Screening And Functional Analysis Of MicroRNA Targeting GnRH Gene

Background: MicroRNAs(miRNAs)are a class of endogenous,functional,19-23 nt non-coding single-stranded small RNAs that play a gene regulatory role in mammalian development.Mature miRNAs bind to the 3 'untranslated region(3' UTR)of target gene mRNAs,inhibiting translation of the target gene protein,or regulating gene expression by inducing mRNA degradation.MiRNAs are involved in processes such as cell proliferation and apoptosis,insulin secretion,and individual development.In mammals,sexual development is regulated by the hypothalamic-pituitary-gonadal axis(HPG axis),and the levels of various hormones secreted by it directly regulate the development and maintain the reproductive function of the body.Hypothalamic GnRH neurons pulsatile secretion of GnRH,is a key event in sexual development.When sexual development started,GnRH neurons secreted GnRH frequency and pulse amplitude were significantly increased.More and more evidences prove that the spatial and temporal expression of genes play a crucial role in the initiation of sexual development.MicroRNAs(miRNAs),on the other hand,regulate gene expression at multiple levels and thus regulate different pathways of sexual development.The laboratory has been committed to studying the genetic and molecular mechanisms of sexual development in mice and found that many microRNAs are involved in the regulation of this important life process.Objective: In GT1-7 cells derived from hypothalamic GnRH neurons,a highly sensitive GnRH expression regulation system was constructed and its ability to regulate GnRH was measured by measuring the change in fluorescence intensity after treatment with regulatory factors.GnRH-targeted miRNAs were screened by dual luciferase reporter system to detect the effect of miRNA on the expression of GnRH in GT1-7 cells under different combination modes.This different combination of effects can simulate the basic physiological state of the organism,to explore miRNA regulation of GnRH,in-depth analysis of its regulatory pathways,so as to improve sexual development gene regulatory network.Methods: Through the CRISPR/Cas9 system,the upstream and downstream homology arms of GnRH and green fluorescent protein(EGFP)gene can be inserted into PUC19 vector by homologous recombination to construct GnRH target vector.According to the GnRH gene location,the most suitable sgRNA was designed by CRISPR/Cas9 system to construct sgRNA vector.In GT1-7 cells,two vectors were co-transfected,and positive monoclonal cells were sorted by flow cytometry and expanded to a green fluorescence-containing reporter system.Seven miRNAs targeted to GnRH were predicted by using Targetscan,mirDB,microRNA target prediction,mirwalk,PicTar and mirnada databases,including mmu-miR203-3p,mmu-miR574-3p,mmu-miR100-3p and mmu-miR370-5p,mmu-miR148a-3p,mmu-miR148b-3p,mmu-miR152-3p.The 3' untranslated region(3'UTR)of GnRH was constructed on the psi-check2 vector and constructed as a GnRH expression vector.The 293 cells were transiently transfected with the miRNA mimics and screened by dual luciferase reporter system GnRH were transfected into GT1-7 cells.The four miRNAs targeting GnRH were further screened by GnRH expression to obtain three miRNAs targeting GnRH.Three miRNA mimics of the same concentration were transfected into GT1-7 cells in different combination modes to detect the effect of Gn RH and other sexual development related genes.Results: A highly sensitive GnRH expression control reporter system has been successfully constructed.After transfection of GT1-7 cells,the change of fluorescence value was detected by flow cytometry.However,after the flow cytometry sorting,fluorescence was not sorted cell.In 293 cells,four miRNAs targeting the GnRH gene were screened by the dual luciferase reporter system,including mmu-miR203-3p,mmu-miR574-3p,mmu-miR100-3p,mmu-miR370-5p Four miRNAs were further tested in GT1-7 cells,and three miRNAs except mmu-miR203-3p were screened out.In order to simulate the basic physiological state of the organism,the detection of mi RNAs on GnRH and sexual development-related genes under different combination modes was examined.The results showed that the expression of GnRH was different between single miRNA transfection,two transfection and three transfection The effect of combined transfection on GnRH was more effective than that of single transfection,but also affected the expression of other sexual development-related genes,but the effect was less than that of GnRH.Conclusion: At different concentrations,miRNAs targeting GnRH gene have different effects on the expression of GnRH,and the effect of miRNA co-transfection on GnRH is more significant than the single transfection at the same concentration.In the detection of developmental related genes,the expression of each gene will have varying degrees of change,but the changes are not as significant as GnRH,probably because of indirect regulation of GnRH effect,need further study.In summary,GnRH genes are regulated by different miRNAs,and miRNAs can regulate the expression of GnRH at multiple levels by combining the roles of miRNAs and further perfect the gene regulatory network.