Systems biology is an approach to comprehensively study complex interactions within a biological system. Most published systems vaccinology studies have utilized whole blood or peripheral blood mononuclear cells (PBMC) to monitor the immune response after vaccination. Because human blood is comprised of multiple hematopoietic cell types, the potential for masking responses of under-represented cell populations is increased when analyzing whole blood or PBMC. To investigate the contribution of individual cell types to the immune response after vaccination, we established a rapid and efficient method to purify human T and B cells, natural killer (NK) cells, myeloid dendritic cells (mDC), monocytes, and neutrophils from fresh venous blood. Purified cells were fractionated and processed in a single day. RNA-Seq and quantitative shotgun proteomics were performed to determine expression profiles for each cell type prior to and after inactivated seasonal influenza vaccination. Our results show that transcriptomic and proteomic profiles generated from purified immune cells differ significantly from PBMC. Differential expression analysis for each immune cell type also shows unique transcriptomic and proteomic expression profiles as well as changing biological networks at early time points after vaccination. This cell type-specific information provides a more comprehensive approach to monitor vaccine responses.
Supplementary Figure from Immune Activity and Response Differences of Oncolytic Viral Therapy in Recurrent Glioblastoma: Gene Expression Analyses of a Phase IB Study
Abstract The Michael J. Fox Foundation’s Parkinson’s Progression Markers Initiative (PPMI) is an observational study to comprehensively evaluate Parkinson’s disease (PD) patients using imaging, biologic sampling, clinical and behavioural assessments to identify biomarkers of PD progression. As part of this study, we obtained 4,756 whole blood samples from 1,570 subjects at baseline, 0.5, 1, 2, and 3 years from enrollment in the study. We isolated RNA and performed whole transcriptome sequencing in this longitudinal cohort. Here, we describe and quantify technical variability associated with this dataset through the use of pooled reference samples, including plate distribution, RNA quality, and outliers. This large, uniformly processed dataset is available to researchers at https://www.ppmi-info.org .
Cystic fibrosis-related (CF-related) diabetes (CFRD) is an increasingly common and devastating comorbidity of CF, affecting approximately 35% of adults with CF. However, the underlying causes of CFRD are unclear. Here, we examined cystic fibrosis transmembrane conductance regulator (CFTR) islet expression and whether the CFTR participates in islet endocrine cell function using murine models of β cell CFTR deletion and normal and CF human pancreas and islets. Specific deletion of CFTR from murine β cells did not affect β cell function. In human islets, CFTR mRNA was minimally expressed, and CFTR protein and electrical activity were not detected. Isolated CF/CFRD islets demonstrated appropriate insulin and glucagon secretion, with few changes in key islet-regulatory transcripts. Furthermore, approximately 65% of β cell area was lost in CF donors, compounded by pancreatic remodeling and immune infiltration of the islet. These results indicate that CFRD is caused by β cell loss and intraislet inflammation in the setting of a complex pleiotropic disease and not by intrinsic islet dysfunction from CFTR mutation.
Many patients with type 1 diabetes (T1D) have residual β cells producing small amounts of C-peptide long after disease onset but develop an inadequate glucagon response to hypoglycemia following T1D diagnosis. The features of these residual β cells and α cells in the islet endocrine compartment are largely unknown, due to the difficulty of comprehensive investigation. By studying the T1D pancreas and isolated islets, we show that remnant β cells appeared to maintain several aspects of regulated insulin secretion. However, the function of T1D α cells was markedly reduced, and these cells had alterations in transcription factors constituting α and β cell identity. In the native pancreas and after placing the T1D islets into a non-autoimmune, normoglycemic in vivo environment, there was no evidence of α-to-β cell conversion. These results suggest an explanation for the disordered T1D counterregulatory glucagon response to hypoglycemia.
