miR-344b-3p靶向Tspo基因参与双酚S诱导的小鼠睾丸合成睾酮功能异常  

miR-344b-3p Targeted Tspo Gene is Involved in Bisphenol S-induced Testosterone Synthesis Abnormal in Mouse

作　　者：孙胜琪 姜晓 周洋西 刘晋祎 杨惠芳 曹佳 SUN Shengqi;JIANG Xiao;ZHOU Yangxi;LIU Jinyi;YANG Huifang;CAO Jia(School of Public Health,Ningxia Medical University,Ningxia Key Laboratory of Environmental Factors and Chronic Disease Control,Yinchuan 750004,China;Institute of Toxicology,Faculty of Military Preventive Medicine,Army Medical University,Chongqing 400037,China)

机构地区：[1]宁夏医科大学公共卫生学院,宁夏环境因素与慢性病控制重点实验室,银川750004 [2]陆军军医大学军事预防医学系毒理学研究所,重庆400037

出　　处：《宁夏医科大学学报》2024年第5期433-439,共7页Journal of Ningxia Medical University

基　　金：国家重点研发计划项目(2022YFC2702902);重庆自然科学基金项目(cstc2021jcyj-msxmX0316)。

摘　　要：目的探讨双酚S(bisphenol S,BPS)影响小鼠睾酮合成异常的作用及机制。方法构建浓度为0、2、10、50 mg·kg^(-1) BPS染毒小鼠模型,采用HE染色观察其睾丸组织的形态、计算机辅助精子分析系统(CASA)分析精子活力、ELISA检测血清睾酮水平的变化。采用RT-qPCR和Western blot检测睾酮生成关键基因mRNA和蛋白的表达水平。利用哺乳动物miRNA靶基因数据库(miRDB)预测筛选出BPS抑制睾酮合成靶基因的上游miRNA,通过RT-qPCR检测BPS染毒小鼠睾丸组织中候选miRNA的表达水平。进一步构建候选miRNA的Inhibitor/Mimics转染TM3细胞模型,并在此基础上进行BPS染毒,检测预测靶基因的表达及蛋白水平变化,确定最终发挥作用的上游miRNA。结果通过构建不同浓度BPS染毒小鼠模型发现,BPS暴露35 d后可导致小鼠睾丸组织病理损伤,精子活力和血清睾酮水平均下降(P均<0.05)。RT-qPCR和Western blot结果提示,Tspo和Cyp11a1是BPS诱导小鼠睾酮生成下降的靶分子(P<0.01)。通过miRDB数据库,筛选出睾酮合成靶基因上游miRNA分别为miR-344b-3p和miR-124-5p。在BPS暴露小鼠模型中发现,BPS染毒组小鼠睾丸组织中miR-344b-3p和miR-124-5p的表达较对照组均上调(P均<0.01)。用Inhibitor/Mimics转染TM3细胞发现,miR-344b-3p的靶基因Tspo得到明显恢复/抑制(P<0.05),在转染Mimics的基础上再进行BPS处理后发现,Tspo的基因表达及蛋白水平进一步降低(P<0.05)。结论BPS可能通过诱导miR-344b-3p抑制其靶基因Tspo的表达参与导致小鼠睾丸合成睾酮功能下降。Objective To explore the role and mechanism of bisphenol S(BPS)affecting testosterone synthesis in mice.Methods Mouse models of BPS toxicity at concentrations of 0,2,10,and 50 mg·kg^(-1) were constructed.HE staining was used to observe the morphology of their testicular tissues,CASA was applied to analyze the sperm viability,and ELISA was used to detect the changes in serum testosterone levels.RT-qPCR and Western blot were used to detect the expression levels of mRNAs and proteins of key genes for testosterone production.The upstream miRNAs of the target genes of testosterone synthesis inhibition by BPS were predicted and screened using the mammalian miRNA target gene database(miRDB),and the expression levels of the candidate miRNAs were detected by RT-qPCR in testicular tissues and TM3 cells of BPS-exposed mice.The Inhibitor/Mimics of candidate miRNAs were further constructed to transfect the TM3 cell model,and on this basis,BPS exposure treatment was performed to detect the expression of the predicted target genes and changes in protein levels to identify the upstream miRNAs.Results By constructing mouse models with different concentrations of BPS exposure,it was found that BPS exposure for 35 days resulted in histopathological damage to the testis,decreased sperm viability and decreased serum testosterone levels in mice(P all<0.05).The combined RT-qPCR and Western blot results suggested that Tspo and Cyp11a1 were the target molecules of BPS-induced decrease in testosterone production in mice(P<0.01).The upstream miRNAs of testosterone synthesis target genes were screened by miRDB database as miR-344b-3p and miR-124-5p,respectively.miR-344b-3p and miR-124-5p expression levels were found to be significantly up-regulated in BPS-treated groups compared with the control group in both in vivo and ex vivo models(P all<0.01).Inhibitors and mimics of transfected miRNAs revealed that miR-344b-3p could regulate Tspo gene expression(P<0.05).Further treatment with BPS found that transfection of mimics could suppress the e

关 键 词：双酚S 睾酮 miR-344b-3p 生殖损害 

分 类 号：R12[医药卫生—环境卫生学]