The Effect Of MiR-152-3p On Thymus Epithelial Cell Proliferation

Thymus is an important central lymphoid organ,an important site for the differentiation,development and maturation of T lymphocytes,and plays an extremely important role in the body’s immune system.Thymus varies greatly with age.It reaches the maximum volume and weight at sexual maturity.Subsequently with its gradual atrophy,thymus is almost replaced by adipose tissue at last.The thymus gland is composed of thymocytes and thymic stromal cells.Thymic epithelial cells（TECs）are the most important thymic stromal cells and important part of the three-dimensional meshwork microenvironment necessary for the development,differentiation and maturation of thymocytes.The decrease of TECs proliferation can lead to the destruction of normal thymic microenvironment,which is the decisive factor of thymus degeneration.However,the molecular mechanism of TECs increment decrease in age-dominated thymus degeneration is still unclear.High-throughput sequencing makes it possible to analyze the transcriptome and genome of a species in detail.micro RNAs（miRNAs）have been found to be involved in the development and degeneration of the thymus.miRNAs are a kind of non-coding RNAs with a length of about 22 nt.With the deepening of research,more and more miRNAs have been found to be related to the regulation of cell proliferation,cell apoptosis,fat metabolism,tumor generation and other aspects.miRNAs play an important regulatory role in TECs,but the effects of miRNAs on TECs related to age and its molecular mechanism need to be further studied.Based on the analysis of previous high-throughput sequencing results in the laboratory,this study screened out age-related differential miRNA（miR-152-3p）from the thymus and TECs sequencing results of mice of different ages.In mice with medullary thymic epithelial cell line（medullary thymic epithelial cell line 1,MTEC1）cells as the research object,through the CCK-8 method,Ed U staining,flow cytometry,real-time fluorescent quantitative PCR,dual luciferase reporter gene experiment,and Western blot techniques,such as from the cell vitality,cell proliferation,cell cycle,etc,to explore the miRNAs to the effect of thymic epithelial cell proliferation and function.The cell viability was detected by cck-8 method,indicating that miR-152-3p inhibited MTEC1 cells.Overexpression of miR-152-3p inhibited the cell viability of MTEC1 cells（P<0.001）.On the contrary,the inhibition of miR-152-3p increased the activity of MTEC1cells.The results of flow cytomgraphy showed that overexpression of miR-152-3p increased the G₁ phase number of MTEC1 cells（P<0.01）and decreased the S phase number（P<0.01）.It was showed G₁ phase arrest and inhibition of cell proliferation.Inhibition of miR-152-3p resulted in decreased G₁ phase number（P<0.01）and increased S and G₂ phases（P<0.01）.It was promoted proliferation.The cell proliferation was detected by Ed U incorporation method.Overexpression of miR-152-3p could inhibit the proliferation of MTEC1 cells,while inhibition of miR-152-3p could promote the proliferation of MTEC1 cells.The above results showed that miR-152-3p inhibited MTEC1 cell proliferation.RT-PCR results showed that overexpression of miR-152-3p could decrease the expression of cycline-related genes such as C-myc（P<0.001）,Ccnd1（P<0.001）,Ccne1（P<0.01）,and Cdk4（P<0.01）.Inhibition of miR-152-3p could increase the expression of C-myc（P<0.001）,Ccnd1（P<0.01）,Ccne1（P<0.01）,and Cdk4（P<0.01）.Through the screening of target gene prediction software and the verification of RT-PCR,it was known that Smad2was negatively regulated with miR-152-3p.The 3’UTR region of Smad2 can bind to miR-152-3p and Smad2 is the direct target gene of miR-152-3p through the double luciferase reporting experiment.The SMAD2 protein level was decreased by Western Blot,which was consistent with the gene expression level.Using transfection to interfere with the fragment of si Smad2,the gene and protein expression of Smad2 were significantly decreased.Inhibition of Smad2 gene expression was found to inhibit the progress of cell cycle and the expression of cell cycle-related genes by flow cytometry and RT-PCR,which was consistent with the results of miR-152-3p.It was indicated that miR-152-3p regulated cell proliferation through targeting Smad2.Conclusion:miR-152-3p with age difference can inhibit the proliferation of thymic epithelial cells by regulating cell cycle-related genes,thus blocking the G₁ phase and inhibiting the proliferation.Smad2 is the target gene of miR-152-3p and can affect cell proliferation and cycle through targeting Smad2.It is speculated that the differential upregulation of miR-152-3p might promote the process of thymic involution.miR-152-3p can be used as a biomarker for thymus involution.It is suggested that miR-152-3p may be a potential target for the treatment of thymic aging.In general,this study provides an important experimental basis for revealing and studying the age difference of thymus degeneration at the molecular level.