Inadequate pancreatic β cell function underlies type 1 and type 2 diabetes mellitus. Strategies to expand functional cells have focused on discovering and controlling mechanisms that limit the proliferation of human β cells. Here, we developed an engraftment strategy to examine age-associated human islet cell replication competence and reveal mechanisms underlying age-dependent decline of β cell proliferation in human islets. We found that exendin-4 (Ex-4), an agonist of the glucagon-like peptide 1 receptor (GLP-1R), stimulates human β cell proliferation in juvenile but not adult islets. This age-dependent responsiveness does not reflect loss of GLP-1R signaling in adult islets, since Ex-4 treatment stimulated insulin secretion by both juvenile and adult human β cells. We show that the mitogenic effect of Ex-4 requires calcineurin/nuclear factor of activated T cells (NFAT) signaling. In juvenile islets, Ex-4 induced expression of calcineurin/NFAT signaling components as well as target genes for proliferation-promoting factors, including NFATC1, FOXM1, and CCNA1. By contrast, expression of these factors in adult islet β cells was not affected by Ex-4 exposure. These studies reveal age-dependent signaling mechanisms regulating human β cell proliferation, and identify elements that could be adapted for therapeutic expansion of human β cells.
Gastric chief cells differentiate from mucous neck cells and develop their mature state at the base of oxyntic glands with expression of secretory zymogen granules. After parietal cell loss, chief cells transdifferentiate into mucous cell metaplasia, designated spasmolytic polypeptide-expressing metaplasia (SPEM), which is considered a candidate precursor of gastric cancer. We examined the range of microRNA (miRNA) expression in chief cells and identified miRNAs involved in chief cell transdifferentiation into SPEM. Among them, miR-148a was strongly and specifically expressed in chief cells and significantly decreased during the process of chief cell transdifferentiation. Interestingly, suppression of miR-148a in a conditionally immortalized chief cell line induced up-regulation of CD44 variant 9 (CD44v9), one of the transcripts expressed at an early stage of SPEM development, and DNA methyltransferase 1 (Dnmt1), an established target of miR-148a. Immunostaining analyses showed that Dnmt1 was up-regulated in SPEM cells as well as in chief cells before the emergence of SPEM in mouse models of acute oxyntic atrophy using either DMP-777 or L635. In the cascade of events that leads to transdifferentiation, miR-148a was down-regulated after acute oxyntic atrophy either in xCT knockout mice or after sulfasalazine inhibition of xCT. These findings suggest that the alteration of miR-148a expression is an early event in the process of chief cell transdifferentiation into SPEM. Gastric chief cells differentiate from mucous neck cells and develop their mature state at the base of oxyntic glands with expression of secretory zymogen granules. After parietal cell loss, chief cells transdifferentiate into mucous cell metaplasia, designated spasmolytic polypeptide-expressing metaplasia (SPEM), which is considered a candidate precursor of gastric cancer. We examined the range of microRNA (miRNA) expression in chief cells and identified miRNAs involved in chief cell transdifferentiation into SPEM. Among them, miR-148a was strongly and specifically expressed in chief cells and significantly decreased during the process of chief cell transdifferentiation. Interestingly, suppression of miR-148a in a conditionally immortalized chief cell line induced up-regulation of CD44 variant 9 (CD44v9), one of the transcripts expressed at an early stage of SPEM development, and DNA methyltransferase 1 (Dnmt1), an established target of miR-148a. Immunostaining analyses showed that Dnmt1 was up-regulated in SPEM cells as well as in chief cells before the emergence of SPEM in mouse models of acute oxyntic atrophy using either DMP-777 or L635. In the cascade of events that leads to transdifferentiation, miR-148a was down-regulated after acute oxyntic atrophy either in xCT knockout mice or after sulfasalazine inhibition of xCT. These findings suggest that the alteration of miR-148a expression is an early event in the process of chief cell transdifferentiation into SPEM. See editorial on page 189. See editorial on page 189. SummaryFollowing