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RNA-seq analysis of mitochondria-related genes regulated by AMPK in the human trophoblast cell line BeWo
Bin Wu1, Albert Gao2, Bin He3
1Department of Reproductive Medicine, Central Hospital Affiliated to Shandong First Medical University, Jinan, Shandong, P.R. China.
Background:
How AMP activated protein kinase (AMPK) signaling regulates mitochondrial functions and mitophagy in human trophoblast cells remains unclear. This study was designed to investigate potential players mediating the regulation of AMPK on mitochondrial functions and mitophagy by next generation RNA-seq.
Methods:
We compared ATP production in protein kinase AMP-activated catalytic subunit alpha 1/2 (PRKAA1/2) knockdown (AKD) and control BeWo cells using the Seahorse real-time ATP rate test, then analyzed gene expression profiling by RNA-seq. Differentially expressed genes (DEG) were examined by Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment. Then protein-protein interactions (PPI) among mitochondria related genes were further analyzed using Metascape and Ingenuity Pathway Analysis (IPA) software.
Results:
Both mitochondrial and glycolytic ATP production in AKD cells were lower than in the control BeWo cells (CT), with a greater reduction of mitochondrial ATP production. A total of 1092 DEGs were identified, with 405 upregulated and 687 downregulated. GO analysis identified 60 genes associated with the term 'mitochondrion' in the cellular component domain. PPI analysis identified three clusters of mitochondria related genes, including aldo-keto reductase family 1 member B10 and B15 (AKR1B10, AKR1B15), alanyl-tRNA synthetase 1 (AARS1), mitochondrial ribosomal protein S6 (MRPS6), mitochondrial calcium uniporter dominant negative subunit beta (MCUB) and dihydrolipoamide branched chain transacylase E2 (DBT).
Conclusions:
In summary, this study identified multiple mitochondria related genes regulated by AMPK in BeWo cells, and among them, three clusters of genes may potentially contribute to altered mitochondrial functions in response to reduced AMPK signaling.
Insights
AMP-activated protein kinase (AMPK) signaling impacts mitochondrial function in human trophoblast cells. This study used RNA-seq to identify key genes regulated by AMPK, revealing potential mechanisms for altered mitochondrial ATP production.
Area of Science:
- Cell Biology
- Mitochondrial Biology
- Molecular Signaling
Background:
- AMP-activated protein kinase (AMPK) signaling's role in human trophoblast mitochondrial function and mitophagy is not well understood.
- Investigating AMPK's regulatory mechanisms is crucial for understanding trophoblast cell physiology.
Purpose of the Study:
- To elucidate how AMPK signaling regulates mitochondrial functions and mitophagy in human trophoblast cells.
- To identify novel genes and pathways involved in AMPK-mediated mitochondrial regulation using next-generation RNA sequencing.
Main Methods:
- Comparison of ATP production between protein kinase AMP-activated catalytic subunit alpha 1/2 (PRKAA1/2) knockdown (AKD) and control BeWo cells via Seahorse analysis.
- RNA sequencing (RNA-seq) to profile gene expression, followed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis.
- Protein-protein interaction (PPI) network analysis using Metascape and Ingenuity Pathway Analysis (IPA) for mitochondria-related genes.
Main Results:
- AKD cells exhibited significantly reduced mitochondrial and glycolytic ATP production compared to controls, with a more pronounced decrease in mitochondrial ATP.
- RNA-seq identified 1092 differentially expressed genes (DEGs), including 405 upregulated and 687 downregulated genes.
- GO analysis highlighted 60 mitochondrion-associated genes. PPI analysis revealed three gene clusters, including AKR1B10, AKR1B15, AARS1, MRPS6, MCUB, and DBT, potentially involved in mitochondrial regulation.
Conclusions:
- AMPK signaling significantly influences mitochondrial function in BeWo cells.
- Multiple mitochondria-related genes are regulated by AMPK, with identified gene clusters potentially mediating altered mitochondrial function under reduced AMPK signaling.
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