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[miR-483-5p aggravates cisplatin-induced premature ovarian insufficiency in rats by targeting FKBP4].

ABSTRACT:

Objective

To investigate the role of FKBP4 protein in cisplatin-induced premature ovarian insufficiency (POI).

Objective

We performed ITRAQ assay of the ovarian tissues from 4 mice with cisplatin-induced POI and 4 control mice, and identified FKBP4 as a significantly down-regulated protein in the oocytes and granulosa cells following cisplatin treatment. TargetScan software was used for target analysis of FKBP4, and qRT-PCR and Western blotting were used to verify the expression levels of miR-483-5p and FKBP4 in the mouse models. Serum samples were collected from patients with POI and healthy women for detecting miR-483-5p level with qRT-PCR. Cell transfection and dual-luciferase assay were performed to determine the relationship between miR-483-5p and FKBP4. In primary granulosa cells and KGN cells, we examined the effect of miR-483-5p alone, miR-483-5p and cisplatin, and miR-483-5p combined with both cisplatin and FKBP4 on cell apoptosis. We also assessed ovarian function in a transgenic mouse model with ovarian miR-483-5p overexpression in comparison wigh wildtype mice using immunofluorescence assay, in situ hybridization and ELISA.

Objective

Ovarian FKBP4 expression was significantly decreased in mice with cisplatin-induced POI. Analysis using TargetScan software indicated that FKBP4 was the potential target of miR-483-5p, which was highly expressed in the ovaries and serum of POI mice and in the serum of patients with POI. In vitro experiments further confirmed that FKBP4 was the target of miR-483-5p. In KGN and primary granulosa cells, FKBP4 overexpression significantly reduced cell apoptosis induced by both cisplatin and miR-483-5p overexpression (P= 0.0045 and 0.0177, respectively). In the transgenic mice with miR-483-5p overexpression in the oocytes, cisplatin induced more severe ovarian damages as compared with those in the wild-type mice.

Objective

miR-483-5p/FKBP4 is a new and important pathway in cisplatin-induced POI, in which cisplatin increases ovarian miR- 483-5p expression to result in targeted downregulation of FKBP4. Up-regulation of miR-483-5p may increase ovarian sensitivity to cisplatin and cause severe ovarian dysfunction. Detection of serum miR-483-5p level may help to predict the occurrence and development of POI.

SUBMITTER: Zhao H

PROVIDER: S-EPMC8267993 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:ObjectiveThis study aims to evaluate the effect of Kuntai capsule on ovarian function in cisplatin-induced premature ovarian insufficiency rats and to explore the mechanism of Kuntai capsule on the ovarian function of rats.MethodsSeventy-four female Sprague-Dawley rats were used for this study. Eight of the rats were randomly assigned to the Control group. The remaining sixty-six rats were utilized to establish the POI model via Cisplatin and then randomly divided into four groups: the model Control group, the Estradiol group, and groups treated with low and high doses of Kuntai capsule. For the 28-day administration, the Control and model Control groups were intragastrically administered with 2.0 mL of 0.9% sodium chloride daily, the Estradiol group with 2.0 mL of Estradiol suspension (0.2mg/kg/d), and the low dose Kuntai capsule group and the high dose Kuntai capsule group with 2.0 mL of Kuntai capsule suspension (0.6g/kg/d, 1.8g/kg/d, respectively). Sex hormone levels, estrous cycle, and ovarian coefficient of the five groups were compared, histological sections analyzed follicle counts, and the protein expressions of growth differentiation factor 9, light chain 3 A-II, and Beclin 1 in the ovarian tissue were detected by Western blotting.ResultsAfter the 28-day administration, the serum Estradiol and Follicle-Stimulating Hormone levels of the group treated with low dose of Kuntai capsule were not significantly different from the Control group, the serum anti-Müllerian Hormone level of the group treated with high dose of Kuntai capsule was significantly higher than the Estradiol group. The estrous cycle of the group treated with low dose of Kuntai capsule was significantly lower than the model Control group. Regarding ovarian coefficient, resting and growing follicles, growth differentiation factor 9, light chain 3 A-II, and Beclin 1 expression, both Kuntai capsule groups outperformed the model Control group with the statistical difference (P<0.05).ConclusionKuntai capsule can improve the estrous cycle and ovarian coefficient of rats with premature ovarian insufficiency, maintain the number of resting and growing follicles, and up-regulate the protein expression of growth differentiation factor 9, light chain 3 A-II, and Beclin 1 of rats' ovaries.

Project description:Ferroptosis is widely present in fibrosis-related diseases. The basic pathology of premature ovarian insufficiency (POI) involves ovarian tissue fibrosis, and there are currently fewer relevant studies addressing the association between ferroptosis and POI. This study aimed to demonstrate that ferroptosis induced by cisplatin (CDDP) caused ovarian tissue fibrosis, leading to POI. Vitamin E (VE), a ferroptosis inhibitor, could repair damaged ovarian function. CDDP was used to establish a rat model of POI, and VE was administered to reverse the reproductive toxicity of CDDP. Ovarian function was assessed by histological section staining, follicle counts, sex hormone levels, as well as fertility assays. The extent of ferroptosis was assessed by transmission electron microscopy (TEM), malondialdehyde (MDA), Perls staining. CCK-8, Ethynyl-2-Deoxyuridine (EdU), and scratch assays were used to determine the effect of CDDP and VE on ovarian granulosa cell (GC) viability. Western blot, quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and immunohistochemistry were performed to evaluate ferroptosis-related molecular changes. Our results showed that CDDP caused follicle development disorders and ovarian tissue fibrosis, the levels of sex hormones suggested impaired ovarian function, and VE could reverse the reproductive toxicity of CDDP. The results of TEM, MDA and Perls staining suggested that the typical mitochondrial signature of ferroptosis was altered in ovarian GCs from the CDDP group, with significantly higher levels of lipid peroxidation and significant iron deposition in ovarian tissue, whereas VE mitigated the extent of ferroptosis. Molecular experiments then confirmed that the ferroptosis-related molecules acetyl CoA synthetase long chain family member 4 (ACSl4), 15-lipoxygenase-1 (ALOX15), solute carrier family 7 member 11 (SLC7A11), and glutathione peroxidase 4 (GPX4) were differentially expressed in each group. In summary, our study preliminarily demonstrated that CDDP may promote GCs to undergo ferroptosis, cause follicle development disorders, ovarian tissue fibrosis, and induce POI by regulating the expression of ACSl4, ALOX15, SLC7A11, and GPX4, while VE improved impaired ovarian function.

