E Bonora1, F Bianco1,2, A Stanzani1,2, F Giancola1,2,3, A Astolfi4, V Indio4, C Evangelisti5, A M Martelli5, E Boschetti1,3, M Lugaresi1, A Ioannou1, F Torresan6, V Stanghellini1, P Clavenzani2, M Seri1, A Moonen7, K Van Beek7, M Wouters7, G E Boeckxstaens7, G Zaninotto8, S Mattioli1, R De Giorgio9. 1. Department of Medical and Surgical Sciences, DIMEC, University of Bologna and St. Orsola-Malpighi Hospital, Bologna, Italy. 2. Department of Medical and Veterinary Sciences, DIMEVET, University of Bologna, Bologna, Italy. 3. Centro di Ricerca Biomedica Applicata, St.Orsola-Malpighi Hospital, Bologna, Italy. 4. Interdepartmental Center for Cancer Research "G. Prodi" (CIRC), University of Bologna, Bologna, Italy. 5. Department of Experimental Medicine, DIMES, University of Bologna, Bologna, Italy. 6. Department of Digestive System, St. Orsola-Malpighi Hospital, Bologna, Italy. 7. Translational Research in GastroIntestinal Disorders (TARGID), Department of Clinical and Experimental Medicine, KU Leuven University, Leuven, Belgium. 8. Division of Surgery, Imperial College London, London, UK. 9. Department of Medical Sciences, Nuovo Arcispedale S.Anna at Cona (Ferrara), University of Ferrara, Ferrara, Italy.
Abstract
BACKGROUND: Achalasia is a rare motility disorder characterized by myenteric neuron and interstitial cells of Cajal (ICC) abnormalities leading to deranged/absent peristalsis and lack of relaxation of the lower esophageal sphincter. The mechanisms contributing to neuronal and ICC changes in achalasia are only partially understood. Our goal was to identify novel molecular features occurring in patients with primary achalasia. METHODS: Esophageal full-thickness biopsies from 42 (22 females; age range: 16-82 years) clinically, radiologically, and manometrically characterized patients with primary achalasia were examined and compared to those obtained from 10 subjects (controls) undergoing surgery for uncomplicated esophageal cancer (or upper stomach disorders). Tissue RNA extracted from biopsies of cases and controls was used for library preparation and sequencing. Data analysis was performed with the "edgeR" option of R-Bioconductor. Data were validated by real-time RT-PCR, western blotting and immunohistochemistry. KEY RESULTS: Quantitative transcriptome evaluation and cluster analysis revealed 111 differentially expressed genes, with a P ≤ 10-3 . Nine genes with a P ≤ 10-4 were further validated. CYR61, CTGF, c-KIT, DUSP5, EGR1 were downregulated, whereas AKAP6 and INPP4B were upregulated in patients vs controls. Compared to controls, immunohistochemical analysis revealed a clear increase in INPP4B, whereas c-KIT immunolabeling resulted downregulated. As INPP4B regulates Akt pathway, we used western blot to show that phospho-Akt was significantly reduced in achalasia patients vs controls. CONCLUSIONS & INFERENCES: The identification of altered gene expression, including INPP4B, a regulator of the Akt pathway, highlights novel signaling pathways involved in the neuronal and ICC changes underlying primary achalasia.
BACKGROUND:Achalasia is a rare motility disorder characterized by myenteric neuron and interstitial cells of Cajal (ICC) abnormalities leading to deranged/absent peristalsis and lack of relaxation of the lower esophageal sphincter. The mechanisms contributing to neuronal and ICC changes in achalasia are only partially understood. Our goal was to identify novel molecular features occurring in patients with primary achalasia. METHODS: Esophageal full-thickness biopsies from 42 (22 females; age range: 16-82 years) clinically, radiologically, and manometrically characterized patients with primary achalasia were examined and compared to those obtained from 10 subjects (controls) undergoing surgery for uncomplicated esophageal cancer (or upper stomach disorders). Tissue RNA extracted from biopsies of cases and controls was used for library preparation and sequencing. Data analysis was performed with the "edgeR" option of R-Bioconductor. Data were validated by real-time RT-PCR, western blotting and immunohistochemistry. KEY RESULTS: Quantitative transcriptome evaluation and cluster analysis revealed 111 differentially expressed genes, with a P ≤ 10-3 . Nine genes with a P ≤ 10-4 were further validated. CYR61, CTGF, c-KIT, DUSP5, EGR1 were downregulated, whereas AKAP6 and INPP4B were upregulated in patients vs controls. Compared to controls, immunohistochemical analysis revealed a clear increase in INPP4B, whereas c-KIT immunolabeling resulted downregulated. As INPP4B regulates Akt pathway, we used western blot to show that phospho-Akt was significantly reduced in achalasiapatients vs controls. CONCLUSIONS & INFERENCES: The identification of altered gene expression, including INPP4B, a regulator of the Akt pathway, highlights novel signaling pathways involved in the neuronal and ICC changes underlying primary achalasia.
Authors: Ambroise Wonkam; Samuel Mawuli Adadey; Isabelle Schrauwen; Elvis Twumasi Aboagye; Edmond Wonkam-Tingang; Kevin Esoh; Kalinka Popel; Noluthando Manyisa; Mario Jonas; Carmen deKock; Victoria Nembaware; Diana M Cornejo Sanchez; Thashi Bharadwaj; Abdul Nasir; Jenna L Everard; Magda K Kadlubowska; Liz M Nouel-Saied; Anushree Acharya; Osbourne Quaye; Geoffrey K Amedofu; Gordon A Awandare; Suzanne M Leal Journal: Commun Biol Date: 2022-04-19