The Function And Regulation Mechanism Of PRKRA In The Chemoresistance Of Pancreatic Cancer

[Background]Pancreatic cancer is one of the most lethal tumors in the digestive system with a 5year survival rate of only 12%.Gemcitabine is the first-line regimen for the chemotherapy of pancreatic cancer.However,due to the unique biological behavior and tumor microenvironment of pancreatic cancer,most patients are prone to chemoresistance,which greatly affects the prognosis of the disease.Therefore,it is of great significance to investigate the mechanism of chemoresistance of pancreatic cancer and discover new therapeutic targets to improve the treatment effect of pancreatic cancer.The protein activator of interferon-induced protein kinase EIF2AK2(PRKRA)is a stress-related protein.It is involved in the physical and pathological processes of cells such as viral infection and stress response and plays an important role in inflammatory diseases and nervous system diseases.In recent years,PRKRA has been confirmed to be involved in the development and progression of tumors,but its role in pancreatic cancer and underlying mechanisms are still unclear.Our previous study found that PRKRA regulated the biological behavior and chemoresistance of pancreatic cancer,but the specific mechanism was unclear.Therefore,this study aims to further explore the role and mechanism of PRKRA in the chemoresistance of pancreatic cancer.[Objective]To investigate the effects of PRKRA on the proliferation,migration and chemoresistance of pancreatic cancer cells.To explore the downstream pathway and specific mechanisms of PRKRA affecting chemoresistance of pancreatic cancer.To evaluate the expression and prognostic value of PRKRA and its downstream pathways in pancreatic cancer.[Methods]Small interfering RNA(siRNA)and overexpression plasmid were used to downregulate or up-regulate the expression level of PRKRA in pancreatic cancer cells.CCK-8 and Transwell assays were used to detect the proliferation,migration,invasion,and chemoresistance of pancreatic cancer cells in vitro.CRISPR/Cas9 and overexpression lentivirus were used to construct pancreatic cancer organoids with stable knockout and overexpression of PRKRA,and the proliferation rate and gemcitabine sensitivity of the organoids were detected.In addition,pancreatic cancer cell lines with stable knockout or overexpression of PRKRA were established,and the effects of PRKRA on the proliferation and chemoresistance of pancreatic cancer in vivo were determined by subcutaneous tumor xenograft model using nude mice.Transcriptome sequencing and bioinformatics analysis were used to screen the downstream differentially expressed genes and explore the potential downstream pathways.RT-qPCR,Western-Blot,and immunofluorescence were used to verify the regulatory effect of PRKRA on downstream target genes.Chromatin immunoprecipitation and dual luciferase reporter gene assay were used to verify the direct mechanism.Finally,the expression levels of PRKRA and its downstream genes in pancreatic cancer and adjacent tissues were evaluated by bioinformatics analysis and immunohistochemistry,and the clinical value of this pathway as a prognostic marker of pancreatic cancer was evaluated.[Results]The results of in vitro showed that the proliferation,migration and invasion ability of pancreatic cancer cells were decreased after PRKRA knockdown,and the sensitivity to gemcitabine was increased.Overexpression of PRKRA increased the proliferation,migration,and invasion ability of pancreatic cancer cells,and decreased the sensitivity to gemcitabine.The results of organoid experiments showed that the proliferation rate of pancreatic cancer organoids was decreased and the diameter of the organoids was significantly reduced after PRKRA knockout.However,after PRKRA overexpression,the proliferation rate and diameter of the organoids increased significantly.The drug sensitivity tests showed that the sensitivity of organoids from different patients to gemcitabine had great heterogeneity,and the IC50 values were positively correlated with the expression of PRKRA.In vivo experiments showed that the growth rate of subcutaneous transplanted tumors in nude mice was decreased after PRKRA knockout.After intraperitoneal injection of gemcitabine,the tumors were significantly reduced.However,overexpression of PRKRA promoted tumor growth and reduced sensitivity to gemcitabine in mice.For the investigation of mechanisms,MMP1,a downstream gene of PRKRA,was identified by transcriptome sequencing,and the downstream pathways and target genes were verified by Western-Blot and RT-qPCR.Using Rescue assay,we demonstrated that PRKRA promoted the proliferation and drug resistance of pancreatic cancer by regulating MMP1 and that PRKRA could regulate the expression of MMP1 through the NF-κB pathway.Bioinformatics analysis,chromatin immunoprecipitation and dual luciferase reporter gene assay suggested that NF-κB could act as a transcription factor to bind to the promoter region of MMP1 and regulate the expression of MMP1.In terms of prognosis,analysis of TCGA and GEO database showed that the expressions of PRKRA and MMP1 in tumor tissues were significantly higher than those in adjacent tissues,which were independent risk factors for poor prognosis of patients.The expression level of PRKRA was related to the pathological grade of tumor.Immunohistochemistry results of clinical surgical tissue samples showed that PRKRA and MMP1 were highly expressed in tumor tissues,and their expression levels were negatively correlated with the prognosis of patients.The combination of PRKRA and MMP1 can be used as a prognostic marker for pancreatic cancer patients.Patients with high expression of both genes have the worst prognosis.[Conclusion]PRKRA can promote the proliferation and chemoresistance of pancreatic cancer cells by activating the NF-κB pathway and enhancing the transcription of MMP1.PRKRA/NFκB/MMP1 pathway can be used as a potential target for the treatment and a prognostic marker of pancreatic cancer.