人脐带间充质干细胞外泌体miR-100对多囊卵巢综合征患者颗粒细胞炎症的影响

目的 探究人脐带间充质干细胞(hUC-MSCs)来源的外泌体miR-100对多囊卵巢综合征(PCOS)患者颗粒细胞炎症的影响。方法 选取2021年3月至2021年12月于石家庄市第四医院生殖医学中心就诊的70例PCOS患者为研究对象(PCOS组),同期70例因输卵管因素助孕治疗的非PCOS患者为对照(对照组),比较两组患者的临床基线资料及IVF妊娠结局相关参数。采用实时荧光定量PCR (RT-qPCR)检测患者颗粒细胞肿瘤坏死因子-α(TNF-α)、干扰素-γ(IFN-γ)的表达。采用二元Logistic回归分析影响PCOS的危险因素。RT-qPCR验证hUC-MSCs外泌体中miR-100的表达水平。合成miR-100模拟物及其对照转染至人卵巢颗粒细胞KGN中，RT-qPCR检测miR-100的表达水平。在脂多糖(LPS)诱导的KGN炎症细胞模型中转染miR-100模拟物及其对照，RT-qPCR检测TNF-α、IFN-γ的mRNA表达水平。采用双荧光素酶报告基因系统检测miR-100对靶基因的调控作用。miR-100过表达后，Western blotting检测KGN炎症模型中Tol...

Effects of human umbilical cord mesenchymal stem cell-derived exosomal miR-100 on inflammation of ovarian granulosa cells in polycystic ovary syndrome

Objective To explore the effects of exosomal miR-100 derived from human umbilical cord mesenchymal stem cells(hUC-MSCs)on the inflammation of granulosa cells(GCs)in polycystic ovary syndrome(PCOS). Methods A total of 70 PCOS patients treated during Mar. 2021 and Dec. 2021 were enrolled as the PCOS group, and another 70 patients treated due to tubal factors were selected as the control group. The clinical characteristics and in vitro fertilization(IVF)outcomes were compared between the two groups. The expressions of pro-inflammatory cytokines TNF-α and IFN-γ in patients’ GCs were detected with real-time quantitative PCR(RT-qPCR). The risk factors influencing the occurrence of PCOS were analyzed with binary Logistic regression analysis. The expression of miR-100 in hUC-MSCs exosomes(exos)was verified with RT-qPCR. After the miR-100 mimics and miR-NC were synthesized and transfected into human ovarian granulosa cells(KGN), the expression of miR-100 was determined with RT-qPCR. After the miR-100 mimics and miR-NC were transfected into lipopolysaccharide(LPS)-induced KGN cells, the mRNA expressions of TNF-α and IFN-γ were detected with RT-qPCR. The target gene of miR-100 was verified with dual-luciferase reporter gene assay. After miR-100 was overexpressed in LPS-induced KGN cells, the effector molecular of Toll-like receptor-4/nuclear factor kappa B(TLR4/NF-κB)signaling pathways, including TLR4, p-IκBa, IκBa, p-p65 and p65, were detected with Western blotting. Results PCOS group had higher body mass index(BMI), luteinizing hormone(LH), estradiol(E₂), testosterone(T), homeostasis model assessment for insulin resistance index(HOMA-IR), antral follicle count(AFC), and number of retrieved oocytes than the control group, whereas lower MII oocyte rate and 2PN fertilization rate(P<0.001, P<0.001, P=0.020, P<0.001, P<0.001, P<0.001, P<0.001, P<0.001, P<0.001). The PCOS group had significantly higher expressions of TNF-α and IFN-γ than the control group(P<0.001). Binary Logistic regression analysis revealed that LH, HOMA-IR, TNF-α and IFN-γ were risk factors influencing the occurrence of PCOS(P=0.031, 0.001, 0.030, 0.005). RT-qPCR showed that miR-100 was enriched in hUC-MSCs-exos. Overexpression of miR-100 significantly inhibited LPS-induced increase of the mRNA expressions of TNF-α and IFN-γ(P=0.041, P<0.001). Dual-luciferase reporter gene assay showed that miR-100 directly targeted TLR4 mRNA 3’UTR and inhibited its expression(P<0.001). Western blotting showed that miR-100 efficiently inhibited LPS-induced elevation of the protein expressions of TLR4, p-IκB and p-p65(P<0.001, P<0.001, P=0.049), but the protein expression of IκB significantly increased(P<0.001). Conclusion The increased expressions of TNF-α and IFN-γ are risk factors influencing the occurrence of PCOS. Exosomal miR-100 derived from hUC-MSCs inhibits the inflammation of granulosa cells via TLR4/NF-κB signaling pathway in PCOS.