Epididymal segment-specific miRNA and mRNA regulatory network at the single cell level

Spermatozoa released from the testis cannot fertilize an egg before becoming mature and motile in the epididymis. Based on three bulk and one single-cell RNA-seq (scRNA-seq) data series, we compared mRNA or miRNA expression between epididymal segment-specific samples and the other samples. Hereby, we identified 570 differentially expressed mRNAs (DE-mRNAs) and 23 differentially expressed miRNAs (DE-miRNAs) in the caput, 175 DE-mRNAs and 15 DE-miRNAs in the corpus, 946 DE-mRNAs and 12 DE-miRNAs in the cauda. In accordance with respective DE-miRNAs, we predicted upstream transcription factors (TFs) and downstream target genes. Subsequently, we intersected target genes of respective DE-miRNAs with corresponding DE-mRNAs, thereby obtaining 127 upregulated genes in the caput and 92 upregulated genes in cauda. Enriched upregulated pathways included cell motility-related pathways for the caput, smooth muscle-related pathways for the corpus, and immune-associated pathways for the cauda. Protein–protein interaction (PPI) network was constructed to extract key module for the caput and cauda, followed by identifying hub genes through cytohubba. Epididymis tissues from six mice were applied to validate hub genes expression using qRT-PCR, and 7 of the 10 genes displayed identical expression trends in mice caput/cauda. These hub genes were found to be predominantly distributed in spermatozoa using scRNA-seq data. In addition, target genes of DE-miRNAs were intersected with genes in the PPI network for each segment. Subsequently, the miRNA and mRNA regulatory networks for the caput and cauda were constructed. Conclusively, we uncover segment-specific miRNA-mRNA regulatory network, upstream TFs, and downstream pathways of the human epididymis, warranting further investigation into epididymal segment-specific functions.

从睾丸释放的精子若未在附睾中成熟并获得运动能力，则无法完成卵子受精。本研究基于3套批量RNA测序（bulk RNA-seq）与1套单细胞RNA测序（scRNA-seq）数据集，对附睾节段特异性样本与其余样本的mRNA或miRNA表达谱进行了比较分析。最终在附睾头（caput）中鉴定出570个差异表达mRNA（DE-mRNAs）与23个差异表达miRNA（DE-miRNAs），在附睾体（corpus）中鉴定出175个DE-mRNAs与15个DE-miRNAs，在附睾尾（cauda）中鉴定出946个DE-mRNAs与12个DE-miRNAs。基于各节段的差异表达miRNA，我们预测了其上游转录因子（TFs）与下游靶基因。随后将各节段差异表达miRNA的靶基因与对应节段的差异表达mRNA取交集，分别得到附睾头的127个上调基因与附睾尾的92个上调基因。上调通路富集分析结果显示：附睾头的上调通路主要参与细胞运动过程，附睾体的上调通路主要涉及平滑肌功能，附睾尾的上调通路则与免疫应答相关。我们构建了蛋白质-蛋白质相互作用（PPI）网络并提取附睾头与附睾尾的关键模块，通过CytoHubba工具鉴定核心基因（hub genes）。本研究选取6只小鼠的附睾组织，通过实时定量逆转录PCR（qRT-PCR）验证核心基因的表达水平，10个候选基因中有7个在小鼠附睾头/尾中呈现一致的表达趋势。借助scRNA-seq数据集分析发现，这些核心基因主要分布于精子细胞中。此外，我们将各节段差异表达miRNA的靶基因与对应PPI网络中的基因取交集，分别构建了附睾头与附睾尾的miRNA-mRNA调控网络。本研究最终揭示了人类附睾节段特异性的miRNA-mRNA调控网络、上游转录因子及下游通路，为后续深入探究附睾节段特异性功能提供了重要依据。