The Impact Of Anaerobic Glycolysis On Regulatory T Cell Proliferation And The Analytical Relationship With Immunotherapy In Renal Clear Cell Carcinoma

Objective:Treg cells(regulatory T cells)is a kind of immunosuppressive T cells,which play a vital role in immune tolerance and immune homeostasis through its immune regulation function,and is also one of the important reasons why tumor cells produce immune escape.ATP provided by anaerobic glycolysis becomes an important source of energy for tumor cells in the aerobic environment of solid tumor cells,and is also one of the important sources of energy for Treg cells.Studies have proved that reducing lactate intake can inhibit anaerobic glycolysis and inhibit the proliferation of Treg cells.The main function of monocarboxylic acid transporter 1(Monocarboxylate Transporter 1,MCT1)is to actively transport lactate from the cell outside the cell.Inhibition of MCT1can reduce the concentration of intracellular lactate,so as to achieve the effect of inhibiting anaerobic glycolysis.However,the series of specific changes in the related signaling pathways after targeted blockade of MCT1 and the changes affecting the signaling pathways after inhibition of anaerobic glycolysis are not clear.We used transcriptomics and metabolome method to analyze this complex process,explore the key pathways,and clarify the changes of various signaling pathways and signaling factors in this process.Explain the molecular mechanism of inhibiting the effect of anaerobic glycolysis on Treg cells.And further compare the impact of patients with renal clear cell carcinoma,to provide basic theoretical evidence for the immunotherapy of renal clear cell carcinoma through Treg cell regulation.The transition from oxidative phosphorylation to anaerobic glycolysis is an important prerequisite for the survival of tumor cells in a hypoxic microenvironment.Regulatory T cells(Treg)cells are a kind of inhibitory T cells,which play a vital role in immune tolerance and immune homeostasis.When Treg cells are increased,they will have adverse effects on the production and development of tumors,so they are often used by renal tumor cells to suppress anti-tumor immunity.Inhibition of anaerobic glycolysis inhibits Treg proliferation and differentiation,but the potential relationship between anaerobic glycolysis and Treg proliferation is unclear.Monocarboxylic acid transporters(MCTs)in cancer cells to transport lactate and clear H~+,inhibition of MCT1 will effectively reduce the concentration of intracellular lactate,and MCT 1 is a key regulation point affecting anaerobic glycolysis,by inhibiting MCT 1 to inhibit the proliferation of Tregs differentiation,combined with renal clear cell carcinoma gene to explore key targeted genes to provide new for tumor clinical immunotherapy direction and reference.Methods:To characterize the potential molecular mechanisms underlying the effects of anaerobic glycolysis on Treg cell proliferation,And the role of MCT1 in Treg cell metabolism,The Treg cells silencing the MCT 1 gene(Si-MCT1)were used as the experimental group,Transcription and metabolomics analysis with the control group transfected with the blank plasmid(Si-NC),The transfection effect was determined by immunoblot assay(Western Blot)and real-time fluorescence assay(Reverse transcription-quantitative polymerase chain reaction,RT-q PCR)for validation;Extracellular lactate concentration was measured by the lactate release assay,To verify whether there are significant differences in lactate absorption production by inhibition of MCT1,Using the cell viability detection experiment(Celltiter)to test whether the amount of ATP(Association of Tennis Professionals)production without aerobic glycolysis is different between the experimental group and the control group,The difference in anaerobic glycolytic capacity between the two groups was described by the absorption of lactate and the amount of ATP production;By using the cell proliferation assay(Cell Counting Kit-8,CCK-8),cell cycle assay and cell apoptosis assay to explore the effect on Treg cell proliferation.Transcriptomics and metabolomics sequencing,experimental and control metabolites,GO and KEGG(Gene Ontology)and Kyoto Encyclopedia of Genes and Genomes)enrichment analysis,and then the enrichment pathway of transcriptomic and metabolomics intersection,select the key pathways and their target genes,the differential expression of the target genes verified again by RT-q PCR experiment.Through the The Cancer Genome Atlas(TCGA)tumor database(https://porta l.gdc.cancer.Gov)used Cox regression to analyze the influence of genes in key pathways on the immune infiltration of Treg cells and the prognosis of patients with renal clear cell carcinoma,Further establish the expression of this gene for patient 1,Regression models for expected survival,3,of 5 years,Draw the Calibration curve to verify the accuracy of the model prediction,By screening the target genes by the CTD database(Comparative Toxicogenomics Database),By Pub Chem(https://pubchem.ncb i.nlm.nih.gov/)web site search for compounds in 2D with 3D structures,To provide a drug synthesis substance reference for tumor immunotherapy.Results:Silencing the MCT1 gene inhibited anaerobic glycolysis to produce ATP by reducing the uptake of lactate,thus inhibiting Treg cell proliferation(P=0.0006),the cell cycle ratio G0/G1:30.24%,while the experimental group G0/G1:68.67%,and the Treg cell replication stalled in G0/G1 phase.Comprehensive analysis of metabolomics ESI+mode and ESI-mode(screening criteria were P<0.05,VIP≥1)identified 15 differential metabolites,including 6 upregulated metabolites,such as lysine,tryptophan-arginine,and9 down-regulated metabolites,such as adenosine,hydrothreonine,etc.Transcriptomics identified 2232 differentially expressed genes(Differentially Expressed Gene,DEG),P<0.05 was used as the standard for this analysis,Found 2159 upregulated genes,There were 73 downregulated genes,Combined transcriptional and metabolomic results,The ATP binding transporter pathway(Adenosine triphosphate Binding Cassette,ABC)pathway is the key pathway to inhibit the Treg cell proliferation under silence the condition of MCT1 gene(P=0.04),All ABC pathway genes were upregulated and verified in RT-q PCR experiments,In the pathway,ABCA5,ABCB10,ABCD2(P<0.001)and ABCC9,ABCA1,ABCA13,ABCB1,ABCC2,The ABCD3 gene(P<0.0001)played the most important role.At the same time,the joint analysis found that lysine biosynthesis,cyclic guanosine monophosphate protein kinase G signaling pathway,vascular smooth muscle contraction are related to the generation of ATP and cell proliferation and differentiation,and it is also an important signaling pathway to inhibit anaerobic glycolysis and affect Treg cell proliferation.According to the TCGA Cancer Genome Atlas database,the expression of genes related to the ABC pathway affected the prognosis of renal clear cell carcinoma,and the ABCD 3 gene was closely related to Treg proliferation and immune invasion of Treg cells in renal clear cell carcinoma(P<0.001).ABCD 3 was used as a key gene for drug compound analysis,and the results showed the largest interaction coefficient of pilinicoic acid and valproic acid(both coefficient are 11),suggesting that the two compounds may become immunotherapy biological agents.Conclusion:The identification of the most differential ABC pathway transporters by transcriptomic and metabolomics analysis also plays a crucial role in the proliferation and immune infiltration of Treg cells.By further screening the target genes in the renal clear cell carcinoma tumor gene bank,multivariate analysis found that the upregulated expression of ABCD3 gene in ABC protein affected Treg cell proliferation and had the function of inhibiting the immune infiltration of Treg cells.Therefore,inhibiting anaerobic glycolysis can affect the proliferation and function of Treg cells,thus ultimately enhancing tumor immune function and inhibiting tumor proliferation and metastasis.Using the CTD database,it may be potential biological agents for immunotherapy of renal clear cell carcinoma.