| N-Hexane is a commonly used industrial organic solvent with cumulative toxicity.Due to its large and feminized and younger occupational exposed population,the effects of n-hexane and its main toxic metabolite 2,5-hexanedione(2,5-HD)on women’s health,especially reproductive health,have received increasing attention.At present,domestic and foreign studies have shown that n-hexane and 2,5-HD can lead to female(female)reproductive function damage,interfere with the survival and apoptosis of ovarian granulosa cells and then damage the ovary may be one of the important toxic mechanisms.Apoptosis involves very complex regulatory mechanisms,in which the progression of the cell cycle is an important condition in determining cell proliferation or apoptosis.Abnormal expression of genes that regulate the normal progression of the cell cycle can lead to cell cycle disorders.As one of the important epigenetic regulatory modalities,miRNAs can regulate the expression of genes at the post-transcriptional level which in turn regulates cellular functions,including regulating the cell cycle.In addition,it has been shown that miRNA regulation plays a key role in the development and function of granulosa cells.Therefore,in this study,we investigated the role of key signaling pathways regulated by miRNAs in 2,5-HD-induced ovarian granulosa cell cycle arrest at the post-transcriptional level through an in vitro exposure model,combined with miRNA and mRNA microarray technology,in order to preliminarily analyze whether miRNA-mediated epigenetic regulation is involved,as well as possible key targets,from a systematic point of view and provide an experimental basis for further revealing the ovariotoxic mechanism of n-hexane.At the same time,it provides a new idea for exploring the intervention target of n-hexane ovarian toxicity,which has important practical significance for formulating reasonable and effective occupational health prevention and control measures and protecting the reproductive health of female workers occupationally exposed to n-hexane.Part Ⅰ Analysis of mRNA and miRNA expression profiles in ovarian granulosa cells after 2,5-HD exposure in vitroObjective: Through bioinformatics analysis of miRNA and mRNA expression profiles after 2,5-HD exposure,the possible regulatory role of miRNAs during2,5-HD-induced toxic injury in ovarian granulosa cells was explored from the post-transcriptional level to further find the key biological effects and signaling pathways that may be mediated by them.Methods:1.21-day-old female SD rats were cultured with primary granulosa cells extracted from the ovaries and exposed to 2,5-HD(0 m M,60 m M)for 24 hours.Using miRNA and mRNA microarray technology,the miRNA and mRNA expression profiles of the two groups of cells were compared and analyzed to screen out differentially expressed mRNAs and mRNAs;2.The target genes of differentially expressed miRNAs were predicted using an online database to construct a miRNA-mRNA co-expression relationship network,and the target mRNAs in the co-expression network were further subjected to functional enrichment analysis,including GO analysis and KEGG analysis;3.Mi RNAs related to the cell cycle and Wnt pathway were screened,from which seven miRNAs were selected,and rat ovarian granulosa cells were exposed to 2,5-HD(0 mmol/L,20 mmol/L,40 mmol/L,and 60 mmol/L)for 24 h.Their expression was detected by RT-PCR to validate the miRNA microarray results.Results:1.According to the criteria of(Fold Change ≥ 2,P ≤ 0.05),a total of 5678 differentially expressed mRNAs were obtained by this sequencing,of which 2982 were up-regulated and 2696 were down-regulated;32 differentially expressed miRNAs,including 13 up-regulated and 19 down-regulated;2.miRNA-mRNA co-expression analysis1)Three databases,miRWalk,miRanda,and Target Scan,were used to predict target genes of DEMs to take an intersection with DEGs,respectively,and to screen mRNAs that showed a negative relationship with the corresponding miRNAs.A total of 262 target mRNAs included in the network were obtained after the results of the three databases were taken to intersect,and 368 miRNA-mRNA co-expression relationship pairs were formed with 27 miRNAs and visualized.2)The results of GO analysis-Biological Process analysis suggested that 3 of the top20 GO items enriched in target mRNAs in the co-expression network were related to the cell cycle(GO0006977,GO0007050,GO0051726),and 2 were related to the canonical Wnt pathway(GO0090090,GO0060070);3)The results of Pathway analysis showed that the target mRNAs in the co-expression network were significantly enriched to 46 signaling pathways,and the top30 pathways were obtained according to the value of-log(P-Value),with Wnt signaling pathway ranked 10 th and cell cycle ranked 23rd;3.Seven miRNAs were randomly selected from the 15 miRNAs that may be involved in regulating the cell cycle and Wnt signaling pathway for RT-PCR validation,and the results showed that the expression levels