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

ABSTRACTSpermatozoa 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.KEYWORDS: Epididymal segmentexpression distributionsingle cell level AcknowledgementsWe would like to acknowledge Linlin Tian from Nanjing Municipal Center for Disease Control and Prevention for the proof reading of this manuscript.Disclosure statementNo potential conflict of interest was reported by the author(s).Supplementary materialSupplemental data for this article can be accessed online at https://doi.org/10.1080/15384101.2023.2280170Author contributionsConceptualization: Ninghong Song and Zengjun Wang; Study design: Ninghong Song and Tong Chen; Analysis and visualization of data: Tong Chen, Liangyu Yao, Wen Liu, Jiaochen Luan and Xiang Zhou; Manuscript writing: Tong Chen, Yichun Wang and Chao Yang; Scientific review: Xuejiang Guo and Chengjian Ji.Data availability statementAll data series used in this study are available in online repository. The names of the repository and accession number of the included data series can be found in Table S1.Ethics statementThis study design was identified and approved by the medical research ethics committee of The First Affiliated Hospital of Nanjing Medical University and was therefore conducted according to ethical standards laid down in the 1964 Declaration of Helsinki and its amendments.Additional informationFundingThis study was funded by the National Natural Science Foundation of China (81871151 and 82071638) and Jiangsu Funding Program for Excellent Postdoctoral Talent (2022ZB730).