parietal cell loss, chief cells transdifferentiate into mucous cell metaplasia, designated spasmolytic polypeptide-expressing metaplasia (SPEM). Induction of SPEM was associated with loss of miR-148a. Loss of miR-148a is an early step in chief cell transdifferentiation. Following parietal cell loss, chief cells transdifferentiate into mucous cell metaplasia, designated spasmolytic polypeptide-expressing metaplasia (SPEM). Induction of SPEM was associated with loss of miR-148a. Loss of miR-148a is an early step in chief cell transdifferentiation. In the stomach mucosa, gastric chief cells are located at the base of oxyntic glands and express secretory zymogens. Chief cells differentiate from mucous neck cells in the lower half of corpus glands without cell division and remain in a fully differentiated state under normal conditions with a lifetime of more than 60 days.1Weis V.G. Petersen C.P. Weis J.A. Meyer A.R. Choi E. Mills J.C. Goldenring J.R. Maturity and age influence chief cell ability to transdifferentiate into metaplasia.Am J Physiol Gastrointest Liver Physiol. 2016; 312: G67-G76Crossref PubMed Scopus (24) Google Scholar Previous studies demonstrated that some transcription factors, including XBP1 and MIST1, are required for the differentiation from mucous neck cells into chief cells and the maintenance of chief cells.2Goldenring J.R. Nam K.T. Mills J.C. The origin of pre-neoplastic metaplasia in the stomach: chief cells emerge from the Mist.Exp Cell Res. 2011; 317: 2759-2764Crossref PubMed Scopus (67) Google Scholar, 3Lennerz J.K.M. Kim S. Oates E.L. Huh W.J. Dherty J.M. Tian X. Bredemeyer A.J. Goldenring J.R. Lauwers G.Y. Shin G.Y. Mills J.C. The transcription factor MIST1 is a novel human gastric chief cell marker whose expression is lost in metaplasia, dysplasia and carcinoma.Am J Pathol. 2010; 177: 1514-1533Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 4Bredemeyer A.J. Geahlen J.H. Weis V.G. Huh W.J. Zinselmeyer B.H. Srivatsan S. Miller M.J. Shaw A.S. Mills J.C. The gastric epithelial progenitor cell niche and differentiation of the zymogenic (chief) cell lineage.Dev Biol. 2009; 325: 211-224Crossref PubMed Scopus (73) Google Scholar On the other hand, parietal cell loss and inflammation induce chief cells to transdifferentiate into mucous cell metaplasia, designated spasmolytic polypeptide-expressing metaplasia (SPEM), with the loss of zymogen granules and the formation of Muc6-containing mucous granules.2Goldenring J.R. Nam K.T. Mills J.C. The origin of pre-neoplastic metaplasia in the stomach: chief cells emerge from the Mist.Exp Cell Res. 2011; 317: 2759-2764Crossref PubMed Scopus (67) Google Scholar, 5Mills J.C. Sansom O.J. Reserve stem cells: differentiated cells reprogram to fuel repair, metaplasia, and neoplasia in the adult gastrointestinal tract.Sci Signal. 2015; 8: re8Crossref PubMed Scopus (95) Google Scholar SPEM is considered a likely precursor lineage for intestinal metaplasia (IM) development,3Lennerz J.K.M. Kim S. Oates E.L. Huh W.J. Dherty J.M. Tian X. Bredemeyer A.J. Goldenring J.R. Lauwers G.Y. Shin G.Y. Mills J.C. The transcription factor MIST1 is a novel human gastric chief cell marker whose expression is lost in metaplasia, dysplasia and carcinoma.Am J Pathol. 2010; 177: 1514-1533Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 6Goldenring J.R. Nam K.T. Wang T.C. Mills J.C. Wright N.A. Spasmolytic polypeptide-expressing metaplasia and intestinal metaplasia: time for reevaluation of metaplasias and the origins of gastric cancer.Gastroenterology. 2010; 138: 2207-2210Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar and these metaplasias are possible precursor lesions of gastric cancer. However, the regulatory mechanisms for the chief cell transdifferentiation process have not been fully elucidated. MicroRNAs (miRNAs) are critical post-transcriptional regulators of gene expression.7Bartel D.P. MicroRNAs: genomics, biogenesis, mechanism, and function.Cell. 2004; 116: 281-297Abstract Full Text Full Text PDF PubMed Scopus (29453) Google Scholar, 8Ha M. Kim V.N. Regulation of microRNA biogenesis.Nat Rev Mol Cell Biol. 2014; 15: 509-524Crossref PubMed Scopus (3563) Google Scholar MiRNAs are involved in the developmental process of various organs as well as cancer progression.9Esquela-Kerscher A. Slack F.J. Oncomirs: microRNAs with a role in cancer.Nat Rev Cancer. 