我们先前在4-乙烯基环己烯二环氧化物(VCD)治疗的SD大鼠卵巢早衰(POF)模型的卵巢组织中筛选了6个差异表达的miRNA,包括miRNA-190a-5p,miRNA-98-5p,miRNA-29a-3p,miRNA-144-5p,miRNA-27b-3p,miRNA-151-5p。在这项研究中,为了研究引起POF发作的机制,我们首先在VCD处理的大鼠POF模型中鉴定了在连续时间点具有较早差异表达的miRNA,并探索了靶miRNA促进POF的机制.SD大鼠注射VCD15天以诱导POF。此外,我们连续15天每天同时采集大鼠血液和卵巢,和黄体生成素(LH),卵泡刺激素(FSH),抗苗勒管激素(AMH),ELISA法检测血清雌二醇(E2)水平。通过qRT-PCR测量大鼠卵巢中的六个miRNA表达。采用双荧光素酶报告基因测定来预测和验证靶miRNA(miRNA-190a-5p)的靶基因(PHLPP1)。Westernblot检测PHLPP1、AKT、p-AKT,FOXO3a,p-FOXO3a,和LHR蛋白对靶基因PHLPP1及其参与原始卵泡过度激活相关通路(AKT-FOXO3a和AKT-LH/LHR)的影响。在VCD建模POF大鼠卵巢期间,miRNA-190a-5p首先显示出显著的差异表达,即,第六VCD治疗,和PHLPP1被证实是它的直接下游目标。从6号开始治疗VCD,miRNA-190a-5p表达上调趋势越显著,PHLPP1和LHRmRNA和蛋白表达下调趋势越明显,伴随着AKT和FOXO3a蛋白更严重的磷酸化,从而持续过度激活大鼠原始卵泡以促进POF的发育。总之,miRNA-190a-5p可能成为POF早期筛查的潜在生物标志物,它可以通过靶向PHLPP1和AKT-FOXO3a和AKT-LH/LHR通路中关键蛋白的表达来持续激活大鼠原始卵泡。
We previously screened 6 differentially expressed miRNAs in ovarian tissues of 4-vinylcyclohexene diepoxide (VCD)-treated premature ovarian failure (POF) model in SD rats, including miRNA-190a-5p, miRNA-98-5p, miRNA-29a-3p, miRNA-144-5p, miRNA-27b-3p, miRNA-151-5p. In this study, to investigate the mechanisms causing the onset of POF, we first identified miRNAs with earlier differential expression at consecutive time points in the VCD-treated rat POF model and explored the mechanisms by which the target miRNAs promote POF. The SD rats were injected with VCD for 15 days to induce POF. Additionally, we collected rat blood and ovaries at the same time every day for 15 consecutive days, and luteinizing hormone (LH), follicle-stimulating hormone (FSH), Anti-Mullerian hormone (AMH), and estradiol (E2) serum levels were detected by ELISA. Six miRNAs expression were measured in rat ovaries by qRT-PCR. Dual-luciferase reporter gene assays were employed to predict and verify the target gene (
PHLPP1) of target miRNAs (miRNA-190a-5p). Western blot was examined to detect the expression levels of
PHLPP1, AKT, p-AKT, FOXO3a, p-FOXO3a, and LHR proteins on the target gene PHLPP1 and its participation in the primordial follicular hyperactivation-related pathways (AKT-FOXO3a and AKT-LH/LHR). During the VCD modeling POF rat ovaries, miRNA-190a-5p was the first to show significant differential expression, i.e., 6th of VCD treating, and
PHLPP1 was verified to be a direct downstream target of it. Starting from the 6th of VCD treatment, the more significant the up-regulation trend of miRNA-190a-5p expression, the more obvious the down-regulation trend of
PHLPP1 and LHR mRNA and protein expression, accompanied by the more severe phosphorylation of AKT and FOXO3a proteins, thus continuously over-activating the rat primordial follicle to promote the development of POF. In conclusion, miRNA-190a-5p may become a potential biomarker for early screening of POF, and it can continuously activate primordial follicles in rats by targeting the expression of
PHLPP1 and key proteins in the AKT-FOXO3a and AKT-LH/LHR pathways.