of rno-miR-3473 increased in all dose groups compared with the control group(P < 0.05),the relative expression levels of rno-miR-214-3p,rno-miR-138-5p,and rno-miR-199a-3p decreased in each dose group(P < 0.05),and the relative expression levels of rno-miR-145-5p and rno-miR-143-3p decreased in the 40 mmol/L and 60 mmol/L dose groups(P < 0.01),while the relative expression levels of rno-miR-196a-5p did not change significantly in each dose group.The results of PCR were generally consistent with those of miRNA microarray.Part Ⅱ Role and possible mechanism of Wnt/β-catenin pathway in 2,5-HD-induced ovarian granulosa cell cycle arrestObjective: Using in vitro exposure experiments,the effects of 2,5-HD on the cycle of rat ovarian granulosa cells were studied,and the role and possible mechanism of the Wnt/β-catenin pathway in it were further explored.Methods:1.After granulosa cells were exposed to 2,5-HD(0 mmol/L,20 mmol/L,40 mmol/L,60 mmol/L)for 24 h,the cycle distribution of granulosa cells and the expression levels of cell cycle-related gene mRNA were detected;2.After granulosa cells were exposed to 2,5-HD(0 mmol/L,20 mmol/L,40 mmol/L,and 60 mmol/L)for 24 h,the expression levels of mRNA and protein of genes related to the Wnt/β-catenin signaling pathway were detected;3.Twenty-four hours after exposure of human ovarian granuloma cells to 2,5-HD(0mmol/L,10 mmol/L,and 20 mmol/L),β-catenin/TCF transcriptional activity was measured by dual-luciferase reporter assay.Results:1.The results of flow cytometry showed that 24 hours after 2,5-HD exposure of ovarian granulosa cells,compared with the control group,the proportion of cells in G0/G1 phase increased in each dose group(P < 0.05),the proportion of cells in S phase decreased in each dose group(P < 0.05),and the proportion of cells in G2/M phase decreased in the 40 mmol/L and 60 mmol/L dose groups(P < 0.05);2.RT-PCR results showed that after 24 hours of 2,5-HD exposure,the mRNA expressions of P21,Cdk2,and Ccnd1 genes,which are closely related to the cell cycle,were significantly changed: the relative expression of P21 mRNA was increased in each dose group compared with the control group(P < 0.05);the relative expression of Cdk2 mRNA was decreased with the increase of exposure dose in each dose group(P < 0.05),and the relative expression of Ccnd1 mRNA was decreased in each dose group in a dose-dependent manner(P < 0.01);3.Expression of mRNA and protein of Wnt/β-catenin signaling pathway related genes1)RT-PCR results showed that the mRNA expression of all eight genes related to the Wnt/β-catenin signaling pathway changed after 24 hours of 2,5-HD exposure:compared with the control group,the relative mRNA expression of these four genes,Fzd1,Lrp6,Tcf3,and Tcf4,decreased with increasing exposure dose in each dose group(P < 0.001),and the relative mRNA expression of these four genes,Fzd6,Lrp5,β-catenin,and Lef1,decreased at doses of 40 mmol/L and 60 mmol/L(P < 0.05);2)Westernblot results showed that after 24 hours of 2,5-HD exposure,β-catenin protein expression levels decreased at doses of 40 mmol/L and 60 mmol/L compared with the control group(P < 0.05),and CCND1 protein expression levels decreased in a dose-dependent manner at each dose(P < 0.05);3)The results of cell immunofluorescence assay showed that the fluorescence expression intensity of β-catenin in the nucleus of granular cells was decreased at all doses compared with the control group after 24 hours of 2,5-HD exposure(P < 0.05),suggesting that 2,5-HD inhibited β-catenin nuclear translocation;4.After COV434 cells were exposed to 2,5-HD for 6 h,the results of the dual fluorescein reporter assay showed that the ratio of TOP/FOP significantly decreased with increasing exposure dose compared with the control group,that is,luciferase activity significantly decreased(P < 0.05),suggesting that 2,5-HD could lead to a decrease in β-catenin/TCF transcriptional activity.Conclusion:Under the conditions of this experiment,the following conclusions were drawn from this study:1.The miRNA and mRNA expression profiles of rat ovarian granulosa cells changed after 2,5-HD exposure,suggesting that epigenetic regulatory mechanisms such as miRNAs may be involved during 2,5-HD-induced ovarian granulosa cell injury.2.The results of functional enrichment analysis of mRNAs in the miRNA-mRNA co-expression network suggested that cell cycle and canonical Wnt signaling pathway regulation in ovarian granulosa cells may be mediated through miRNAs after 2,5-HD exposure.3.In vitro exposure to 2,5-HD can lead to cell cycle arrest in G0/G1 phase in ovarian granulosa cells.4.2,5-HD may inhibit the nuclear translocation of β-catenin by inducing Wnt/β-catenin pathway inhibition in ovarian granulosa cells,resulting in reducedβ-catenin/TCF transcriptional activity,reducing the expression of genes such as CCND1,a representative target of this pathway,which in turn induces cell cycle arrest in G0/G1 phase. |
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