2006; 6: 259-269Crossref PubMed Scopus (6186) Google Scholar, 10Calin G.A. Croce C.M. MicroRNA signatures in human cancers.Nat Rev Cancer. 2006; 6: 857-866Crossref PubMed Scopus (6605) Google Scholar Dysregulation of miRNAs has been reported in human gastric cancer11Ueda T. Volinia S. Okumura H. Shimizu M. Taccioli C. Rossi S. Alder H. Liu C.G. Oue N. Yasui W. Yoshida K. Sasaki H. Nomura S. Seto Y. Kaminishi M. Calin G.A. Croce C.M. Relation between microRNA expression and progression and prognosis of gastric cancer: a microRNA expression analysis.Lancet Oncol. 2010; 11: 136-146Abstract Full Text Full Text PDF PubMed Scopus (732) Google Scholar and Helicobacter pylori–induced gastritis,12Matsushima K. Isomoto H. Inoue N. Nakayama T. Hayashi T. Nakayama M. Nakao K. Hirayama T. Kohno S. MicroRNA signatures in Helicobacter pylori-infected gastric mucosa.Int J Cancer. 2011; 128: 361-370Crossref PubMed Scopus (166) Google Scholar, 13Zabaleta J. MicroRNA: a bridge from H pylori infection to gastritis and gastric cancer development.Frontiers in Genetics. 2012; 3: 294Crossref PubMed Scopus (40) Google Scholar contributing to gastric epithelial cell proliferation. We previously reported an analysis of miRNAs in laser capture microdissected human chief cells, SPEM cells, and IM cells, suggesting that miR-30a down-regulation and miR-194 up-regulation were related to metaplasia progression through regulation of the transcription factors HNF4γ and NR2F2.14Sousa J.F. Nam K.T. Petersen C.P. Lee H.J. Yang H.K. Kim W.H. Goldenring J.R. miR-30-HNF4gamma and miR-194-NR2F2 regulatory networks contribute to the upregulation of metaplasia markers in the stomach.Gut. 2016; 65: 914-924Crossref PubMed Scopus (37) Google Scholar However, it remains unclear whether miRNAs are involved in the initiation of SPEM development. Chief cell transdifferentiation and the transition to SPEM cells occur through series of ordered events. Our group and others have identified a number of events that chief cells undergo to transdifferentiate from zymogen secreting cells into mucous secreting metaplastic cells. Acute oxyntic atrophy induces an early loss of the chief cell maturation-specifying transcription factor, Mist1,15Weis V.G. Sousa J.F. LaFleur B.J. Nam K.T. Weis J.A. Finke P.E. Ameen N.A. Fox J.G. Goldenring J.R. Heterogeneity in mouse SPEM lineages identifies markers of metaplastic progression.Gut. 2013; 62: 1270-1279Crossref PubMed Scopus (75) Google Scholar, 16Ramsey V.G. Doherty J.M. Chen C.C. Stappenbeck T.S. Konieczny S.F. Mills J.C. The maturation of mucus-secreting gastric epithelial progenitors into digestive-enzyme secreting zymogenic cells requires Mist1.Development. 2007; 134: 211-222Crossref PubMed Scopus (148) Google Scholar, 17Petersen C.P. Meyer A.R. De Salvo C. Choi E. Schlegel C. Petersen A. Engevik A.C. Prasad N. Levy S.E. Peebles R.S. Pizarro T.T. Goldenring J.R. A signalling cascade of IL-33 to IL-13 regulates metaplasia in the mouse stomach.Gut. 2018; 67: 805-817Crossref PubMed Scopus (55) Google Scholar and up-regulation of the specific splice variant of CD44, CD44 variant 9 (CD44v9).17Petersen C.P. Meyer A.R. De Salvo C. Choi E. Schlegel C. Petersen A. Engevik A.C. Prasad N. Levy S.E. Peebles R.S. Pizarro T.T. Goldenring J.R. A signalling cascade of IL-33 to IL-13 regulates metaplasia in the mouse stomach.Gut. 2018; 67: 805-817Crossref PubMed Scopus (55) Google Scholar, 18Wada T. Ishimoto T. Seishima R. Tsuchihashi K. Yoshikawa M. Oshima H. Oshima M. Masuko T. Wright N.A. Furuhashi S. Hirashima K. Baba H. Kitagawa Y. Saya H. Nagano O. Functional role of CD44v-xCT system in the development of spasmolytic polypeptide-expressing metaplasia.Cancer Science. 2013; 104: 1323-1329Crossref PubMed Scopus (64) Google Scholar, 19Meyer A.R. Engevik A.C. Willet S.G. Williams J.A. Zou Y. Massion P.P. Mills J.C. Choi E. Goldenring J.R. Cystine/glutamate antiporter (xCT) is required for chief cell plasticity after gastric injury.Cell Mol Gastroenterol Hepatol. 2019; 8: 379-405Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar CD44v9 is an activator and a stabilizer of xCT, a cystine transporter, which promotes adaptation to reactive oxygen species and cellular stress.18Wada T. Ishimoto T. Seishima R. Tsuchihashi K. Yoshikawa M. Oshima H. Oshima M. Masuko T. Wright N.A. Furuhashi S. Hirashima K. Baba H. Kitagawa Y. Saya H. Nagano O. Functional role of CD44v-xCT system in the development of spasmolytic polypeptide-expressing metaplasia.Cancer Science. 