Study On The Mechanism Of LncRNA TRG-AS1 Promoted The Progression Of Hepatocellular By Targeting MiR-4500 Regulated BACH1 And Glycolysis Pathway

Background:Hepatocellular carcinoma（HCC）is the most common primary liver tumor,with an incidence rate of fifth in the world and the mortality rate in third place.In recent years,the incidence rate of HCC has shown a significant growth trend due to the changes of diet and living environment.China has become the highest incidence rate of HCC in the world.At present,there is still a lack of specific clinical treatment for hepatocellular carcinoma,and the limitations and side effects of traditional treatment methods such as surgery,radiotherapy and chemotherapy lead to low survival rates and a decline in quality of life.Therefore,it is necessary to clarify the comprehensive pathological process and corresponding molecular mechanism of hepatocellular carcinoma,Finding and discovering new therapeutic strategies or targets has become a hot issue in cancer medicine research.The progression of HCC is a dynamic process involving a variety of gene expression and cytokine activation.Among them,Bach1 gene expression and glycolysis pathway activation are one of the key factors leading to the progression of HCC.Targeted regulation of Bach1 gene mutation and glycolysis pathway can provide a new direction for the research and development of anti-HCC drugs.Noncoding RNA has a variety of biological functions.It is a kind of therapeutic and prognostic molecules with important application value.Its regulatory effect on HCC has attracted more and more attention.Previous studies showed that lncRNA TRG-AS1 was significantly highly expressed in HCC cells.Through bioinformatics prediction software,it was found that lncRNA TRG-AS1 could combine with mir-4500 to form Cerna regulatory network,and mir-4500 could target Bach1,Then,whether lncRNA TRG-AS1 and mir-4500 regulate Bach1 and glycolysis pathway through the formation of Cerna network to promote the progress of HCC is the core issue of this subject.In recent years,lncRNA TRG-AS1 has become the focus of researchers because of its rich biological functions.However,the role of lncRNA TRG-AS1in the progression of HCC and its effect on Bach1 gene and glycolytic pathway of HCC cells are not clear.Therefore,paying attention to the regulatory mechanism of the Cerna network of lncRNA TRG-ASland miRNA in HCC provides a new idea for the diagnosis and treatment of HCC.Part Ⅰ The expression of lncRNA TRG-AS1 and Bach1 in hepatocellular carcinoma and its clinical significanceObjective:To study the relationship between the expression of chlna-1 and the prognosis of hepatocellular carcinoma.Methods:Based on TCGA data set,the expression of lncrna trg-asl and Bach1 in HCC and adjacent tissues were analyzed by gene expression profiling interactive analysis（GEPIA）（cancer PKU.CN）;Kaplan Meier survival curve was drawn.Results:1.Lncrna trg-as1 and Bach1 were highly expressed in hepatocellular carcinoma;2.The expression of lncrna trg-asl and Bach1 is correlated in hepatocellular carcinoma.Conclusion:Based on TCGA,this study found that lncrna trg-asl and Bach1 were highly expressed in HCC tissues,and selected the candidate gene of lncrna trg-asl,so as to lay a foundation for further study of the functional mechanism of lncrna trg-asl in the occurrence and development of HCC.Part Ⅱ lncRNA TRG-AS1 promotes the proliferation,metastasis and EMT of hepatoma cells and affects the process of oxygen glycolysisObjective:To investigate the effect and regulation of lncRNA TRG-AS1 on biological functions such as proliferation,migration and invasion of HCC cells and aerobic glycolysis,so as to provide new ideas and potential targets for the treatment of HCC diseases.Methods:the expression of lncrnatrg-asl in hepatocellular carcinoma cell lines（HCCLM3,MHCC97-H,Huh-7,mhcc97-1 and SK-Hep-1）and thle-2 in normal hepatocytes were detected by RT qPCR.Sh-trg-asl#1/#2 was transfected into hepatoma cells HCCLM3 and Huh-7.The changes of cell proliferation were detected by clone formation test and edu;Transwell detected the changes of cell invasion and metastasis;RT qPCR,Western blotting and immunofluorescence staining were used to detect the expression of metastasis related proteins E-cadherin and N-cadherin;Glucose intake and lactic acid production were measured;ELISA and RT-qPCR were used to detect the activity and expression of key enzymes in the process of glycolysis.Results:1.The expression of lncrna trg-asl was significantly increased in hepatoma cells;2.Knockdown of lncrna trg-asl can inhibit the proliferation,metastasis,invasion and EMT process of hepatoma cells;3.Knockdown of lncrna trg-asl can inhibit gluts and inhibit the grape absorption of hepatoma cells;4.Knockdown of lncrna trg-asl can inhibit the activity and expression of ldhA and reduce the production of lactic acid;5.Knockdown of lncrna trg-asl can increase the activity and expression of CS,the key enzyme of tricarboxylic acid cycle,so as to promote oxidative phosphorylation of hepatoma cells.Conclusion:lncrna trg-as1 exerts the function of tumor promoting gene and affects the process of glycolysis in hepatoma cells.The part Ⅲ the role and mechanism of lncrnatrg-asl targeting mir-4500 in regulating Bach1 and glycolysis pathway to promote the progression of HCCObjective:to reveal the molecular mechanism of lncrna trg-asl and mir-4500 Cerna regulatory network in the progression of HCC,and to explore the clinical value of lncrna trg-asl as a biomarker for early diagnosis of HCC from the aspects of target gene regulation,signal pathway and molecular mechanism network.Methods:target genes were predicted by bioinformatics analysis.The interaction between trg-asl,mir-4500 and BTB domains and CNC homologue 1（Bach1）was confirmed by rip,RNA pull-down and luciferase reporter analysis.Further study the effects of mir-4500 and Bach1 on the proliferation,metastasis and invasion of hepatoma cells at the cellular level.The final response experiment verified the role of lncrnatrg-asl regulation targeting mir-4500 in regulating Bach1 to promote the progression of HCC and the mechanism of glycolysis.Results:1.Lncrna trg-asl targeted binding to mir-4500 in hepatoma cells;2.Mir-4500 was significantly down regulated in hepatoma cells.Overexpression of mir-4500 inhibited the proliferation,invasion,migration and EMT of hepatoma cells;3.Bach1 is regulated by trg-asl and mir-4500;4.Bach1 acts as a tumor promoting gene in HCC;5.Lncrna trg-as1 is involved in the progression of HCC by targeting mir-4500/Bach1 axis;6.Lncrna trg-as1 regulates gluts by targeting mir-4500/Bach1 axis and affects the absorption of HCC cells;7.Lncrna trg-as1 regulates ldhA activity and expression by targeting mir-4500/Bach1 axis,thereby affecting the production of lactic acid;8.Lncrna trg-as1 regulates the activity and expression of the key enzyme CS of the tricarboxylic acid cycle by targeting the mir-4500/Bach1 axis,thereby affecting the oxidative phosphorylation of hepatoma cells.Conclusion:trg-as1 regulates Bach1 as a molecular sponge of mir-4500.Mir-4500 silencing or Bach1 overexpression in lncrnatrg-asl knockdown cells completely saved cell proliferation,migration,invasion,EMT progression and glycolysis.Lncrnatrg-as1 targets mir-4500 to regulate Bach1 and glycolysis pathway and promote the progression of HCC.