The Role Of PRKAA1/AMPKα1 And Preliminary Study On Its Mechanism In Pancreatic Cancer

Objective: Pancreatic cancer is an aggressive digestive tract tumor with poor prognosis.Most patients have no obvious symptoms in the early stage,and often develop to the late stage before being diagnosed.Due to its special tumor microenvironment,traditional treatment also has poor efficacy and high mortality.PRKAA1 is a catalytic subunit of 5 ’-prime-AMP-activated protein kinase(AMPK).Existing studies have shown that PRKAA1 gene is closely related to the occurrence,development and prognosis of tumors.This study will start with PRKAA1 gene expression in pancreatic cancer,study its relationship with clinicopathological characteristics and prognosis of pancreatic cancer patients,explore the changes of pancreatic cancer cell phenotype after PRKAA1 gene knockdown,and preliminarily explore its molecular mechanism in the progression of pancreatic cancer.Methods: Firstly,we obtained RNAseq data of pancreatic cancer and corresponding clinical information from The Cancer Genome Atlas(TCGA)and GTEx databases,analyzed the expression of the PRKAA1 gene in digestive tract tumors,as well as its expression in normal pancreatic tissue and pancreatic cancer tissue.q RT-PCR and Western Blot were used to detect the expression difference of PRKAA1 in pancreatic cancer cells and normal pancreatic cells.We collected 75 surgical specimens of pancreatic cancer patients and 20 normal pancreatic tissues from our hospital,performed immunohistochemical staining to analyze the expression of PRKAA1 in the tissues,and collected clinical pathological data of patients for correlation analysis.Kaplan-Meier survival curves were plotted using data from the TCGA database(TCGA cohort)and our hospital patients(our hospital cohort)to compare the survival differences between high and low expression groups of the PRKAA1 gene.Cell immunofluorescence was used to determine the localization of the PRKAA1 gene in pancreatic cancer cells.CRISPR-Cas9 knockout stable cell lines were established for CCK-8,colony formation,wound healing,Transwell invasion assays,and flow cytometry to verify the effect of PRKAA1 gene knockout on the phenotypes of pancreatic cancer cells,and Western Blot was used to further verify the effect of PRKAA1 gene knockout on the expression of proliferation,migration,and apoptosis-related proteins in pancreatic cancer cells.In addition,to explore the preliminary mechanism of PRKAA1 regulation of pancreatic cancer progression,we also detected the changes in the expression of PI3 K,p-PI3 K,Akt,and p-Akt proteins after knocking down PRKAA1.Results: Analysis of data downloaded from TCGA and GTEx revealed that PRKAA1 gene is highly expressed in digestive tract tumors,including pancreatic cancer.Immunoblotting and q RT-PCR experiments showed that PRKAA1 expression was increased in pancreatic cancer cell lines CFPAC-1 and PANC-1 compared to normal pancreatic epithelial cells h TERT-HPNE(P<0.01).Immunohistochemistry demonstrated higher expression of PRKAA1 gene in poorly differentiated pancreatic cancer tissues.Clinical data from 75 patients were collected and followed up,and Pearson χ2 test was used to analyze the correlation between PRKAA1 expression and clinical pathological variables of pancreatic cancer patients.The results showed that PRKAA1 expression level was correlated with age(P=0.001),T stage(P<0.001),and M stage(P=0.003).Univariate regression analysis showed that high expression of PRKAA1(HR=1.991,95% CI: 1.206-3.288,P=0.007),CA19-9(HR=1.001,95% CI:1.000-1.002,P=0.014),T stage(HR=1.959,95% CI: 1.241-3.094,P=0.004),and M stage(HR=4.498,95% CI: 2.548-7.939,P=0.001)were prognostic factors for pancreatic cancer patients.Multivariate regression analysis was performed with factors that had P<0.05,and it was found that M stage(HR=4.453,95% CI:2.244-8.837,P=0.001)and high expression of PRKAA1 protein(HR=1.879,95% CI:1.067-3.310,P=0.029)were independent risk factors for poor prognosis of pancreatic cancer patients.Survival analysis results from both cohorts showed that high expression of PRKAA1 indicated poor prognosis of pancreatic cancer patients,including TCGA cohort(HR=1.591,95% CI: 1.053-2.404,P=0.0276)and our hospital cohort(HR=2.030,95% CI: 1.207-3.415,P=0.0013).Two cell lines with relatively high expression of PRKAA1 were selected for subsequent experiments.Firstly,CRISPR-Cas9 knockout stable cell lines were established,and phenotypic tests such as colony formation,CCK-8,and flow cytometry were performed to detect apoptosis,wound healing,and Transwell invasion.It was observed that the proliferation and migration ability of CFPAC-1 and PANC-1 cells were significantly inhibited,while the apoptosis ability was significantly enhanced(P< 0.05).Cellular immunofluorescence showed that PRKAA1 was mainly localized in the cytoplasm.Changes in the expression of PI3 K,phosphorylated PI3K(p-PI3K),AKT,phosphorylated AKT(p-AKT),and other proteins were detected in PRKAA1 knockout cells.The results showed that the expression of these proteins was reduced in PRKAA1 knockout group compared with the negative control group.The p-PI3K/PI3 K ratio and p-AKT/AKT ratio were significantly decreased(P< 0.05).Conclusion:1.The expression of PRKAA1 is significantly increased in pancreatic cancer,and its high expression is associated with age,lymph node metastasis,distant metastasis and poor survival;2.Down-regulation of PRKAA1 could regulate the proliferation,migration and apoptosis of CFPAC-1 and PANC-1 cells;3.PRKAA1 may promote the progression of pancreatic cancer through PI3K/Akt signaling pathway.