2013; 104: 1323-1329Crossref PubMed Scopus (64) Google Scholar, 19Meyer A.R. Engevik A.C. Willet S.G. Williams J.A. Zou Y. Massion P.P. Mills J.C. Choi E. Goldenring J.R. Cystine/glutamate antiporter (xCT) is required for chief cell plasticity after gastric injury.Cell Mol Gastroenterol Hepatol. 2019; 8: 379-405Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 20Ishimoto T. Nagano O. Yae T. Tamada M. Motohara T. Oshima H. Oshima M. Ikeda T. Asaba R. Yagi H. Masuko T. Shimizu T. Ishikawa T. Kai K. Takahashi E. Imamura Y. Baba Y. Ohmura M. Suematsu M. Baba H. Saya H. CD44 variant regulates redox status in cancer cells by stabilizing the xCT subunit of system xc(-) and thereby promotes tumor growth.Cancer Cell. 2011; 19: 387-400Abstract Full Text Full Text PDF PubMed Scopus (822) Google Scholar The increased cellular stress is associated with an increase in autophagy, which is necessary for breaking down the zymogen granules.21Willet S.G. Lewis M.A. Miao Z.F. Liu D. Radyk M.D. Cunningham R.L. Burclaff J. Sibbel G. Lo H.G. Blanc V. Davidson N.O. Wang Z.N. Mills J.C. Regenerative proliferation of differentiated cells by mTORC1-dependent paligenosis.EMBO J. 2018; 37Crossref PubMed Scopus (84) Google Scholar Importantly, the process of transdifferentiation can be arrested at different stages,17Petersen C.P. Meyer A.R. De Salvo C. Choi E. Schlegel C. Petersen A. Engevik A.C. Prasad N. Levy S.E. Peebles R.S. Pizarro T.T. Goldenring J.R. A signalling cascade of IL-33 to IL-13 regulates metaplasia in the mouse stomach.Gut. 2018; 67: 805-817Crossref PubMed Scopus (55) Google Scholar, 19Meyer A.R. Engevik A.C. Willet S.G. Williams J.A. Zou Y. Massion P.P. Mills J.C. Choi E. Goldenring J.R. Cystine/glutamate antiporter (xCT) is required for chief cell plasticity after gastric injury.Cell Mol Gastroenterol Hepatol. 2019; 8: 379-405Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 21Willet S.G. Lewis M.A. Miao Z.F. Liu D. Radyk M.D. Cunningham R.L. Burclaff J. Sibbel G. Lo H.G. Blanc V. Davidson N.O. Wang Z.N. Mills J.C. Regenerative proliferation of differentiated cells by mTORC1-dependent paligenosis.EMBO J. 2018; 37Crossref PubMed Scopus (84) Google Scholar suggesting that chief cell transdifferentiation occurs through a set of stepwise events that are coordinated and maintained in a defined order. Here we investigated the influence of miRNAs on the initiation of chief cell transdifferentiation into SPEM cells. We performed miRNA profiling specifically on mouse chief cells and compared miRNA expression with that in conditionally immortalized mouse chief cell and SPEM cell lines. Interestingly, several miRNAs were highly expressed in normal chief cells but down-regulated in SPEM cells. Among them, miR-148a was the most highly expressed miRNA by more than 10-fold in chief cells. Loss of miR-148a was associated with the early initiation of chief cell transdifferentiation. In addition, the loss of miR-148a led to up-regulation of an early SPEM marker, CD44 variant 9, and one of its target genes, DNA methyltransferase 1 (Dnmt1). The loss of miR-148a was found early during the chief cell transdifferentiation process, preceding up-regulation of CD44v9. These findings suggest that miR-148a is an early regulator in reprogramming chief cells during transdifferentiation into SPEM. In a previous study, our group reported the miRNA profile of human SPEM and IM in comparison with chief cells from normal stomach.14Sousa J.F. Nam K.T. Petersen C.P. Lee H.J. Yang H.K. Kim W.H. Goldenring J.R. miR-30-HNF4gamma and miR-194-NR2F2 regulatory networks contribute to the upregulation of metaplasia markers in the stomach.Gut. 2016; 65: 914-924Crossref PubMed Scopus (37) Google Scholar In those studies, we identified miRNAs related with human IM progression; however, no miRNAs related to SPEM development were confirmed. We therefore sought to investigate miRNAs during the initiation of SPEM development by using mouse models. First, to profile miRNAs from mouse chief cells, we crossed Mist1CreERT2/+mice with R26RmTmG reporter mice. After tamoxifen injection, immunostaining analyses of these mouse stomachs showed that most of green fluorescent protein (GFP)-positive cells were chief cells at the base of glands, with only occasional labeled cells in the isthmus regions (Figure 1A).1Weis V.G. Petersen C.P. Weis J.A. Meyer A.R. Choi E. Mills J.C. Goldenring J.R. Maturity and age influence chief cell ability to transdifferentiate into metaplasia.Am J Physiol Gastrointest Liver Physiol. 