RESUMO
Polycystic ovary syndrome (PCOS) is one of the most common gynaecological endocrine disorders, and more than 60% of PCOS patients have varying degrees of insulin resistance (IR). The regulatory role of microRNAs (miRNAs) at post-transcriptional levels in human cumulus cells relating to IR in PCOS remains unclear. In this case-control study, 26 PCOS patients with IR (PCOS-IR) and 24 patients without IR (PCOS-control) were enrolled. We determined the differentially expressed miRNA and mRNA using next-generation sequencing technology, and these miRNAs and mRNAs were validated by quantitative real-time polymerase chain reaction (PCR). These miRNA regulating pathways (e.g., MAPK pathway) were analysed by bioinformatics analysis, and the Rap1b was demonstrated to be targeted by miR-612 based on quantitative real-time PCR, western blot and luciferase activity assay. A total of 59 known miRNAs and 617 differentially expressed genes were identified that differentially expressed between PCOS-IR and PCOS-control cumulus cells. Moreover, the potential regulating roles of miRNAs and their targeting genes in pathophysiology of IR and PCOS were analysed by gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation, and several key processes were enriched, such as MAPK activity. Furthermore, Rap1b, a regulator of the MAPK pathway, was demonstrated to be suppressed directly by miR-612 in PCOS-IR cumulus cells based on negative expression correlation validation, dual luciferase activity assay and reduction of Rap1b expression after miR-612 mimics transfection. Our results suggested that miRNAs and their targeted pathways in ovarian cumulus cells may play important roles in the aetiology and pathophysiology of PCOS with IR.
Assuntos
Células do Cúmulo/metabolismo , Resistência à Insulina , Sistema de Sinalização das MAP Quinases , MicroRNAs/metabolismo , Síndrome do Ovário Policístico/metabolismo , Estudos de Casos e Controles , Biologia Computacional , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , RNA Mensageiro/metabolismoRESUMO
Intrauterine adhesions (IUA) are associated with the loss of stem cells in the endometrium. Menstrual bloodderived stem cells (MenSCs) can be isolated from the menstrual blood and differentiated into endometrial cells. To check the transplantation feasibility of MenSCs for the treatment of severe IUA, MenSCs were isolated from menstrual blood, cultured in Dulbecco's modified Eagle's medium (DMEM), identified by immunocytochemistry and flow cytometry, differentiated into endometrial cells in vitro, and finally transplanted into the axillary subcutaneous tissue of nonobese diabetic/severe combined immunodeficiency (NODSCID) mice to create endometrial tissue. Additionally, the cloning efficiency and POU domain class 5 transcription factor 1 (OCT4) positivity of MenSCs from patients with severe IUA were compared with those from healthy women. Immunocytochemistry and flow cytometry results showed that 95.1±0.8% cells were OCT4positive, 0.9±0.4% were cluster of differentiation (CD)45positive, 1.8±0.9% were STRO1positive and 1.0±0.4% were human leukocyte antigenantigen D relatedpositive. Following differentiation in vitro, the results of immunocytochemistry, reverse transcriptionpolymerase chain reaction and western blot analysis showed that the expression of cytokeratin (CK) and vimentin (VIM) was increased in MenSCs compared with that in control subjects. Subsequent to transplantation in mice administered with sequential 17ßestradiol and progesterone, CK, VIM, estrogen receptor and progesterone receptor were expressed in the transplantation regions, suggesting that MenSCs could differentiate into endometrial tissues in vivo. The cloning efficiency and OCT4 positivity of MenSCs from patients with severe IUA was significantly decreased. In conclusion, to the best of our knowledge, this is the first study in which MenSCs could differentiate into endometrial cells in vitro and create endometrial tissue in NODSCID mice in vivo, with impaired cloning efficiency and OCT4 expression of MenSCs from patients with IUA. This study will provide a theoretical basis for the treatment of IUA with MenSCs.