Whole transcriptomes analyses of organoids derived from Mist1Cre/ERT2 mice corpus with oxidative stress
Purpose: To explore the molecular mechanisms of Mist1+ cells that are specifically resistance to oxidative stress, and the mechanism that oxidative stress in regulating the stemness of Mist1+ cells, RNA sequencing was performed to analyze the geome-wide change of Mist1+ cells under oxidative stress (high fat diet or Gpx4 knockout) compared with control mice, and further the difference between these two Mist1+ cells. Methods: Total mRNA was extracted from Mist1+ cells which were isolated from corpus of Mist1Cre/ERT2;RFPLSL mice (8 weeks) fed with normal diet and high fat diet. Besides, we formed Mist1+ organoids from Mist1Cre/ERT2;RFPLSL mice and  Gpx4F/F;Mist1Cre/ERT2;RFPLSL mice respectively, organoids were passaged, and total mRNA was extracted from organoids in passage 5, 10 and 15. Then RNA quality was assessed using an Agilent Bioanalyzer 2100 and the sample reads were sequenced using Illumina NovaSeq 6000 platform. Results: Our results revealed differentially expressed genes in Mist1+ cells from high fat diet-fed mice compared with normal diet-fed mice. Further KEGG analysis enriched multiple pathways/biological processes including ROS, endocytosis, oxidative phosphorylation, Ras signaling, and ferroptosis. Further GSEA analysis showed that genes associated with stem cell maintenance and differentiation were significantly enriched in the high fat diet group, suggesting that Mist1+ cells enhance their stemness and transcriptional activity in response to oxidative stress induced by a high-fat diet. As for Gpx4 knockout-induced oxidative stress, we found that Gpx4 knockout in Mist1+ cells rewired multiple signaling pathways that drive cell growth, especially Hippo signaling pathway that is closely related with gastric cancer initiation and progression. Conclusion: Our study present the detailed transcripts analysis of Mist1+ cells under oxidative stress (high fat diet or Gpx4 knockout). Based on RNA-seq transcriptome characterization, we conclude a mechanism for Mist1+ cells that are resistance to oxidative stress and act as tumor initiation cells under oxidative stress. Overall design: Total mRNA was extracted from Mist1+ cells which were isolated from corpus of Mist1Cre/ERT2;RFPLSL mice (8 weeks) fed with normal diet and high fat diet. Besides, we formed Mist1+ organoids from Mist1Cre/ERT2;RFPLSL mice and  Gpx4F/F;Mist1Cre/ERT2;RFPLSL mice respectively, organoids were passaged, and total mRNA was extracted from organoids in passage 5, 10 and 15. Then RNA quality was assessed using an Agilent Bioanalyzer 2100 and the sample reads were sequenced using Illumina NovaSeq 6000 platform.