2016; 312: G67-G76Crossref PubMed Scopus (24) Google Scholar We sorted GFP-positive cells from 2 mice and performed miRNA sequencing (Figure 1B). Forty-three miRNAs were highly expressed in mouse chief cells (read values >500 in both mice) (Table 1). Among them, miR-148a-3p was the most highly expressed miRNA (read values >150,000) in chief cells, more than 10-fold higher than other highly expressed miRNAs such as miR-375-3p, let-7 family (let-7b-5p, let-7c-5p, let-7f-5p and let-7a-5p), and miR-200b-3p (read values >8000). Interestingly, these miRNAs have already been reported as down-regulated in human gastric cancer tissues and related to gastric cancer progression.22Qiu X. Zhu H. Liu S. Tao G. Jin J. Chu H. Wang M. Tong N. Gong W. Zhao Q. Qiang F. Zhang Z. Expression and prognostic value of microRNA-26a and microRNA-148a in gastric cancer.J Gastroenterol Hepatol. 2017; 32: 819-827Crossref PubMed Scopus (30) Google Scholar, 23Tsukamoto Y. Nakada C. Noguchi T. Tanigawa M. Nguyen L.T. Uchida T. Hijiya N. Matsuura K. Fujioka T. Seto M. Moriyama M. MicroRNA-375 is downregulated in gastric carcinomas and regulates cell survival by targeting PDK1 and 14-3-3zeta.Cancer Res. 2010; 70: 2339-2349Crossref PubMed Scopus (391) Google Scholar, 24Tang H. Deng M. Tang Y. Xie X. Guo J. Kong Y. Ye F. Su Q. Xie X. miR-200b and miR-200c as prognostic factors and mediators of gastric cancer cell progression.Clin Cancer Res. 2013; 19: 5602-5612Crossref PubMed Scopus (155) Google Scholar, 25Motoyama K. Inoue H. Nakamura Y. Uetake H. Sugihara K. Mori M. Clinical significance of high mobility group A2 in human gastric cancer and its relationship to let-7 microRNA family.Clin Cancer Res. 2008; 14: 2334-2340Crossref PubMed Scopus (205) Google ScholarTable 1MicroRNAs Highly Expressed in Mouse Chief CellsAccessionChromosomeStrandStartEndGene IDSample 1Sample 2Entrez IDmmu-miR-148a-3pchr6–5121982851219849MI0000550_1157846.9654154942.4573387166mmu-miR-375-3pchr1–7494723574947256MI0000792_135248.2008421476.74894723900mmu-let-7b-5pchr15+8553775585537776MI0000558_125949.9115427815.42445387245mmu-let-7c-5pchr16+7759991777599938MI0000559_115987.6691222193.04491387246mmu-let-7c-5pchr15+8553704685537067MI0000560_114821.5940920835.45521723966mmu-let-7f-5pchrX+1.48E+081.48E+08MI0000563_113039.9796715870.56644387253mmu-miR-7a-5pchr13–5849420258494224MI0000728_211223.585396231.293692723902mmu-miR-200b-3pchr4–1.55E+081.55E+08MI0000243_19422.30292710591.6593387243mmu-let-7a-5pchr13–4863360848633629MI0000556_28746.25684312009.56271387244mmu-miR-7a-5pchr7+8603318186033203MI0000729_18740.2055884823.681713723884mmu-let-7a-5pchr9+4134481541344836MI0000557_18363.61536611563.89665723965mmu-let-7f-5pchr13–4863325848633279MI0000562_28230.52646411138.08047387252mmu-miR-192-5pchr19+62648576264877MI0000551_17084.1203187057.18444387187mmu-miR-21-5pchr11–8639762286397643MI0000569_25868.1457957696.271615387140mmu-miR-26a-5pchr10+1.26E+081.26E+08MI0000706_15001.5377083946.313897723962mmu-miR-26a-5pchr9+1.19E+081.19E+08MI0000573_15001.5377083946.313897387218mmu-let-7i-5pchr10–1.22E+081.22E+08MI0000138_24517.5661155195.810144387251mmu-miR-99a-5pchr16+7759918577599206MI0000146_13504.2500293589.631416387229mmu-miR-30d-5pchr15–6817281968172840MI0000549_22757.1202972825.520615387228mmu-miR-7b-5pchr17+5638244056382462MI0000730_12657.3011111373.046719723883mmu-miR-200a-3pchr4–1.55E+081.55E+08MI0000554_12465.2256642397.499834387242mmu-miR-200c-3pchr6–1.25E+081.25E+08MI0000694_12239.8773182418.092972723944mmu-miR-182-5pchr6–3011596230115986MI0000224_22006.9653591888.583689387177mmu-miR-1a-3pchr18–1078548310785504MI0000652_11755.90612363.24694981723959mmu-miR-125a-5pchr17+1796778117967804MI0000151_11663.6485761174.480463387235mmu-let-7g-5pchr9+1.06E+081.06E+08MI0000137_11585.0042571562.052254387249mmu-miR-183-5pchr6–3011971130119732MI0000225_21533.5813692018.018941387178mmu-miR-151-3pchr15–7308525073085270MI0000173_11308.2331851660.600129387169mmu-miR-215-5pchr1+1.87E+081.87E+08MI0000974_11215.9759673101.305644387211mmu-miR-127-3pchr12+1.11E+081.11E+08MI0000154_21188.753604600.1103493387146mmu-miR-143-3pchr18–6180885361808873MI0000257_11025.412731771.4655906387161mmu-miR-378-3pchr18–6155749261557512MI0000795_11020.8753452281.30322723889mmu-miR-423-5pchr11–7689162476891646MI0004637_21001.214241177.423751519mmu-miR-27b-3pchr13+6340206863402088MI0000