| Objective: POF(premature ovarian failure)is a reproductive system disease,refers to the women’s ovarian failure,whose age are under 40 year-old,accompanied by 4-6 months of menopause,FSH(follicle stimulating hormone)>40 IU/L in twice detections that are consecutive but with an interval of more than 4 weeks.However,in about 90% POF cases,The etiology remains unclear.for it is complex,at least relating to the factors of genetics,autoimmunity,Metabolism&infection,and iatrogenic factors.Over the past few decades,non-coding part of the genome has been recognized for the significance to human development,physiology,and disease.Microribonucleotides(microRNAs,miRNAs)are a class of single-stranded non-coding nucleotide molecules that are about 19-25 nucleotides in length,which can regulate the expression of target gene at the post-transcriptional level by paired with the 3’ untranslated region(UTR)of mRNA and trigger the RNA’s degradation or translational repression.It is estimated that miRNAs regulate the translation of more than60% protein-coding genes,are involved in the regulation of proliferation,differentiation,apoptosis,tumorigenesis and hematopoiesis.miRNAs play an important role in the regulation of ovary,but there are few reports in POF.This study focuses on POF-related miRNAs,and has a deep understanding of the expression and function of miRNAs,in order to contribute to the etiology study of idiopathic POF,and this may provide new ideas for the diagnosis and treatment of the infertile patients with ovarian hypofunction.Methods: 1.Mice were used as the research object in this study.the POF mouse model was constructed by intraperitoneal injection of cisplatin.Continuously monitor vaginal exfoliated cells,So that it could be judged if the estrous cycle of the mice become disturbed;HE staining was used to observe if the morphology of the ovary changes;Elasa monitor the AMH(anti-mullerian hormone)levels,body weight and mental state,in order to judge whether the mouse POF model was successfully constructed.2.Detected the differential expressions of miR-199 a,miR-183,miR-181 a,miR-92 a and miR-21 in ovarian tissue of mice between the model group(POF group)and the normal group by real-time fluorescent PCR(qPCR)technology.3.Predicted the target genes regulated by miR-199 a,miR-183,miR-181 a,miR-92 a and miR-21 by miRNA target gene prediction database;And GO enrichment analysis predicts the biological properties of target genes;DO enrichment analysis predicts the relationship between target genes and diseases;KEGG enrichment analysis predicts the relationship between target genes and the regulation of signaling pathways.4.RT-PCR detected the expression levels of target genes SMAD7、FOXO1、SIRT1mRNA predicted.5.All data were analyzed by SPSS25.0software.Results:1.The mouse POF model was successfully constructed.It was characterized by: the disturbed estrous cycle;decreased anti-Mullerian hormone(AMH)levels;weight loss;poor mental status;altered ovarian histology.2.The qPCR results of the relative expression of two miRNA groups showed:compared with the ovarian tissue of the normal group,the expressions of miR-199 a,miR-183,miR-92 a and miR-21 in the ovarian tissue of the model group were reduced.The amount of miR-181 a decreased,while the expression of miR-181 a increased(p < 0.05),and the difference was statistically significant.3.After bioinformatics analysis and review of relevant literature,the following conclusions can be drawn:The target gene of miR-199 a is SIRT1;the target gene of miR-183 is FOXO1;the target gene of miR-181 a is SIRT1 and SMAD7;the target gene of miR-92 a is SMAD7;The target gene of miR-21 is SMAD7.GO enrichment analysis showed that the predicted target genes of these five miRNAs were mostly enriched in biological processes such as reproductive structure development,positive regulation of cell migration,and regulation of apoptosis signaling pathway;DO enrichment analysis showed that female reproductive diseases and ovarian-related diseases ranked relatively high,indicating that the predicted target genes of these five miRNAs may have regulatory roles in ovarian-related diseases;KEGG enrichment analysis showed that most of the top-ranked signaling pathways were involved in the regulation of follicle development,atresia and granulosa cell apoptosis,and the predicted target genes of these five miRNAs might be involved in regulation.4.Compared with SMAD7,FOXO1 and SIRT1 mRNA in the ovarian tissue of the normal group,it was showed that the expression of SIRT1 mRNA in the model group decreased;The expression of SMAD7 mRNA and FOXO1 mRNA in the model group increased,and the difference between the two groups was statistically significant(p<0.05).Conclusion:1.The POF mouse model was successfully constructed;2.Compared with the ovarian tissue of the normal group,the expression levels of miR-199 a,miR-183,miR-92 a and miR-21 in the ovarian tissue of the POF group decreased,while the expression level of miR-181 a was increased(p<0.05),it could be suggested miR-181 a to be used as a diagnostic marker for POF,while further research is needed;3.Preliminary forecast that the expression of miR-199 a may be involved in the occurrence and development of premature ovarian failure by targeting SIRT1,while the expression of miR-183 may be involved in by targeting SMAD7 and SIRT1,the expression of miR-92 a may be involved in by targeting SMAD7,and the expression of miR-21 may be involved in by targeting SMAD7;The differential mRNA level expression of SIRT1,FOXO1 and SMAD7 in the ovarian tissue of mice with premature ovarian failure may be provided a new idea for exploring the etiology and clinical diagnosis and treatment of premature ovarian failure. |
