Hypoxia-inducible Factor-1α Regulates The Expression Of Pyrroline-5-carboxylate Reductase And Promotes The Proliferation And Migration Of Lung Adenocarcinoma A549 Cells

Objective:Hypoxia is a common pathophysiological phenomenon in the process of tumor occurrence and development,and is the result of the imbalance of cell oxygen consumption and availability.Under hypoxic conditions,and proline metabolism reprogramming can meet the exuberant demand of tumor cells for energy,and at the same time lead to the enhancement of metastasis potential of cancer cells.Hypoxia-inducible factor-1α（HIF-1α）is an important regulatory factor for the body to perceive and adapt to hypoxic environment,and is closely related to the increase of distant metastasis and low survival rate of various tumors.The interaction between HIF-1αand tumor metabolic pathways has a significant impact on the occurrence and development of tumors.However,the relationship between HIF-1αand the regulation of proline metabolism in tumor cells under hypoxic conditions is still unclear.This project aims to evaluate the prognostic value of high expression of proline anabolic-related enzyme（pyrroline-5-carboxylate reductase,PYCR1）in cancer patients through meta-analysis,and to explore the relationship between the expression of PYCR1 in lung adenocarcinoma and the prognosis of patients.An in vitro hypoxia model of lung adenocarcinoma A549 cells was established to explore the effects of HIF-1αand PYCR1 on the proliferation and migration capacity of lung adenocarcinoma A549 cells,as well as the possible internal mechanism between HIF-1αand PYCR1 under hypoxic conditions,so as to provide more reliable evidence for basic research and clinical work.Methods:（1）Exploring the clinical prognostic value of PYCR1 in various cancers.Thorough database searches were conducted in Pub Med,EMBASE and Cochrane libraries,Rev Man 5.3 software for statistical analysis and meta-analysis of clinical studies of PYCR1 and cancer.In order to further explore the relationship between the expression of PYCR1 in lung adenocarcinoma and the prognosis of patients,the UALCN database（http://ualcan.path.uab.edu/cgibin/TCGAEx Result New2.pl?genenam=PYCR1&ctype=LUAD）and Kaplan-Difference analysis was performed on the Meier Plotter database（http://kmplot.com/analysis/index.php?p=service）.（2）The A549 cell line was cultured continuously for 5h,10h,and 15h in a hypoxic box（1%O₂,5%CO₂）respectively,and a normoxia control group was set up.RT-q PCR was used to determine the expression of HIF-1αand PYCR1 m RNA.Then,under hypoxic conditions,HIF-1αinhibitor BAY87-2243 and HIF-1αagonist DMOG were used to intervene HIF-1αin advance,and then hypoxic treatment was conducted for 10 hours.Determination of HIF-1 by immunoblotting and RT-q PCRαAnd the expression of PYCR1,the change of A549 cell proliferation ability was measured by CCK8 test,and the change of A549 cell migration ability was measured by scratch test.Results:（1）Eight articles were selected,and 728 cancer patients were enrolled.The cancer types include lung,stomach,pancreatic ductal adenocarcinoma,hepatocellular carcinoma,and renal cell carcinoma.The meta-analysis results showed that the expression of PYCR1 was higher in the clinical stage III–IV group than that in the clinical stage I–II group（OR=1.67,95%CI:1.03–2.71）,higher in the lymph node metastasis group than in the non-lymph node metastasis group（OR=1.57,95%CI:1.06–2.33）,and higher in the distant metastasis group than in the non-distant metastasis group（OR=3.46,95%CI:1.64–7.29）.However,there was no statistical difference in PYCR1expression between different tumor sizes（OR=1.50,95%CI:0.89–2.53）and degrees of differentiation（OR=0.82,95%CI:0.54–1.24）.The results of differential analysis showed that the expression level of PYCR1 in lung adenocarcinoma was significantly higher than that of normal tissues,and patients with high PYCR1 expression were negatively correlated with the overall survival time of lung adenocarcinoma,indicating that patients with high PYCR1 expression had a poor prognosis.（2）Compared with the normoxia group,the m RNA expression of HIF-1αand PYCR1was rapidly up-regulated at first,and then gradually degraded with the increase of hypoxia time.Select 10h as the time point of continuous hypoxia.Compared with the hypoxic control group,BAY87-2243 decreased the cell proliferation and migration ability（P<0.05）,and the expression of HIF-1αand PYCR1 decreased（P<0.05）;After DMOG treatment,the proliferation and migration of cells were up-regulated（P<0.05）,and the expression of HIF-1αand PYCR1 were up-regulated（P<0.05）.Conclusion:The expression of PYCR1 is related to cancer metastasis,and the higher the expression of PYCR1,the worse the cancer prognosis.Patients with high PYCR1expression are negatively correlated with overall survival time of lung adenocarcinoma,and PYCR1-mediated molecular events and biological processes may be potential mechanisms for lung adenocarcinoma metastasis.Under hypoxic conditions,HIF-1αregulates the expression of PYCR1 and promotes the proliferation and migration of lung adenocarcinoma A549 cells.