142_2860.560016781.7616481387221mmu-miR-30c-5pchr1+2329855323298575MI0000548_1852.9982668620.7028672723964mmu-miR-30c-5pchr4–1.2E+081.2E+08MI0000547_2848.4606843617.0253704387227mmu-miR-92a-3pchr14+1.15E+081.15E+08MI0000719_2739.56800381023.717545751549mmu-let-7e-5pchr17+1796733017967351MI0000561_1680.5837996718.5145194387248mmu-let-7d-5pchr13–4863144748631468MI0000405_2603.4510756628.792413387247mmu-miR-30a-5pchr1+2327911323279134MI0000144_1586.8142113556.7203464387225mmu-miR-320-3pchr14+7084336470843385MI0000704_2580.7647177527.3028558723838mmu-miR-25-3pchr5–1.39E+081.39E+08MI0000689_1552.0292875714.1023224723926mmu-miR-200a-5pchr4–1.55E+081.55E+08MI0000554_2547.491972686.8912993387242 Open table in a new tab To investigate miRNAs related to metaplasia development, we examined miRNA expression profiles for conditionally immortalized mouse chief cell (ImChief) and SPEM cell (ImSPEM) lines, previously established from Immortomice.26Weis V.G. Petersen C.P. Mills J.C. Tuma P.L. Whitehead R.H. Goldenring J.R. Establishment of novel in vitro mouse chief cell and SPEM cultures identifies MAL2 as a marker of metaplasia in the stomach.Am J Physiol Gastrointest Liver Physiol. 2014; 307: G777-G792Crossref PubMed Scopus (22) Google Scholar ImChief cells express chief cell markers such as pepsinogen C (Pgc) and Mist1 and produce characteristic zymogen granules, although they do not express gastric intrinsic factor (GIF). In contrast, ImSPEM cells express SPEM-specific markers such as Tff2 and He4 and some intestinalized markers such as Cftr and PigR. We extracted total RNAs from ImChief cells and ImSPEM cells and performed miRNA sequencing. We detected 87 miRNAs down-regulated (P < .01, with read values for ImChief cells >500 and fold-change >5) and 7 miRNAs up-regulated (P < .01, with read values for ImSPEM cells >500 and fold-change >5) in ImSPEM cells compared with ImChief cells (Tables 2 and 3). From these 2 different sequencing studies, we identified 15 miRNAs that were both highly expressed in sorted chief cells and down-regulated in ImSPEM cells compared with ImChief cells (Figure 1C) as candidate miRNAs related to SPEM development. This group of miRNAs included miR-148a-3p, miR-200 family members (miR-200a-3p, miR-200a-5p, miR-200b-3p, and miR-200c-3p), miR-30 family members (miR-30a-5p and miR-30c-5p), and let-7 family members (let-7f-5p and let-7i-5p).Table 2MicroRNAs Down-regulated in ImSPEM Cells Compared With ImChief CellsFeatureImSPEM cells_1ImSPEM cells_2ImSPEM cells_3ImChief cells_1ImChief cells_2ImChief cells_3ImSPEM cells/ImChief cellsmmu-miR-141-5p0001005157615930mmu-miR-141-3p60.575.584147862151753220628.50.000423mmu-miR-200c-3p7638326.51331512489.50.000469mmu-miR-205-5p1451481972330993554703187240.00054mmu-miR-205-3p5534571517558500.000834mmu-miR-203-3p811114932634660650.00173mmu-miR-672-5p1241015143013900.001825mmu-miR-676-3p2226827358560.00264mmu-miR-429-3p60697318745.522299.523313.50.003139mmu-miR-146a-5p136.5163.5172.528169.567458.5421720.003429mmu-miR-200a-3p42.540.54692971274913597.50.003619mmu-miR-200b-3p2392693163791548814.5565260.005752mmu-miR-135b-5p616171674.672275.832806.670.005772mmu-miR-200a-5p125362410677450.00821mmu-miR-183-5p1153136213081096001147571738200.009601mmu-miR-183-3p1114131045114716450.009904mmu-miR-182-5p1500612308142061002426113651814537170.011557mmu-miR-96-5p981129265377080108210.012358mmu-miR-193b-3p6710894300622595077.50.026009mmu-miR-31-3p2482481569144626320.027271mmu-miR-31-5p6738572475521824072179162440710.031059mmu-miR-582-3p32214884490512410.033779mmu-miR-421-3p1671922264175498159070.038837mmu-miR-147-3p2618284276045990.044172mmu-miR-222-3p2279148922613295950742470260.046119mmu-miR-210-5p394664684121712120.047864mmu-miR-27b-3p5848065172.574847.51248916124568415845840.048662mmu-miR-181b-5p563509.5742.6710536.8312984.1713485.670.04905mmu-miR-181a-5p7652748789691428881403942031230.049564mmu-miR-148a-3p8590.59828.59697.5140196181813223713.50.051522mmu-miR-148a-5p6766781008114015440.057151mmu-miR-298-5p7706307941071210690150970.060111mmu-miR-21a-3p4801124901738510894167390.071535mmu-miR-101b-3p142.52111821863.522802870.50.076347mmu-miR-27b-5p8285100965124812670.076724mmu-miR-126a-5p2124063833065424855320.077929mmu-miR-1843a-5p851031171180118914620.079614mmu-miR-365-3p64129103107191716580.081185mmu-miR-221-3p2572297331182730932974396370.086699mmu-miR-29b-3p151.672742111543.3323172887.330.094354mmu-miR-30c-5p28013116393629133