The Influence,and Mechanism Of Signal Transducer And Activator Of Transcription 5A(STAT5A) On Glucose Metabolism In Hepatollular Carcinoma

Background: Signal transducer and activator of transcription 5(STAT5)has a wide range of physiological and pathological functions in human.STAT5 can be activated by various cytokines such as prolactin(Prl),erythropoietin,growth hormone(GH)and epidermal growth factor(EGF).In liver,studies have found that the GH-STAT5 signaling pathway plays an important role in the control of lipid metabolism pathway: liver-specific knockout of STAT5 increases the expression of CD36 and PPAR(Peroxisome Proliferator Activated Receptor),resulting in fatty liver,impaired glucose tolerance,impaired insulin resistance and impaired liver regeneration.In addition,loss of STAT5 in the liver may also increase the expression of TGF-? and STAT3 to accelerate liver fibrosis and tumor formation.Besides,liver-specific knockdown of STAT5 A regulates the expression of NOX4(reactive oxygen species(ROS)-generating enzyme NOX4),which results in decreased expression of the apoptotic proteins PUMA and BIM and in turn promotes proliferation of hepatoma cells.STAT5 contains two subtypes of STAT5 A and STAT5 B.Both subtypes are encoded by a linked gene linked to human chromosome 17,and have 95% similarity in DNA structure.However,due to differences in transactivation region,STAT5 A and STAT5 B have specific biological functions in physiological and pathological processes.This study aims to explore the role of STAT5 A subtypes in the progression of liver cancer,especially the effect of glucose metabolism in liver cancer.At the same time,we hope to find new potential therapeutic targets from the perspective of inhibiting glucose metabolism.Methods: First,we analyzed and clarified the expression of STAT5 A in the tumor tissue of patients with HCC.The Kaplan-Meier method was used to analyze the relationship between STAT5 A expression and patients prognosis,as well as the multivariate survival analysis.In terms of biological function,a stable STAT5A-overexpressing Huh7 cell line was constructed,and CCK8,flow cytometry,and subcutaneous tumor-bearing experiments in nude mice were used to explore the effect of STAT5 A on proliferation and cell cycle.Then,Kit was used to examine the effect of STAT5 A expression on cell glucose consumption and lactic acid metabolism.Furthermore,with GC-MS technology,specific analysis of changes in glycolysis and each metabolite on the TCA cycle was performed.In terms of mechanism,q-PCR and Wectern blot techniques were used to analyze the possible pathways of STAT5 A and the role of glycometabolism targets.Results:1.The relationship between the expression of STAT5 A and the prognosis of patients.According to the analysis of tissue microarray results of 148 patients,the expression of STAT5 A in HCC was significantly reduced compared with matched non-tumor tissues.Kaplan-Meier survival analysis showed that patients with low expression of STAT5 A had higher RFS and OS.Multivariate survival analysis also had similar results: STAT5 A expression and whether there was envelope,AFP expression and tumor size could be independent risk factors affecting patients RFS and OS.2.The effect of STAT5 A on proliferation and cell cycle in vitro and in vivo.(1)After detecting the expression of STAT5 A in commonly used hepatoma cell lines,Huh7 was selected to construct a stable over-expressing cell line,while the STAT5 A knock-out cell line was correspondingly with si RNA.CCK8 proliferation experiments showed that the proliferation of cells was significantly inhibited after overexpression of STAT5 A,while the proliferation of cells after STAT5 A knockdown was significantly enhanced.Subcutaneous tumor-bearing experiments in nude mice also confirmed that STAT5 A could inhibit cell proliferation in vivo.Flow cytometry analysis also revealed that over-expression of STAT5 A could inhibit the progression of cells,arresting cells at G0/G1 phase.(2)Regarding glucose metabolism,kit was used to detect glucose and lactate.It was found that overexpression of STAT5 A could significantly reduce glucose consumption and lactate production.Further analysis of specific metabolites suggested that over-expression of STAT5 A could reduce major products of glycolysis and TCA cycle.3.Regulation of AKT by STAT5A(1)In terms of pathways,STAT5 A was found to significantly regulate AKT phosphorylation,which in turn inhibits STAT5 A phosphorylation after inhibiting AKT.Paired patient tissue microarray results also confirmed that STAT5 A expression was negatively correlated with p-AKT expression.(2)q PCR detection revealed that STAT5 A can affect the expression of multiple genes at the transcriptional level.Similar results are found at the RNA level in patients ' tissues.Further WB experiment found that STAT5 A significantly affects the expression of HK2 protein levels.The reversal of HK2 protein expression after addition of AKT inhibitors confirmed that the AKT pathway plays an important role in the regulation of STAT5A-HK1 regulation.(3)mi RNA-23 a regulates expression of STAT5 A.(4)In addition,we also confirmed that STAT5 A has a significant positive regulation on another important transcription factor,HNF4 A,and that HNF4 A could directly bind to PI3 K.Conclusion: We first clarified the low expression of STAT5 A in the tumor tissues of patients with HCC was related with poor prognosis.Besides,STAT5 A significantly inhibited cell proliferation and cell cycle progression in vitro and in vivo,which meaned an important anti-cancer effect.We also demonstrated that overexpression of STAT5 A could significantly inhibit the glycolysis process and TCA cycle.In terms of mechanism,we verified the relationship between STAT5 A and p-AKT and identified the key gene for downstream glucose metabolism,HK2.In addition,we also found that mi R-23 a could regulate STAT5 A and the STAT5A-HNF4A-PI3K-AKT signaling pathway was also confirmed,but the specific mechanism is still unclear.