Han Xiang, Shao Wang, Yue Zhou, Xing Liu, Shen Lianju, Long Chunlan, Zhang Deying, He Dawei, Lin Tao, Wei Guanghui
Chongqing Key Laboratory of Child Urogenital Development and Tissue Engineering, Chongqing 400014, China.
Department of Urology, Children's Hospital of Chongqing Medical University, Chongqing 400014, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2019 Apr 30;39(4):456-463. doi: 10.12122/j.issn.1673-4254.2019.04.12.
To investigate the transcriptome profile of genital tubercles (GTs) in male SD rats and explore the mechanism of hypospadias induced by Di (2-ethylhexyl) phthalate (DEHP).
Forty time-pregnant SD rats were randomly divided into 4 equal groups, namely GD16 group and GD19 group (in which the male GTs were collected on gestation day[GD]16 and GD19 for RNA-seq, respectively), control group and DEHP exposure group (with administration of oil and 750 mg/kg DEHP by gavage from GD12 to GD19, respectively).In the control and DEHP exposure groups, the GTs were collected from the male fetuses on GD19.5, and scanning electron microscopy and HE staining were used to observe the morphological changes.The differentially expressed genes (DEGs) in the GTs were screened using lllumina HiSeq 2000 followed by GO and KEGG enrichment analyses to characterize the transcriptome profile.Immunofluorescence assay was performed to verify the DEGs (Mafb) identified by RNA-seq results.Immunofluorescence assay and Western blotting were used to examine the expression levels of Mafb in the penile tissue.
A total of 1360 DEGs were detected in the GTs between GD16 group and GD19 group by RNA-seq.Among these genes, 797 were up-regulated and 563 were down-regulated.These DEGs were mainly enriched in the cell adhesion plaque signaling pathway, axon guidance signaling pathway, and extracellular matrix receptor signaling pathway.Compared with that in GD16 group, Mafb was significantly up-regulated in GD19 group, which was consistent with the sequencing results.Mafb and β-catenin were significantly down-regulated in DEHP-exposed group compared with the control group ( < 0.01).
Mafb expression increases progressively with the development of GTs in male SD rats.DEHP exposure causes significant down-regulation of Mafb and β-catenin, suggesting that β-catenin signaling pathway that affects Mafb is related to DEHP-induced hypospadias in SD rats.
研究雄性SD大鼠生殖结节（GTs）的转录组图谱，探讨邻苯二甲酸二（2-乙基己基）酯（DEHP）诱导尿道下裂的机制。
将40只孕龄期SD大鼠随机分为4组，即GD16组和GD19组（分别在妊娠第16天和第19天收集雄性GTs用于RNA测序）、对照组和DEHP暴露组（分别从妊娠第12天至第19天通过灌胃给予油剂和750 mg/kg DEHP）。在对照组和DEHP暴露组中，于妊娠第19.5天收集雄性胎儿的GTs，采用扫描电子显微镜和HE染色观察形态学变化。使用Illumina HiSeq 2000筛选GTs中的差异表达基因（DEGs），随后进行GO和KEGG富集分析以表征转录组图谱。进行免疫荧光分析以验证RNA测序结果鉴定出的DEGs（Mafb）。采用免疫荧光分析和蛋白质免疫印迹法检测阴茎组织中Mafb的表达水平。
通过RNA测序在GD16组和GD19组的GTs中总共检测到1360个DEGs。在这些基因中，797个上调，563个下调。这些DEGs主要富集于细胞黏附斑信号通路、轴突导向信号通路和细胞外基质受体信号通路。与GD16组相比，Mafb在GD19组中显著上调，这与测序结果一致。与对照组相比，DEHP暴露组中Mafb和β-连环蛋白显著下调（<0.01）。
雄性SD大鼠GTs的发育过程中Mafb表达逐渐增加。DEHP暴露导致Mafb和β-连环蛋白显著下调，提示影响Mafb的β-连环蛋白信号通路与DEHP诱导的SD大鼠尿道下裂有关。
