Sex Difference In Thymus Mi Rna Expression And MiR-16-5p On Proliferation Of Thymic Epithelial Cells In Mice

Thymic involution is attributed to one of the main causes of reduction in immune function and immunosenescence.However,the thymus regression shows not only age-depended involution but also a sex-biased involution during the process of thymus aging.MicroRNAs(miRNAs)are a class of short non-coding RNAs,which regulate gene expression at transcription or post-transcription level,study has indicated that miRNAs play important roles in a wide range of physiological and pathological processes,including the animal's individual development,cell differentiation and the formation of organs.Although the age-related miRNAs expression in mice thymus has been well demonstrated,but the miRNAs expression associated with sex difference and their potential influence on the thymus development are still not clear.Study also indicated that the effect of sex hormone on thymic involution is mediated by sex hormone directly act on TECs,because TECs expression of sex hormone receptors is required for thymic involution and normal thymic development.In order to invistitated the meanchism and importance of miRNAs expression in mice thymus for regulating thymus involution,high-throughput sequencing was used to further investigate the miRNAs expression changes under the affect of 17?-Estradiol in mice TECs.The main research results are as follows:1.The microarray results indicated that a total of 625 unique miRNAs present on the agilent mice microarray.77 miRNAs expression were found to be aberrantly expressed in mice thymus,59 and 28 miRNAs were defined as the sex-and age-specific miRNAs,respectively,10 mi RNAs expression were defined as the both sex-and age-specific miRNAs in mice thymus.When removed the lower signal intensity in theses miRNAs expressions,it was further found that 7 and 18 mi RNAs exhibited sex-and age-associated differential expression,respectively.There are 3 miRNAs expression were defined as the both sex-and age-specific miRNAs.Therefore,a total of 25 differential expressions of miRNAs were further obtained in this study.2.Further analysis showed that,in the age-effect group miRNAs expression there were 7 miRNAs that exhibited old age-biased expression(12 month expression > 3 month expression)and 11 miRNAs exhibited young age-biased expression(3 month expression > 12 month expression).In the sex-effect group miRNAs expression,5 miRNAs exhibited female-biased expression(female expression >male expression)and 2 miRNAs exhibited male-biased expression(male expression > female expression).3.The expression of miR-181c-5p,miR-20b-5p,miR-98b-5p,miR-329-3p,miR-341-5p and miR-2137 were showed significant age difference in mice thymus by qPCR.The expression of mi R-27b-3p,miR-140-3p and miR-378a-3p were showed significant sex difference in mice thymus by qPCR.4.GO and KEGG results indicated that many overlapping pathways was enrichment by both the age-or sex-associated miRNAs expression in mice thymus.In addition,it is worthy to note that some pathways are regulated just by the age-or sex-associated miRNAs expression in mice thymus.All the enrichment pathway function indicated that these miRNAs expression involves the cell proliferation,cell apoptosis,cell differntion,disease infection,tumor,immunes,stresses and sex hormone regulations,these factors play important roles for regulating thymus involution.5.The old-biased miRNAs expression in mice thymus mainly associated with the physiological stress.Sex hormone play a direct regulation roles for sex-related mi RNAs expression.The expression of miR-27b-3p and mi R-378a-3p were confirmed as the estrogen-responsive miRNAs expression in mice thymus in vivo.6.High-throughput sequencing miRNAs expression in TECs indicated that,a total of 3,961 mature miRNAs were detected in mouse medullar thymic epithelial cells(MTEC1)cells both from the E2 and control groups,respectively,by high-throughput sequencing,and 69 differential expressions of miRNAs were identified in TEC cells.Remove the low express levels of miRNAs from TEC cells,we finally obtained 6 differential expression of miRNAs in E2 group cells as compared with the control group cells,of which 2 upregulated and 4 downregulated in TEC cells.7.E2 can regulates the specific miRNAs expression in TECs via Er?,and these miRNAs expression has a key role for regulation cells proliferation.8.The E2 inducted of mi R16-5p was choosed to further analysis by which functions inhibiting cells proliferation in TECs.The CCk-8 and Edu assay showed that overexpression of miR-16-5p significantly decrease the TEC cell proliferation activity,whereas,inhibit the expression of mi R-16-5p in TEC cells significantly increased the cell proliferation activity.The flow cytometry analysis result indicated that mi R-16-5p inhibited cell proliferation in TEC cells by causing cells arrest at G1/S check-point.Western blot and qPCR showed that Cyclin D1 and IGFBP3 was the potential target genes by miR-16-5p in TEC cells.9.Luciferase reporter experiments indicated that the expression of miR-16-5p inhibites Cyclin D1 expression by targeting 1731~1737 and 1808~1814 sequence of cyclin D1 3 'UTR,respectively,and can also inhibites IGFBP3 expression by targeting 360~366 sequence of IGFBP3 3 'UTR,indicated that miR-16-5p regulating TECs cells proliferation is via direct targeting Cyclin D1 and IGFBP3.In summary,our study indicated that the sex-different involution in mice thymus has a significant effect on the miRNAs expression in the thymus tissue level,these specific miRNAs expression has a direct link with the sex hormone regulations in vivo.In the cells level,our study indicated that E2 through ER? could specifically regulate the miRNAs expression inTECs,theses miRNAs expression play important roles for regulating cells function and proliferation,indicated that different sex hormone in vivo result in different TECs function changes may be an important reason for explaining why thymus involution has a sex difference.In this study,we explored the importance for miRNAs mediated thymus involution both from the tissues and cell levels in mice thymus.Due to miRNAs are strong regulation factors for gene expression at the post-transcription level transcription,at the molecular levels,our study provide important experimental basis for further study sex-related thymus involution.