.6730398.33408780.098128mmu-miR-92a-3p55854445.335511.6742970.6747225.3366885.670.098942mmu-miR-194-5p1651362421465207219480.098997mmu-miR-425-5p3464364993521420447890.102365mmu-miR-19b-3p301.5369.5309.52310.173555.53663.50.102895mmu-miR-221-5p6072973393409360050630.102966mmu-miR-374b-5p5510013074491410250.106224mmu-miR-126a-3p5349763875616650.110353mmu-miR-17-5p1011301308301150.831259.830.111397mmu-miR-328-3p160152.5210.515131110.51946.50.114442mmu-miR-130b-3p119112.5176629.51266.516560.114724mmu-miR-19a-3p57.563.555.5363.83552.5615.50.115222mmu-miR-48412411134148992059226144650.117461mmu-miR-20a-5p8411384570774887.330.125934mmu-miR-23b-3p1793.530922818.51750618406.5250820.126306mmu-miR-30a-5p22727.52477126421.5164380193723.5225365.30.126691mmu-miR-30e-5p10724.510780.511758.5705818967998109.330.128744mmu-miR-192-5p293827853313.5202302144427916.50.129852mmu-let-7f-5p22306.0528289.7735971.93174340.2270159221880.10.129908mmu-miR-210-3p84694912176351787089300.130102mmu-miR-22-3p10718610997814691973775892566210229610.135529mmu-miR-107-3p182.5186.33225.671261.8314181703.830.135617mmu-let-7i-5p7772940112263.55912379414.577297.50.136384mmu-miR-191-5p136181631018084975681003121510970.137579mmu-miR-98-5p514.271025.471211.274932.677444.47119.930.141099mmu-miR-34b-5p444.55486423035.54005.543810.143101mmu-miR-93-5p4297479459263100032748407990.143639mmu-let-7b-3p63871085394438110.143893mmu-let-7j980.5966.5132661968255.58213.50.144408mmu-miR-99b-3p104119123625822.58540.150337mmu-miR-30a-3p357445506.52347.52619.53139.50.161414mmu-miR-24-3p3038398232871661722764242530.161973mmu-miR-27a-3p46846406.57145.5312673634043419.50.164249mmu-miR-34b-3p3474055672219250532990.164402mmu-miR-342-3p17915916289973214090.164474mmu-miR-1839-5p1942352931243142716420.16744mmu-miR-130a-3p26132714.5331013909.517149.5202560.168323mmu-miR-140-3p6167988073466468250390.168423mmu-miR-29a-3p5652.55820699928115.538135.542291.830.170177mmu-miR-450b-5p3102903301263180421950.176739mmu-miR-34c-5p19682.52012026912100916.51279931448270.178507mmu-miR-351-5p23161802199992109920151210.178593mmu-miR-34c-3p1263273171091148816330.182811mmu-miR-28a-3p157207307942126414000.186079mmu-miR-28a-5p6117119293248405643240.193584mmu-miR-186-5p5336569764012343630215360280.194404mmu-let-7d-3p3875896472297203039510.196062 Open table in a new tab Table 3MicroRNAs Up-regulated in ImSPEM Cells Compared With ImChief CellsFeatureImSPEM cells_1ImSPEM cells_2ImSPEM cells_3ImChief cells_1ImChief cells_2ImChief cells_3ImSPEM cells/ImChief cellsmmu-miR-10b-5p255958.5248754.526392569.5107.5118.52601.144mmu-miR-199a-5p1767171525366818188.0625mmu-miR-199a-3pmmu-miR-199b-3p280169777968474858115.9869mmu-miR-344d-3p587491675361187.65mmu-miR-10a-5p60241.555707.5592733125.53297.55008.515.328mmu-miR-155-5p887130415931641992356.327759mmu-miR-181c-5p5615906572132673282.237624 Open table in a new tab Because it was by far the most highly expressed miRNA species in chief cells, we focused our subsequent studies on miR-148a. To examine the distribution of miR-148a expression, we performed in situ hybridization analyses for miR-148a (Figure 2). In normal stomach, miR-148a was strongly expressed in the bases of corpus glands, deep to Griffonia simplicifolia lectin II (GSII)-positive mucous neck cells, and surface cells and mucous neck cells showed no or very low expression of miR-148a (Figure 2A). MiR-148a expression was localized in the cytoplasm, especially in the basal side of cells. In contrast to the corpus, the antrum and the duodenum showed little or no expression of miR-148a. Importantly, dual immunostaining with in situ hybridization demonstrated that miR-148a–positive cells expressed the chief cell markers Mist1 and GIF (Figure 2B). Examination of Mist1CreERT2/+;R26RtdTomato/+ mice also showed that Mist1-positive chief cells have strong miR-148a expression (Figure 2C). These data suggest that miR-148a is strongly and specifically expressed in chief cells in the gastric corpus. Next, to evaluate the alteration of expression of miR-148a during the development of SPEM, we examined in situ hybridization analyses in 2 mouse models of acute oxyntic atrophy (administration of either DMP-777 or L635). Compared with normal chief cells, miR-148a was down-regulated in GSII-positive SPEM cells in mice after 10 days of DMP-777 treatment or 3 days of L635 treatment (Figure 3A). Importantly, miR-148a was down-regulated in chief cells without GSII expression after
