Effect Of 17β-Estradiol On Expression Of LncRNA In Mouse Thymic Epithelial Cells

The thymus is an important central immune organ and an important site for the maturation and differentiation of T cells.However,after the majority of mammals have matured,the thymus will gradually degenerate with age.Studies have shown that the destruction of the thymic microenvironment is closely related to the degeneration of the thymus,while thymic epithelial cells(TEC)are also an important part of the thymic microenvironment,and the increase of apoptosis and proliferation of TECs are important factors for the change of thymus microenvironment.Therefore,the research has shown that promote the proliferation of TECs can delay the degradation of the thymus and even promote the degradation of thymic regeneration.In fact,in many mammals,age-related thymus degeneration at least partly attributable to the sex steroid hormone secretion after sexual maturity,while estrogen is the main sex steroid hormone,studies have confirmed the degeneration of thymus following estrogen-induced maturation.Estrogen receptors exist in thymic epithelial cells and it is thought that the proliferation and function of thymic epithelial cells are closely related to the sensitivity of thymic epithelial cells to sex hormones.In recent years,research on lncRNA gradually rise,lncRNA is a class of RNA molecule with a transcript longer than 200 nt,they are not encoded proteins,but on a variety of levels in the form of RNA(epigenetic regulation,transcriptional regulation and transcription regulation,etc.)control gene expression level.However,the effect of E2-regulated lncRNAs expression on the proliferation of TECs in TECs cells has not been reported.In view of the important role played by E2,TECs,and lncRNAs in thymic tissue,it is speculated that the expression of E2-regulated lncRNAs in TECs may play an important role in the regulation of cell proliferation and thus regulate the degeneration of thymus.In this study,the mouse thymic epithelial cell line(MTEC1)was used as the research object to investigate the effects of differentially expressed lncRNAs on proliferation of MTEC1 cells and its mechanism of action after 17β-estradiol treatment of MTEC1.In this study,different concentrations(1,10,25,50 nmol/L)of 17β-estradiol were used to treat MTEC1 at different times(6,12,24,48 h),using the CCK-8 method,Edu incorporation method,Flow cytometry,high-throughput sequencing(RNA-seq),real-time fluorescence quantitative PCR,molecular cloning,overexpression,silencing,etc.,to explore the optimal treatment conditions for E2,and to examine the effects of E2 on cell proliferation,cell viability,and cell cycle.Based on the high-throughput sequencing data,the expression profile of differential lncRNAs in MTEC1 cells was analyzed,and the target molecule lncRNA-Gm12840 was screened to study the effect of lncRNA-Gm12840 on the proliferation of MTEC1 cells and its mechanism of action.The results are as follows:(1)17β-estradiol can significantly reduce the activity of MTEC1 cells in a time-and dose-dependent manner,taking into account the toxic and side effects of 17β-estradiol.The best treatment condition for E2 is 50 nmol/L E2 treatment MTEC1 cells for 24 h.(2)Flow cytometry results showed that there was a significant G2 arrest after E2 treatment of MTEC1 cells.Therefore,it is speculated that E2 can inhibit the proliferation of MTEC1 cells.(3)High-throughput sequencing showed that P < 0.05 was considered as the threshold for differential gene screening,and there were 962 lncRNAs that met the conditions,with 335 up-regulated and 627 down-regulated.By further analysis,P < 0.05,│log2│≥ 1 as the screening criteria,the differentially expressed lncRNAs satisfying the conditions totaled 537,of which 202 were up-regulated and 335 were down-regulated.There were 395 significantly enriched GO terms for significant GO enrichment analysis(P < 0.05).Many of these significantly enriched GO terms are related to cell proliferation,cell cycle,and apoptosis.The KEGG enrichment analysis showed that there were 16 significantly enriched KEGG pathways(P < 0.05),of which Jak-STAT,ribosome,cytokine-cytokine receptor interaction,autoimmune thyroid disease,protein export,proteasome,regulation of autophagy Bar-signal pathways are involved in cell proliferation,metabolism and regulation of cell homeostasis.(4)Overexpression of lncRNA-Gm12840 can significantly enhance the viability of MTEC1 cells and promote cell proliferation.It can regulate the cell cycle progression of MTEC1 by regulating the expression of cell cycle-related genes.Conclusion: 17β-estradiol can significantly reduce the activity of MTEC1 cells and arrest cells in the G2 phase.The results of RNA-seq showed that the differentially expressed lncRNAs may play an important role in the proliferation of MTEC1 through Jak-STAT and cytokine-cytokine receptor interaction.In summary,this study provides a new direction for exploring the relationship between lncRNA and thymic epithelial cell proliferation.