| The thymus is the primary immune organ for T cell development,and plays a crucial role in the immune system of body.With the growth of the age,the thymus gradually degenerates,especially obvious in the sexual maturity.In addition to the factors of age and gender,the increase in sexual steroids produced after sexual maturity is also important in thymic involution.It is significant to study mouse thymic involution for the development of measures of promoting thymic regeneration.However,there are limited reported the effecf age,sex,and gonadal steriods on thymus involution in molecular level.With the rapid development of high-throughput sequencing technology and bioinformatics processing technology,it is possible to perform a detailed analysis of the transcriptome of a species.In this study,the miRNAs,lncRNAs and mRNAs regulatory networks of 1-month-old and 3-month-old female and male mice were studied.The effects of age,sex and sex steriods differences on thymus involution were to be deeply revealed at molecular level,and a new method to inhibit thymus involution or promote thymus regeneration was intended to be found.The thymus tissue of 1 month old male mice(1M),1 month old female mice(1F),3 month old male mice(3M),3 month old male castrated mice(3Mx),and 3 months old female mice(3F)and 3 month old female ovariectomized mice(3Fx)were studied.High throughput sequencing,bioinformatics analysis and RT-q PCR were used to study the regulatory relationship among miRNAs,lncRNAs and mRNAs during mouse thymic degeneration and investigate the effects of miRNAs,lncRNAs and mRNAs on the age,sex and sex steroid hormones of mouse thymic degeneration.Subsequently,the biological function of the screened age and sex-related miR-183-5p in medullary thymic epithelial cell line 1(MTEC1)cells was studied by CCK-8 assay,flow cytometry,molecular cloning,double luciferase reporter gene,RT-q PCR and Western blot and so on.The regulatory mechanism of miR-183-5p in the process of thymus involution in mice was studied.The main research results are as follows:1.lncRNA sequencing was performed on mouse thymus of different ages and genders,then a total of 132.2G sequencing data was obtained.As a result,18116 lncRNAs were identified in the thymus,including 8546 novel lncRNAs.In 3F vs 1F,1794 differentially expressed lncRNAs were identified;In 3M vs 1M,235 differentially expressed lncRNAs were identified;In 3M vs 3F,136 differentially expressed lncRNAs were identified;In 1M vs 1F,1472 differentially expressed lncRNAs were identified;In 3M vs 3Mx,165 differentially expressed lncRNAs were identified;In 3F vs 3Fx,333 differentially expressed lncRNAs were identified.Significantly enriched signaling pathways for predicted target genes of differentially expressed lncRNAs are associated with cell proliferation,growth,development and metabolism.2.The results of mRNA sequencing of mouse thymus of different ages and genders showed that 2127 differentially expressed mRNAs were identified in 3F vs 1F.In 3M vs 1M,206 differentially expressed mRNAs were identified;In 3M vs 3F,124 differentially expressed mRNAs were identified;In 1M vs 1F,1349 differentially expressed mRNAs were identified;In 3M vs 3Mx,208 differentially expressed mRNAs were identified;In 3F vs 3Fx,144 differentially expressed mRNAs were identified.Significantly enriched signaling pathways of differentially expressed mRNAs are associated with cell proliferation,transcriptional regulation,immune response and metabolism.3.The results of miRNA sequencing of mouse thymus of different ages and genders showed that 265 differentially expressed miRNAs were identified in 3M vs 1M;256 differentially expressed miRNAs were identified in 3F vs 1F;262 differentially expressed miRNAs were identified in 1M vs 1F;253 differentially expressed miRNAs were identified in 3M vs 3F;273 differentially expressed miRNAs were identified in 3M vs 3Mx;239 differentially expressed miRNAs were identified in 3F vs 3Fx.Among them,fourteen miRNAs have differences in age,sex,and gonadal steroids.For target gene prediction and signal pathway analysis,the results showed that differential miRNAs can participate in the regulation of thymus involution through Ras signaling pathway,PI3K-Akt signaling pathway,Wnt signaling pathway and so on.4.The detection results of cell proliferation showed that overexpression of miR-183-5p can significantly decrease the viability of MTEC1 cells,while the viability of MTEC1 cells can significantly increase after inhibition of miR-183-5p expression;the flow cytometry assay indicated that miR-183-5p can cause arrest and inhibition cell proliferation during G1;the consequences of RT-q PCR and Western blot suggested that Lrp6 and Smad4 may be key target genes for cell proliferation.5.The results of double luciferase reporter gene assay showed that miR-183-5p can inhibit Lrp6 expression and Smad4 expression by targeting 203?210 sequence of 3' UTR and 627?632 sequence of 3' UTR,respectively,which indicated that miR-183-5p affects MTEC1 cell proliferation by regulating the expression of the target genes Lrp6 and Smad4.Conclusion:1.Determination of the age and sex-related differential expression profiles of lncRNAs,miRNAs and mRNAs in mouse thymus,which can provide a database for studying thymic degeneration at the molecular level.2.miR-183-5p can inhibit thymic epithelial cell proliferation by targeting Lrp6 and Smad4.3.The Wnt signaling pathway regulated by Lrp6 may be an important pathway for miR-183-5p to regulate thymic epithelial cell proliferation. |
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