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Transcriptome and Proteome Profiling of Primary Human Gastric Interstitial Cells of Cajal Predicts Pacemaker Networks.
Journal of Neurogastroenterology and Motility 2023 April 31
BACKGROUND/AIMS: Interstitial cells of Cajal (ICC) are specialized gastrointestinal (GI) pacemaker cells required for normal GI motility. Dysfunctions in ICC have been reported in patients with GI motility disorders, such as gastroparesis, who exhibit debilitating symptoms and greatly reduced quality of life. While the proteins, calcium-activated chloride channel anoctamin-1 (ANO1) and the receptor tyrosine kinase (KIT), are known to be expressed by human ICC, relatively little is known about the broad molecular circuitry underpinning human ICC functions. The present study therefore investigates the transcriptome and proteome of ANO1-expressing, KITlow /CD45- /CD11B- ICC obtained from primary human gastric tissue.
METHODS: Excess human gastric tissue resections were obtained from sleeve gastrectomy patients. ICC were purified using fluorescence-activated cell sorting (FACSorting). Then, ICC were characterized by using immunofluorescence, real-time polymerase chain reaction, RNA-sequencing and mass spectrometry.
RESULTS: Compared to unsorted cells, real-time polymerase chain reaction showed the KITlow /CD45- /CD11B- ICC had: a 9-fold ( P < 0.05) increase in ANO1 expression; unchanged KIT expression; and reduced expression for genes associated with hematopoietic cells (CD68, > 10-fold, P < 0.001) and smooth muscle cells (DES, > 4-fold, P < 0.05). RNA-sequencing and gene ontology analyses of the KITlow /CD45- /CD11B- cells revealed a transcriptional profile consistent with ICC function. Similarly, mass spectrometry analyses of the KITlow /CD45- /CD11B- cells presented a proteomic profile consistent with ICC activities. STRING-based protein interaction analyses using the RNA-sequencing and proteomic datasets predicted protein networks consistent with ICC-associated pacemaker activity and ion transport.
CONCLUSION: These new and complementary datasets provide a valuable molecular framework for further understanding how ICC pacemaker activity regulates smooth muscle contraction in both normal GI tissue and GI motility disorders.
METHODS: Excess human gastric tissue resections were obtained from sleeve gastrectomy patients. ICC were purified using fluorescence-activated cell sorting (FACSorting). Then, ICC were characterized by using immunofluorescence, real-time polymerase chain reaction, RNA-sequencing and mass spectrometry.
RESULTS: Compared to unsorted cells, real-time polymerase chain reaction showed the KITlow /CD45- /CD11B- ICC had: a 9-fold ( P < 0.05) increase in ANO1 expression; unchanged KIT expression; and reduced expression for genes associated with hematopoietic cells (CD68, > 10-fold, P < 0.001) and smooth muscle cells (DES, > 4-fold, P < 0.05). RNA-sequencing and gene ontology analyses of the KITlow /CD45- /CD11B- cells revealed a transcriptional profile consistent with ICC function. Similarly, mass spectrometry analyses of the KITlow /CD45- /CD11B- cells presented a proteomic profile consistent with ICC activities. STRING-based protein interaction analyses using the RNA-sequencing and proteomic datasets predicted protein networks consistent with ICC-associated pacemaker activity and ion transport.
CONCLUSION: These new and complementary datasets provide a valuable molecular framework for further understanding how ICC pacemaker activity regulates smooth muscle contraction in both normal GI tissue and GI motility disorders.
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