Absence Of Slc26a9 Results In The Disorder Of Gastric Epithelial Cell Differentiation,Which Is The Key Event To Development Of Chronic Atrophic Gastritis

Objective:Parietal cell loss is the critical and initial step of gastric carcinogenesis,and previous study showed that Slc26a9 loss impairs parietal cell function and survival.the down-regulation of Slc26a9 gene leads to the development of gastric cancer.As an earlier event of gastric cancer,chronic atrophic gastritis has a special urgency and important scientific value for early diagnosis and timely intervention of gastric cancer by clarifying its occurrence and malignant transformation mechanism.This study aims to clarify the important role of Slc26a9 gene in the development of chronic atrophic gastritis by using gastric tissue-specific Slc26a9 knockout mice and the clinical correlation between Slc26a9 and patients with chronic atrophic gastritis.Methods:1)The mRNA and protein expression of SLC26A9 in human normal gastric mucosa and gastric mucosa of patients with chronic atrophic gastritis was detected by qRT-PCR and IHC respectively;2)Stomach-specific Slc26a9 knockout mice were purchased form Cyagen Biosciences Inc.?Guangzhou,China?.PCR amplification and WB were used to detect Slc26a9knockout in the stomach of mice;3)From one month after birth,the body weight of Slc26a9^fl/fl and Slc26a9^fl/fl/Atp4b-Cre mice was measured every month,and the survival rate of the mice was observed;4)Gastric histology of Slc26a9^fl/fl and Slc26a9^fl/fl/Atp4b-Cre mice from 8 days to 18months after birth was observed by H&E staining;5)Immunohistochemistry was used to identify the molecular markers of gastric mucosal atrophy and metaplasia in Slc26a9^fl/fl and Slc26a9^fl/fl/Atp4b-Cre mice.6)Genes related to the differentiation of gastric mucosa,gastric stem cells and gastric cancer stem cell were analyzed,the mRNA expression of IL-1?,IL-6,IL-11,IL-17 and IL-33 in the gastric mucosa of Slc26a9^fl/fl and Slc26a9^fl/fl/Atp4b-Cre mice was detected by qRT-PCR;targeted genes of gastric stem cells Lrig 1,Lgr 5 and Mist 1 in Slc26a9^fl/fll/fl and Slc26a9^fl/fl/Atp4b-Cre mice from 1 month to 14 months after birth was identified by in situ hybridization and immunohistochemistry;7)Genes related to the tight junction and EMT were analyzed,targeted genes of tight junction Claudin 18.2 and Muc5ac,and E-cadherin,ZO-1 and Vimentin were investigated by immunohistochemistry in Slc26a9^fl/fl and Slc26a9^fl/fl/Atp4b-Cre mice at6-months-old;8)Proliferation and apoptosis were detected by Ki67 and TUNNEL staining respectively;targeted protein of apoptosis Cyt-C,Endo G and AIF were identified by WB;electron microscopy was used to observe the mitochondria of parietal cells;9)Genes related to TGF-?signaling pathway was analyzed,target genes of TGF-?signaling pathway was investigated by gene chip analysis,targeted genes of TGF-?signaling were observed by immunohistochemistry in 6-month-old Slc26a9^fl/fl and Slc26a9^fl/fl/Atp4b-Cre mice;10)Proteins of EMT-related were analyzed,EMT-related proteins including E-cadherin,ZO-1,Vimentin and Snail was investigated by immunohistochemistry in 14-month-old Slc26a9^fl/fl and Slc26a9^fl/fl/Atp4b-Cre mice;targeted gene of gastric cancer stem cell CD44 was observed by immunohistochemistry in 1-14 month-old Slc26a9^fl/fl and Slc26a9^fl/fl/Atp4b-Cre mice.Results:1)SLC26A9 is highly expressed in the normal gastric epithelium,but significantly downregulation in the chronic atrophic gastritis;2)Stomach-specific Slc26a9 knockout mice were successfully prepared and identified;3)Slc26a9 gene deletion had no effect on the growth and survival of mice;4)Slc26a9 gene deletion leads to the occurrence of spontaneous chronic atrophic gastritis,intestinal metaplasia and gastric malignant lesions in mice;5)Deletion of Slc26a9 gene results in the disorder of gastric mucosal epithelial cells differentiation markers,alteration of molecular markers for chronic atrophic gastritis and intestinal metaplasia in mice;6)Deletion of Slc26a9 gene result in disorder of gastric mucosal stem cells differentiation in mice;deletion Slc26a9 gene led to upregulation of inflammatory factors,including IL-1?,IL-6,IL-11and IL-17,indicating the occurrence of spontaneous inflammation in mice;targeted genes of gastric stem cells Lrig 1,Lgr 5and Mist 1 in Slc26a9^fl/fl and Slc26a9^fl/fl/Atp4b-Cre mice were gradually down regulated from 1 month to 14 months;7)Deletion of Slc26a9 gene altered the expression of tight junction related proteins in mice and led to spontaneous EMT;8)Deletion Slc26a9 gene led to overproliferation and apoptosis inhibition;targeted proteins of apoptosis Cyt-C,Endo G and AIF were down regulated in gastric mucosa of Slc26a9^fl/fl/Atp4b-Cre mice;compared with Slc26a9^fl/fll/fl mice,mitochondria of parietal cells in Slc26a9^fl/fl/Atp4b-Cre mice gastric mucosa remains normal;9)Deletion Slc26a9 gene caused alteration in multiple signaling pathways in mice including TGF-?signaling pathway,targeted genes of TGF-?signaling pathway TGF-?,p-smad2 and p-smad3 were downregulated in Slc26a9^fl/fl/Atp4b-Cre mice;10)Deletion Slc26a9 gene led to spontaneous EMT in gastric mucosa of 14-month-age Slc26a9^fl/fl/Atp4b-Cre mice and up regulated expression of cancer stem cell marker CD44.Conclusion:Deletion of Slc26a9 caused spontaneous chronic atrophic gastritis and malignant disease in the stomach of mice,and SLC26A9 is down-regulated in human chronic atrophic gastritis;Slc26a9 gene is essential for maintaining the steady state of gastric mucosal epithelial differentiation in mice,deletion of Slc26a9 caused stem cell differentiation disorder and spontaneous inflammation,leading to chronic atrophic gastritis and malignant pathological processes;deletion of the Slc26a9 gene leads to dysregulation of differentiation,overproliferation and apoptosis inhibition in mice by affecting the TGF-?signaling pathway,which caused development of chronic atrophic gastritis.