Next Generation Sequencing Facilitates Quantitative Analysis of testis Transcriptomes in Xenopus laevis

Purpose: Circular RNAs (circRNAs) are implicated in multiple developmental anomalies. The goals of this study are to research whether circRNAs involve in the effects in atrazine (AZ)-exposed X. laevis Methods: At Nieuwkoop-Faber (NF) stage 47 (13 d post-hatch), mixed sex X. laevis tadpoles exposed to 100 µg/L AZ for 180 days. Then total RNAs from individual testis was isolated. RNA library construction and circRNA sequencing was analysis Results: CircRNAs were detected and identified with Find_circ software. We found that treatment of AZ led to 405 circRNAs differentially expressed including 44 upregulated and 361 downregulated compared with froglets in the control groups. Two hundreds and eighty two differentially expressed circRNAs played miRNA sponges roles. Kyoto Encyclopedia of Genes and Genomes pathway analysis of miRNA targets showed that AZ-affected circRNAs are mainly involved in 19 pathways Conclusions: This study for the fisrt time provides evidence that AZ can alter circRNAs which play a role in AZ-induced testicular degeneration of developing male X. laevis through regulation of expressions of functional genes in the testes of X. laevis. Overall design: Examing circRNAs of testes of X. laevis from 2 conditions including AZ-treated and control, each with 3 replicates, using Illumina HiSeq 4000

研究目的：环状RNA（circular RNAs, circRNAs）参与多种发育异常的发生过程。本研究旨在探究环状RNA是否介导阿特拉津（atrazine, AZ）暴露对非洲爪蟾（X. laevis）的影响效应。
研究方法：在Nieuwkoop-Faber（NF）47期（孵化后13天），将混合性别非洲爪蟾蝌蚪暴露于100 μg/L阿特拉津中180天。随后分离单个个体睾丸的总RNA，构建RNA文库并开展环状RNA测序分析。
研究结果：使用Find_circ软件检测并鉴定环状RNA。与对照组幼蛙相比，阿特拉津处理组共筛选得到405个差异表达环状RNA，其中44个表达上调、361个表达下调。其中282个差异表达环状RNA可发挥miRNA海绵（miRNA sponge）的调控作用。对miRNA靶基因进行京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）通路富集分析显示，受阿特拉津影响的环状RNA主要参与19条信号通路。
研究结论：本研究首次提供证据表明，阿特拉津可通过调控非洲爪蟾睾丸内功能基因的表达，改变环状RNA的表达谱，进而参与阿特拉津诱导的发育中雄性非洲爪蟾睾丸退行性病变。
整体实验设计：设置阿特拉津处理组与对照组两个实验组，分别采集非洲爪蟾睾丸组织进行环状RNA检测，每组设置3个生物学重复，采用Illumina HiSeq 4000测序平台完成测序。