most of them don’t have any apparent symptom or sign in the early time of this disease.However,if the patients received the surgery,their prognosis is still not optimistic.The 5-year overall survival is about 11%.Therefore,it is essential to elucidate the molecular mechanism of pancreatic cancer progression for predicting the patients’ prognosis and choosing effective treatment.Our group found some genes of high frequency mutation in pancreatic cancer patients with poor prognosis,and the gene of PPP1R13B is a key gene whose protein is ASPP1(Apoptosis stimulating protein of P53 1).There is no associated research on it in pancreatic cancer.So in this study,we intend to clarify the relation between ASPP1 expression level and the prognosis in pancreatic cancer patients by clinicopathologic data.Meanwhile we also want to investigate how the expression level change of ASPP1 affects the malignant behaviors in pancreatic cancer cells and animal experiment.Finally,we use RNASeq to find the possible downstream molecular mechanisms.Method1.Immunohistochemistry was applied to evaluate the expression level of ASPP1 in paratumor tissue and tumor tissue.Then analyze the relationship between the expression level of ASPP1 and pancreatic cancer patients’ prognosis.2.Knockdown and overexpress ASPP1 in pancreatic cells.Use cell proliferation,colony formation assay,cell migration,drug resistance of cell,cell cycle and cell apoptosis to clarify the effect of ASPP1 expression on pancreatic cancer malignant behavior.And nude mice xenograft model was used to verify the tumor formation and proliferation of ASPP1.3.RNASeq was used to find the differential expression genes,and enrichment analysis was used to find the possible downstream mechanism.4.Use western blot,qRT-PCR and flow cytometry to verify the possible downstream mechanism.Results1.The expression level of ASPP1 was significantly high in para-tumor tissue.And pancreatic cancer patients in ASPP1 low expression group had a poor prognosis compared to the high expression group.2.We knocked down and overexpressed ASPP1 in AsPC-1 and MIA PaCa-2a pancreatic cells.And we found down-regulation of ASPP 1 can promote cell proliferation,increase the number of colony formation,increase the drug resistance of gemcitabine,inhibit cell apoptosis and regulate cell cycle,but it did not affect the capacity of migration.And ASPP1 could affect tumor proliferation in vivo.3.From the results of differential expression genes enrichment analysis,we found that DNA repair and the pathway of cell cycle are of most significance.4.Western blot and qRT-PCR verified the expression of protein and mRNA associated with cell cycle are changed.And the result of flow cytometry after cell synchronization also demonstrated cell cycle is regulated by the expression of ASPP 1.And the level of phosphorylation of RB and transcription factors E2F was upregulated.Conclusions1.The expression level of ASPP1 is high in para-tumor,compared to tumor tissue.And pancreatic cancer patients in the low expression group of ASPP1 in tumor tissue are of poor prognosis.2 Down-regulation of ASPP1 could promote cell proliferation and tumor growth in vivo.And it also increases drug resistance of gemcitabine,inhibit cell apoptosis and regulate cell cycle.3.Down-regulation ASPP1 promotes cell proliferation may be mediated by the following potential mechanisms.Cell cycle arrest was inhibited,then transcription factors E2F level upregulated. |
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