TNNT1 Regulating JAK2/STAT3 Signal Axis To Promote The Progression Of Gastric Cancer

Background:Gastric cancer(GC)is one of the most common malignant tumours in the world and,and the current treatment of gastric cancer suffers from poor efficacy.The molecular mechanisms underlying the development of gastric cancer are of great importance to the treatment of gastric cancer.Troponin T1(TNNT1)is one of the three subunits encoding the troponin complex.Recent studies has been reported that TNNT1 may be involved in the regulation of a variety of tumours,but there are no report on whether TNNT1 regulates the progression of GC.In this study,the relationship between the expression of TNNT1 in GC and the malignant grade of the tumour was verified by bioinformatics analysis,and then the possible regulatory relationship of GC was verified by experiments at the cell,tissue and animal level.Finally,the specific molecular mechanism that may lead to the regulation of GC by TNNT1 was explored and verified by experiments.This study may help in the diagnosis and treatment of GC in the future.Methods:1.Bioinformatics analysis and verification of TNNT1: 1)using TCGA database,TIMER 2.0 database and Kaplan-Meier Plotter database to analyse the expression and prognosis of TNNT1 gene in GC tissues;2)using immunohistochemistry,q RT-PCR and Western blot to detect the expression of TNNT1 in fresh GC tissues to verify the bioinformatics analysis,and to analyse the correlation between immunohistochemical and prognostic data score and pathological stage.2.The effect of TNNT1 expression on the biological function of GC cells: 1)The GC cell model of stable expression of TNNT1 interference group/overexpression group was constructed by recombinant lentivirus vector,and the transfection efficiency was detected by q RT-PCR and Western blot.2)CCK-8 method,plate cloning test and correlation between the expression of KI67 and TNNT1 were used to analyse the effect of TNNT1 expression on the proliferation of GC cells.3)Scratch test,Transwell migration and invasion test were used to verify the effect of TNNT1 expression on the migration and invasion of GC cells.4)Nude mouse tumour formation assay and immunohistochemical analysis were used to investigate the effect of TNNT1 expression on GC cell proliferation in vivo.3.To explore the molecular mechanism of TNNT1 regulation of GC progression:1)screen the possible molecular mechanism of TNNT1 regulation of GC progression by GSEA enrichment analysis and verify the hypothesis at the protein level;2)use pathway inhibitors to treat overexpressed model cells and analyse the effects of pathway inhibitors on the biological function of model cells by CCK-8 and Transwell migration and invasion experiments.3)Verify the effects of pathway inhibitors on protein expression in model cells.4.Preliminary exploration of TNNT1-related prognostic molecules: 1)Pearson correlation test to extract genes co-expressed with TNNT1 gene and analyse the expression difference;2)SVM-RFE machine learning algorithm and LASSO regression analysis to screen TNNT1-related prognostic molecules.Results:1.The up-regulated expression of TNNT1 in gastric cancer is related to the pathological stage and prognosis of GC: 1)The pan-cancer analysis of TIMER 2.0database shows that the expression of TNNT1 is up-regulated in many kinds of tumours,and the analysis of TCGA database and Kaplan-Meier Plotter database shows that the expression of TNNT1 is up-regulated in GC,and the high expression of TNNT1 is related to the poor prognosis of GC patients.2)The results of immunohistochemistry,q RT-PCR and Western blot confirmed the results of bioinformatics analysis and suggested that the high expression of TNNT1 was associated with the poor pathological stage of GC.2.TNNT1 promotes the proliferation,migration and invasion of GC cells: 1)CCK-8 assay and plate cloning test showed that TNNT1 could promote the proliferation of GC cells,and the correlation analysis showed that the expression of TNNT1 was positively correlated with the expression of KI67.2)Scratch test,Transwell migration and invasion assay showed that TNNT1 could promote the migration and invasion of GC cells.3.TNNT1 regulates the JAK2/STAT3 axis to promote the progression of GC cells: 1)GSEA enrichment analysis suggests that the up-regulated expression of TNNT1 in GC may activate the JAK-STAT pathway.Western blot experiments show that the expression level of JAK2/STAT3 signalling axis-related proteins changes with the change in TNNT1 expression level.2)CCK-8 assay,Transwell migration and invasion assay showed that the proliferation,migration and invasion ability of overexpressed model cells decreased after treatment with JAK kinase inhibitor AG490.3)Western blot results showed that the expression of JAK2/STAT3 signalling axis-related proteins was downregulated in overexpressed model cells treated with JAK kinase inhibitor AG490.4.Preliminary investigation of TNNT1-related prognostic molecules TMEM191 A,PPP1R14BP2,RTN4 R,EFNA3 and TUBB4 A may be important prognostic molecules involved in the regulation of GC progression by TNNT1.Conclusion:1.The expression of the TNNT1 gene is upregulated in GC and is closely associated with the prognosis of GC patients.The expression of TNNT1 is also significantly correlated with stage,T stage and N stage.2.The expression of the TNNT1 gene was upregulated in GC cells.In vitro experiments showed that TNNT1 gene could promote the proliferation,migration and invasion of GC cells.In vivo experiments showed that TNNT1 gene could promote the proliferation of subcutaneous tumours in nude mice,and the expression of TNNT1 was positively correlated with the expression of KI67.3.GSEA enrichment analysis and in vitro experiments showed that the TNNT1 gene affects the proliferation,migration and invasion of GC cells by regulating the JAK2/STAT3 signalling pathway.4.TMEM191 A,PPP1R14BP2,RTN4 R,EFNA3 and TUBB4 A may be important molecules involved in the regulation of GC progression by TNNT1.