雄性大鼠生殖结节中相关lncRNA和mRNA可能参与邻苯二甲酸二丁酯诱导的尿道下裂形成过程

LncRNA and mRNA in the genital tubercle of male rats may be involved in the formation of di-n-butyl phthalate-induced hypospadias

目的:探讨雄性大鼠生殖结节中长链非编码RNA(long non-codingRNA,lncRNA)和mRNA在邻苯二甲酸二丁酯(din-butyl phthalate,DBP)诱导雄性大鼠尿道下裂形成中的可能作用。方法:在孕鼠孕13~18 d时每天按体重800 mg/kg DBP溶于1 m L玉米油灌胃,对照组每天给予1 m L玉米油。于孕19 d时取出胎鼠,分别取下灌药组发生尿道下裂胎鼠和对照组雄性胎鼠的生殖结节,提取RNA,构建RNA测序文库测序。对测序结果的lncRNA进行筛选、差异分析和功能预测,再随机筛选8条lncRNA用q RT-PCR验证其表达量。结果:与对照组相比,尿道下裂组生殖结节中有598个差异mRNA,427个差异lncRNA,q RTPCR结果与测序结果一致。在生物过程、细胞组成、分子功能方面,差异表达mRNA富集程度最高的分别是红细胞生长、血红蛋白合成、结合珠蛋白结合。分布最多的的信号通路为病毒性心肌炎通路。差异lncRNA功能最多分布在生长激素释放激素受体的结合;分布最多的信号通路为金葡菌感染通路。结论:邻苯二甲酸二丁酯诱导的尿道下裂雄性大鼠与正常雄性大鼠相比,其生殖结节中lncRNA和mRNA的差异表达显著,lncRNA可能通过与mRNA相互作用参与尿道下裂的形成过程。

Objective：To investigate the expression of long non-coding RNA（lncRNA）and mRNA in the genital tubercle of hypospadiac rats induced by di-n-butyl phthalate（DBP）. Methods：We placed male and female rats with a ratio of 1∶1 in the same cage and then checked if there was white glue frosting vaginal suppository in the next day to determine the female rats mating successfully,confirmed this day as the first day of pregnancy. By giving rats 800 mg/kg DBP dissolved in 1 mL corn oil in the 13 to 18 days of pregnancy to establish model of hypospadias,the pregnant rats in the control group were given only 1 mL/d corn oil. Fetal rats were taken out from pregnant rats for 19 days of pregnancy. The meatus of urethra was observed with an anatomical microscope to find if male fetal rats developed hypospadias. Then,we removed the genital tubercle of male fetal rats and extracted total RNA in it. RNA sequencing library was constructed and then sequenced with sequencing machine. The sequencing result of lncRNA was analyzed and screened,and function prediction was performed. Finally,we chose eight lncRNAs randomly to validate their level of expression by qRT-PCR. Results：The sequencing results showed that compared with the control group,the hypospadias group had 598 differential expression mRNA,427 differential expression lncRNAs. qRT-PCR results were consistent with the results of sequencing. In the biological process,cell component and molecular function,the most differential expressions of mRNA were related to erythrocyte development,extracellular exosome and haptoglobin binding. The most mRNAs were related to the signaling pathways of viral myocarditis. LncRNAs function prediction showed that the most lncRNAs played in combination of growth hormone releasing hormone receptor;The most lncRNAs were related to the signaling pathways of staphylococcus aureus infection. Conclusion：Compared with normal male rats,the male rats of hypospadias induced by DBP have changed the level of expression of lncRNA and mRNA in the genital tubercle significantly. LncRNA may involved in the formation process of hypospadias and interfered development of genital tubercle by interacting with mRNA.