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Single-cell RNA-seq analysis of regulatory role of Gm4665 in spermatogenesis

ABSTRACT: Purpose: To understand the likely mechanisms behind the effects of candidate lncRNA knockdown, we applied single-cell RNA-seq (scRNA-seq) to decipher transcriptional alterations of affected germ cells induced by Gm4665 knockdown. Methods: We removed somatic cells and isolated RFP+ germ cells infected with AAV9-RFP from shCtrl or Gm4665-depleted testes by FACS, before performing scRNA-seq with the Chromium system (10x Genomics). For scRNA-seq, the gene cell barcode matrix was filtered based on the number of genes detected per cell (any cells with fewer than 500 or more than 4000 genes per cell were filtered out) and the percentage of mitochondrial UMI counts (any cells with more than 10% of mitochondrial UMI counts were filtered out). Results: We separately analyzed the differentially expressed genes (DEG) induced by Gm4665 knockdown in rST and elST populations. This demonstrated that Gm4665 knockdown resulted in increased expression of 190 genes and decreased expression of 328 genes in rST, and increased expression of 135 genes and decreased expression of 35 genes in elST. Conclusions: Single-cell transcriptional analysis of male germ cells indicated the functional requirement of Gm4665 for multiple aspects of spermatid differentiation during spermiogenesis.

ORGANISM(S): Mus musculus

PROVIDER: GSE161511 | GEO | 2020/11/16

REPOSITORIES: GEO

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Project description:Pancreatic cancer is a complex disease with a desmoplastic stroma, extreme hypoxia, and inherent resistance to therapy. Understanding the signaling and adaptive response of such an aggressive cancer is key to making advances in therapeutic efficacy and understanding disease progression. Redox factor-1 (Ref-1), a redox signaling protein, regulates the DNA binding activity of several transcription factors, including HIF-1. The conversion of HIF-1 from an oxidized to reduced state leads to enhancement of its DNA binding. In our previously published work, knockdown of Ref-1 under normoxia resulted in altered gene expression patterns on pathways including EIF2, protein kinase A, and mTOR. In this study, single cell RNA sequencing (scRNA-seq) and proteomics were used to explore the effects of Ref-1 on metabolic pathways under hypoxia.Results: We also integrated the scRNA data analysis with the proteomic analysis and found that the differentially expressed genes and pathways identified from the scRNA-seq data are highly consistent to the significant proteins observed in the proteomics data, especially for the upregulated cell cycle and transcription pathways and downregulated metabolic, apoptosis and signaling pathways under hypoxia. Conclusion: The scRNA-seq and proteomics data consistently demonstrated down-regulated central metabolism pathways in APE1/Ref-1 knockdown vs scrambled control under both normoxia and hypoxia conditions. Experimental Methods: scRNA-seq comparing pancreatic cancer cells expressing less than 20% of the Ref-1 protein was analyzed using left truncated mixture Gaussian model. Matched samples were also collected for bulk proteomic analysis of the four conditions. scRNA-seq data was validated using proteomics and qRT-PCR. Ref-1’s role in mitochondrial function was confirmed using mitochondrial function assays and qRT-PCR. Results: We also integrated the scRNA data analysis with the proteomic analysis and found that the differentially expressed genes and pathways identified from the scRNA-seq data are highly consistent to the significant proteins observed in the proteomics data, especially for the upregulated cell cycle and transcription pathways and downregulated metabolic, apoptosis and signaling pathways under hypoxia. Conclusion: The scRNA-seq and proteomics data consistently demonstrated down-regulated central metabolism pathways in APE1/Ref-1 knockdown vs scrambled control under both normoxia and hypoxia conditions.

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Single-cell RNA-seq analysis of regulatory role of Gm4665 in spermatogenesis

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