Effects Of Arg1,Cyp1b1 Mediated By MicroRNA On Proliferation And Apoptosis Of Mouse Thymic Epithelial Cells

The thymus is an important immune organ that produces T cells and plays an extremely important role in the body’s immune system.With the increase of age,thymus gradually atrophied and degenerated,especially after sexual maturity.In addition to age and gender factors,the increase of sex steroid hormones after sexual maturity is also an important factor for thymus degeneration.The study of thymus degeneration in mice is of great significance to find out the measures to promote thymus regeneration.However,there are few reports on the mechanism of thymic degeneration at molecular level.With the rapid development of high-throughput sequencing technology and bioinformatics processing technology,it has become possible to analyze the transcriptome of a species in full detail.In this project,by studying the mutual regulatory network of mRNAs in mouse epithelial cells,the molecular level is used to reveal the effects of different mRNAs on thymus degeneration,and to find new methods to inhibit thymus degeneration or promote thymus regeneration.The thymic epithelial cells of 1-month-old male mice(1M),1-month-old female mice(1F),3-month-old male mice(3M)and 3-month-old female mice(3F)were studied in his paper.High-throughput sequencing,bioinformatics analysis,RT-q PCR and other methods were used to analyze and verify the differential expression of mRNAs in the mouse thymus,and the regulatory relationship between the mRNAs in the process of mouse thymus degeneration was studied.Subsequently,Arg1 and Cyp1b1 were selected to predict their upstream microRNAs,which was verified by double Luciferase Report System.CCK-8,flow cytometry,molecular cloning,RT-q PCR,Western blot and co-IP were used to study the effects of microRNAs and Arg1,Cyp1b1 on the proliferation,apoptosis and enzyme function of thymic epithelial cells.We sequenced,analyzed and verified the mouse thymic epithelial cell lines after overexpressed and inhibited CYP1B1,and obtained a large number of mRNAs difference data.The main findings are as follows:1.This experiment identified mRNAs that were differentially expressed in t thymic epithelial cells of mice of different ages and genders(3F vs 1F,1585;3M vs 1M,4857;1M vs 1F,4556;3M vs 3F,1439).2.mmu-mi R-340-5p and mmu-mi R-126b-5p can target and regulate Arg1 in MTEC1 cells.3.Overexpression of Arg1 can significantly improve the vitality of MTEC1 cells,inhibit apoptosis,and promote the cell cycle process,and promote the proliferation of MTEC1 cells;after inhibition of Arg1 expression,the activity of MTEC1 cells decreased significantly.Inhibition of arg1 inhibited the cell cycle process of MTEC1 cells,and then inhibited cell proliferation.4.Arg1 can regulate BAX,Caspase-3,Bcl-2,Caspase-9,CCND1,Cyclin B,Cyclin E,P53 at the gene level,but can only regulate Bcl-2,CCND1,Cyclin B,Cyclin E,P53 at the post-transcription level.There is a direct interaction between the protein ARG and the protein CCND1,and there may be an indirect interaction with the proteins Bcl-2,Cyclin B,Cyclin E,and P53.5.mmu-mi R-429-3p and mmu-mi R-200c-3p can target and regulate Cyp1b1 in MTEC1 cells.6.Overexpression of Cyp1b1 for 48 h can significantly reduce the activity of MTEC1 cells,inhibit the proliferation of MTEC1 cells,block the cell cycle process of MTEC1 cells,and then inhibit the proliferation of MTEC1 cells;while inhibition of Cyp1b1 expression for48 h can significantly improve the activity of MTEC1 cells,inhibit Cyp1b1 to promote the cell cycle process of MTEC1 cells,and then promote the proliferation of MTEC1 cells.7.Cyp1b1 can regulate BAX,Bcl-2,Caspase-3,Caspase-8,Caspase-9,CCND1,CDK4,Cyclin B,Cyclin E,P53 at the gene level,and can only regulate Bcl-2,Caspase-3,Caspase-8,Caspase-9,CCND1,CDK4 at the post-transcription level.There is an interaction between protein CYP1B1 and protein CDK4,and there may be indirect interactions with proteins BAX,Bcl-2,Caspase-3,Caspase-8,Caspase-9,CCND1.8.Compared with the control group,1028 differentially expressed mRNAs were identified in the Cyp1b1 overexpression group,including 522 up-regulated and 506 down regulated.Compared with the control group,1109 differentially expressed mRNAs were identified in the Cyp1b1 inhibition group,including 533 up-regulation and 576 downregulation.Compared with the Cyp1b1 inhibition group,1175 differentially expressed mRNAs were identified in the Cyp1b1 overexpression group,including 623 up-regulated and552 down regulated mRNAs.10.Cyp1b1 may affect the proliferation and apoptosis of TECs by regulating the cell senescence-related pathway.11.Arg1 and Cyp1b1 may interact indirectly through UBC,Cyp1a2,Nr1h3.Conclusion:1.The differentially expressed mRNAs in thymic epithelial cells of mice in different ages and genders were identified,which provided a database for the future study of thymus degeneration at the molecular level.2.mmu-mi R-340-5p and mmu-mi R-126b-5p can target and regulate Arg1 in MTEC1cells;mmu-mi R-429-3p and mmu-mi R-200c-3p can target and regulate Cyp1b1 in MTEC1 cells.3.There is a direct interaction between protein ARG and protein CCND1,and there is an interaction between protein CYP1B1 and protein CDK4.