The Mechanism Of AKR1C3 Promoting The Proliferation,Invasion And Metastasis Of Hepatocellular Carcinoma Cells

Background:Primary liver cancer is one of the most common malignant tumors of the gastrointestinal tract worldwide,with the sixth highest incidence and the fourth highest mortality rate of all malignant tumors.Hepatocellular carcinoma(HCC)is the most prevalent type of primary liver cancer,accounting for more than 75%of all cases.Therefore,there is an urgent need to find new diagnostic and prognostic markers for HCC,to investigate the molecular mechanisms of rapid proliferation and invasive metastasis,and to explore more effective therapeutic targets for HCC.AKR1C3 is a member of the aldo-keto reductase superfamily and is widely expressed in the gastrointestinal tract,liver,prostate and breast tissue,where it is involved in physiological processes such as steroid metabolism and androgen synthesis.Previous studies have shown that AKR1C3 is associated with various malignancies such as gastric cancer,prostate cancer and triple-negative breast cancer,but it has rarely been studied in liver cancer.Our group has shown that high expression of AKR1C3 is a poor prognostic factor for HCC and promotes the development of HCC,but its molecular regulatory mechanism has not been fully elucidated.Objective:To explore the significance of AKR1C3 in the diagnosis and prognosis of HCC,to analyze the molecular mechanisms by which AKR1C3 promotes the proliferation,invasion and metastasis of hepatocellular carcinoma cells,to provide a new theoretical basis for the development of HCC in the clinic,and to provide new therapeutic targets for the comprehensive prevention and treatment of HCC.Methods:1.Download the high-throughput sequencing data of 364 HCC tissues and 50 adjacent tissues from The Cancer Genome Atlas(TCGA)database,and 212 HCC tissues and 177 adjacent tissues from the International Cancer Genome Consortium(ICGC)database.A microarray containing 80 HCC and adjacent tissues was purchased,and 25 fresh HCC and adjacent tissues diagnosed in the First Affiliated Hospital of Zhengzhou University from January 2020 to June 2020 were collected.The differential expression of AKR1C3 in HCC tissues and adjacent tissues was detected by bioinformatics analysis,immunohistochemical staining,real-time fluorescence quantitative PCR and Western Blot.2.Follow-up information of HCC patients from TCGA and ICGC databases was downloaded,and patients were divided into high and low expression groups based on median AKR1C3 expression for Kaplan-Meier(K-M)survival analysis.AKR1C3 expression and several clinical indicators of the patients were subjected to multifactorial COX regression analysis and column plots(Nomogram)were drawn to determine whether AKR1C3 could be an independent prognostic factor for HCC patients.3.After measuring the expression of AKR1C3 in various hepatocellular carcinoma cell lines,the AKR1C3 interference/overexpression system of hepatocellular carcinoma cells were constructed separately using lentiviral vectors,and cell proliferation assays,clone formation assays,scratch assays and Transwell invasion and migration assays were performed to detect changes in the proliferation,invasion and metastatic ability of AKR1C3 interference/overexpression cells in vitro.4.Functional enrichment analysis of AKR1C3 was performed to investigate its possible regulation of signaling pathways.Changes in the content of phosphorylated ERK protein,a key enzyme for fatty acid synthesis,downstream of interference/overexpression of AKR1C3 were detected using Western Blot,and changes in lipid content in the interfering/overexpressing cells were confirmed using Nile Red staining.Results:1.Analysis of the sequencing results of HCC and paraneoplastic tissues from TCGA and ICGC revealed that AKR1C3 expression was up-regulated in HCC tissues and the difference was statistically significant,and AKR1C3 could better distinguish HCC from adjacent tissues.Real-time fluorescence quantitative PCR,Western Blot and immunohistochemical staining again confirmed that AKR1C3 was up-regulated at both transcriptional and translational levels in HCC tissues.2.Survival analysis(K-M method)showed that patients in the TCGA and ICGC datasets had lower survival rates in the high AKR1C3 expression group compared to the low expression group,with statistically significant differences(P=0.0037,0.0063).Multi-factor COX regression analysis showed that high expression of AKR1C3 was an independent predictor of poor patient prognosis(P=0.005,HR=1.706,95%confidence interval:1.171-2.488).Column plots constructed using AKR1C3 and patients’ clinical characteristics were more accurate in predicting patient survival at 3 and 5 years.3.The cell proliferation assay(CCK-8 method)and clone formation assay showed that the proliferation of hepatoma cells MHCC97-H and SMCC-7721 was significantly inhibited in vitro after AKR1C3 interference,while the proliferation of Hep G2 and Li-7 cells was significantly enhanced after overexpression of AKR1C3.24h and 48h scratch assays confirmed that the AKR1C3 interference system Transwell invasion and migration assay showed that the invasion and transfer ability of AKR1C3-interfering system was significantly reduced,while the invasion and transfer ability was enhanced after overexpression of AKR1C3.The subcutaneous tumorigenesis assay in immunodeficient mice confirmed that the size and volume of subcutaneous tumors in the AKR1C3-interfered group were significantly smaller than those in the control group.4.Functional enrichment analysis suggested that AKR1C3 might activate ERK signaling pathway and fatty acid metabolism in HCC.Further Western Blot experiments confirmed that phosphorylated ERK,SREBP1,ACLY,ACC,FASN and other proteins were significantly reduced in AKR1C3-interfered system;after overexpression of AKR1C3,phosphorylated ERK,SREBP1,ACLY,ACC,FASN and ACLY,ACC and FASN were increased after overexpression of AKR1C3;Nile red staining experiments demonstrated that lipid synthesis was significantly reduced in the AKR1C3-interfering system and increased in the overexpressing system.Conclusions:AKR1C3 expression is upregulated in HCC tissues and has certain diagnostic and prognostic significance.AKR1C3 may promote the proliferation,invasion and metastasis of hepatocellular carcinoma cells by activating the ERK signaling pathway and fatty acid synthesis.