Project description:BackgroundPremature ovarian insufficiency (POI) patients are predisposed to metabolic disturbances, including in lipid metabolism and glucose metabolism, and metabolic disorders appear to be a prerequisite of the typical long-term complications of POI, such as cardiovascular diseases or osteoporosis. However, the metabolic changes underlying the development of POI and its subsequent complications are incompletely understood, and there are few studies characterizing the disturbed metabolome in POI patients. The aim of this study was to characterize the plasma metabolome in POI by using ultrahigh-performance liquid chromatography-mass spectrometry (UHPLC-MS/MS) metabolomics and to evaluate whether these disturbances identified in the plasma metabolome relate to ovarian reserve and have diagnostic value in POI.MethodsThis observational study recruited 30 POI patients and 30 age- and body mass index (BMI)-matched controls in the Center for Reproductive Medicine, Department of Gynecology and Obstetrics, Nanfang Hospital, Southern Medical University, from January 2018 to October 2020. Fasting venous blood was collected at 9:00 am on days 2-4 of the menstrual cycle and centrifuged for analysis. An untargeted quantitative metabolomic analysis was performed using UHPLC-MS/MS.ResultsOur study identified 48 upregulated and 21 downregulated positive metabolites, and 13 upregulated and 48 downregulated negative metabolites in the plasma of POI patients. The differentially regulated metabolites were involved in pathways such as caffeine metabolism and ubiquinone and other terpenoid-quinone biosynthesis. Six metabolites with an AUC value > 0.8, including arachidonoyl amide, 3-hydroxy-3-methylbutanoic acid, dihexyl nonanedioate, 18-HETE, cystine, and PG (16:0/18:1), were correlated with ovarian reserve and thus have the potential to be diagnostic biomarkers of POI.ConclusionThis UHPLC-MS/MS untargeted metabolomics study revealed differentially expressed metabolites in the plasma of patients with POI. The differential metabolites may not only be involved in the aetiology of POI but also contribute to its major complications. These findings offer a panoramic view of the plasma metabolite changes caused by POI, which may provide useful diagnostic and therapeutic clues for POI disease.

Project description:Premature ovarian insufficiency (POI) is defined as a primary ovarian defect characterized by absent menarche (primary amenorrhea) or premature depletion of ovarian follicles before the age of 40 (secondary amenorrhea) with hypergonadotropism and hypoestrogenism. Premature ovarian insufficiency has few known genetic causes but in familial cases a genetic link is often suspected. A large consanguineous family with three female affected with POI was investigated. All samples including 3 affected and 5 unaffecd underwent whole genome SNP genotyping using Affymetric Axiom_GW_Hu_SNP array. Linkage analysis was carried out using HomozygosityMapper and Allegro softwares.Linkage analysis mapped the disease phenotype to long arm of chromosome 20. Sequence data analysis of potential candidate genes failed to detect any pathogenic variant. Affymetrix SNP arrays were performed according to the manufacturer's directions on DNA extracted from peripheral blood samples. DNA of eight individuals including three affected subjects was used for homozygosity mapping. Genotyping was performed using the Affymetrix Axiom_GW_Hu_SNP array. Briefly, 250 ng genomic DNA was digested with Digestion Master Mix containing 2 µl NE buffer 2 (10X), 0.5 µl BSA (100X; 10 mg/ml) and 1 µl Nsp1. Digested DNA sample was ligated to Nsp1 adaptor using T4 DNA ligase and amplified by 2 µl of TITANIUM Taq DNA polymerase (50X) and 100 µM PCR primer. PCR products were purified on a Clean-Up plate (Clontech Lab, Madison, USA) and eluted by RB buffer. Purified PCR products were fragmented using Fragmentation Reagent (0.05U/µl DNase 1) for 35 minutes at 37°C followed by labeling of fragmented samples with Labeling Master Mix (30 mM GeneChip DNA Labeling Reagent, 30 U/µl Terminal Deoxynucleotidyl Transferase) for 4 hours at 37°C. Labeled samples were hybridized to Axiom_GW_Hu_SNP array by mixing the sample with Hybridization Master Mix, denatured on thermoblock and loaded on to Array. Array was then placed in a hybridization oven (GeneChip Hybridization Oven 640, USA) for 16-18 hours. After hybridization, array was washed and stained on an automated Fluidic Station 450 followed by scanning on GeneChip Scanner 3000 7G using GeneChip Operating Software (GCOS).

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[miR-483-5p aggravates cisplatin-induced premature ovarian insufficiency in rats by targeting FKBP4].

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