双酚A对小鼠睾丸间质细胞毒性及miR-203-3p表达的影响
Effects of bisphenol A on mouse Leydig cells and miR-203-3p expression
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摘要:
背景 双酚A（BPA）作为一种重要的增塑剂原料被广泛应用，其对机体健康产生的损伤作用越来越被人们所重视；而生殖毒性是损伤作用中的重要一方面，研究BPA所导致的生殖损伤及其机制已成时下热点。微小RNA（miRNA）的生物学作用及早期损伤标志的意义也被人们所重视。
目的 研究BPA对小鼠睾丸间质细胞毒性及miR-203-3p表达的影响。
方法 通过不同浓度的BPA（0、1、10、100、1 000 μmol/L）处理小鼠睾丸间质细胞（TM3）24 h后，运用CCK-8法检测细胞活力情况，采用流式细胞术检测细胞凋亡指标；应用realtime PCR对miR-203-3p的表达水平进行检测。然后通过TargetScan数据库及miRDB数据库对miR-203-3p的靶基因进行预测，并通过DAVID数据库对靶基因情况进行GO分析及Pathway分析。
结果 随着染毒浓度增加，各组的T M 3细胞活力出现不同程度的下降趋势，但仅1 000 μmol/L浓度组与对照组差异具有统计学意义（P < 0.05）；与对照组相比，1 000 μmol/L组的细胞总凋亡率（31.33±7.12）%升高（P < 0.05）；miRNA的检测结果显示，与对照组相比，miR-203-3p在1、100 μmol/L两个浓度组出现升高（均P < 0.05）；而当浓度为1 000 μmol/L时，该miRNA表达水平出现了下降（P < 0.05）。通过两个数据库生物信息学预测的其可能调控靶基因为278个；通过David数据库进行GO分析的富集结果显示，排在第一位的生物过程、分子功能和细胞组分分别是转录DNA模板、蛋白结合和核；Pathway分析富集的信号通路排在首位的是FoxO信号通路。
结论 不同浓度的BPA可以导致小鼠睾丸间质细胞出现不同程度的损伤现象，这种损伤现象可能与miR-203-3p的改变有关，且该miRNA可能成为重要的早期损伤检测指标。
Abstract:Background Bisphenol A (BPA) is widely used as an important plasticizer raw material, and its adverse health effects have raised concerns. BPA induced reproductive toxicity and related mechanism of action are research hotspots. The biological effects of microRNAs (miRNA) and their significance as potential early damage markers have attracted much attention.
Objective This study is conducted to investigate the cytotoxicity of BPA on mouse Leydig cells and the expression of miR-203-3p.
Methods Mouse Leydig cells (TM3) were treated with BPA at different concentrations (0, 1, 10, 100, 1 000 μmol/L) for 24 h, and detected for cell viability by CCK-8, apoptosis by flow cytometry, and the expression levels of miR-203-3p by real-time PCR. The target genes of miR-203-3p were predicted by TargetScan database and miRDB database, and the GO analysis and Pathway analysis of the target genes were conducted using DAVID database.
Results With higher exposure concentrations, the viability of TM3 cells in each group showed decreasing trends, but only the viability change in the 1 000 μmol/L group had a significant reduction compared with that in the control group (P < 0.05). The apoptosis rate in the 1 000 μmol/L group(31.33±7.12)% was significantly higher than that in the control group (P < 0.05). The results of miRNA detection showed that compared with the control group, miR-203-3p expression increased at 1 μmol/L and 100 μmol/L BPA (Ps < 0.05), but decreased at 1 000 μmol/L BPA (P < 0.05). There were 278 possible regulatory target genes predicted by TargetScan database and miRDB database. According to the results of GO enrichment analysis using David database, transcriptional DNA template ranked first in biological process, protein binding ranked first in molecular function, and nucleus ranked first in cell components. According to the results of Pathway analysis, Fox0 was the most abundant pathway.
Conclusion Different concentrations of BPA could cause varied degrees of damage to mouse Leydig cells, which may be related to the changes of miR-203-3p expression. Moreover, this miRNA may be an important early damage marker.
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