Micro-RNA-124-5p promotes insulin producing cell differentiation through regulating transcriptional factor NKX6.1.

Aims: Differentiating human embryonic stem cells into pancreatic ß cells has been proposed as a practical approach to managing diabetes. There have been several protocols attempting to generate ß-like cells or insulin-producing cells (IPCs), but their low efficiency is a common issue. The expression level of Nkx6.1 is crucial for maintaining pancreatic ß cell identity, while the proportion of PDX1 and Nkx6.1 double positive cells were not satisfied in the present protocols, leading to relative low efficiency in the differentiation into IPCs. This study aims to identify the mechanism underlying the regulation of Nkx6.1 during IPC differentiation and provide new insights for diabetes therapy. Methods: In the current study, human embryonic stem cell (hESC) line H1 was used to perform IPC specifications. Immunofluorescence, flow cytometry, and qPCR were conducted to analyze gene expression. In addition, insulin and C-peptide were measured through glucose-stimulated insulin secretion (GSIS) assays and ELISA. Results: We found that the transcription factor NKX6.1, a crucial inducer of early pancreatic development and IPC generation, was downregulated by micro-RNA-124-5p (miR-124-5p) in hESCs during IPC differentiation. Also, we observed that miR-124-5p was upregulated and bound to the 3' untranslated region (3' UTR) of NKX6.1 in pancreatic progenitor (PP), which subsequently suppressed PP differentiation. Moreover, inhibiting miR-124-5p induced the generation of IPCs. Conclusion: The current study results demonstrated an important role for miR-124-5p in regulating NKX6.1 expression, which appears to be a practical strategy for producing IPCs.

Role of Kruppel-Like Factor 5 in Deoxycholic Acid-Mediated Intestinal Transdifferentiation of Esophageal Squamous Epithelium.

Barrett's esophagus (BE) is an acquired condition in which normal squamous epithelium is replaced with metaplastic columnar epithelium as a consequence of gastroesophageal reflux disease. BE is known as a precursor of esophageal adenocarcinoma. Currently, the molecular mechanism underlying epithelial metaplasia in BE patients remains unknown. Therefore, we investigated the role of Krüppel-like factor 5 (KLF5) signaling in the initiation of BE-associated metaplasia. Sprague-Dawley (SD) rats were used to create a surgical model of bile reflux injury. Immunohistochemistry was performed to analyze human and mouse esophageal specimens. Human esophageal squamous epithelial (HET-1A) cells were treated with bile acid and used in transfection experiments. Quantitative real-time PCR and western blot analysis were performed to detect the expression of KLF5, CDX2, MUC2 and villin. Epithelial tissue from both the rat BE model and human BE patients strongly expressed KLF5, CDX2, MUC2, and villin. Bile acid treatment also increased the expression of KLF5, CDX2, MUC2 and villin in esophageal epithelial cells in a time-dependent manner. Moreover, siRNA-mediated knockdown of KLF5 blocked the expression of CDX2, MUC2 and villin, but transfection of a KLF5 expression vector into esophageal epithelial cells promoted their transdifferentiation into columnar-like cells, as demonstrated by increased expression of the intestinal markers CDX2, MUC2 and villin. Thus, in addition to its function as a transcription factor, KLF5 may be linked to an increased risk of BE development.

BMP4 promotes a phenotype change of an esophageal squamous epithelium via up-regulation of KLF4.

INTRODUCTION: Barrett's esophagus is a metaplastic lesion. However, the cellular and molecular mechanisms involved are poorly understood. The aim of this study was to investigate the roles of KLF4 and BMP4 in the pathogenesis of Barrett's epithelium. MATERIALS AND METHODS: Immunohistochemistry was used to analyse the expression of KLF4, BMP4, CDX2, MUC2 and MUC5AC in human esophageal specimens. Human esophageal squamous epithelial cells were subjected to bile acid treatment and used in transfection experiments. Quantitative real-time PCR and Western blot analysis were used to detect the expression of KLF4, BMP4, CDX2, MUC2 and MUC5ac. RESULTS: In human tissues, Barrett's epithelium strongly expressed BMP4, p-Smad1/5/8 and KLF4. Furthermore, bile acids increased the expression of BMP4, KLF4, p-Smad1/5/8, CDX2, MUC2 and MUC5ac in esophageal epithelial cells in a time-dependent manner. Moreover, we found that BMP4 up-regulated the expression of KLF4, CDX2, MUC2 and MUC5ac, but Noggin, a specific BMP4 antagonist, can block the expression of KLF4, CDX2, MUC2 and MUC5ac induced by BMP4. However, BMP4 cannot induce the expression of CDX2, MUC2 and MUC5ac in cells with KLF4 siRNA, and Noggin cannot block the expression of KLF4, CDX2, MUC2 and MUC5ac in cells transfected with the KLF4 expression vector. CONCLUSION: Our results demonstrate that BMP4 promotes a phenotype change of an esophageal squamous epithelium via up-regulation of KLF4.

Deoxycholic acid (DCA) confers an intestinal phenotype on esophageal squamous epithelium via induction of the stemness-associated reprogramming factors OCT4 and SOX2.

Barrett's esophagus (BE) is essentially a metaplasia in which the normal stratified squamous epithelium is replaced by columnar epithelium. This study focuses on the involvement of OCT4 and SOX2, 2 key cell-reprogramming factors, in the deoxycholic acid (DCA)-induced expression of the intestinal hallmarks Cdx2 and MUC2 using both in vivo and in vitro models. Up-regulated expression of OCT4 and down-regulated expression of SOX2 were observed in BE compared with normal esophagus and esophagitis. Consistent with the data in vivo, DCA induced time-dependent expression of OCT4 at both the mRNA and protein levels and decreased nuclear expression of SOX2 in Het-1A cells. Down-regulation of OCT4 expression by siRNA abrogated DCA-induced expression of Cdx2 and MUC2, whereas siRNA against SOX2 significantly upregulated the expression of both Cdx2 and MUC2. Our data indicate that both OCT4 and SOX2 play important roles in the development of BE triggered by bile acid reflux.

Prevalence, histologic and clinical characteristics of heterotopic gastric mucosa in Chinese patients.

AIM: To determine the prevalence, demographic, clinical and histopathologic features of heterotopic gastric mucosa (HGM) in Chinese patients. METHODS: Patients referred to three endoscopy units were enrolled in this study. The macroscopic characteristics of HGM were documented. Biopsies were obtained and observed using hematoxylin and eosin staining. Helicobacter pylori colonization was examined by Whartin-Starry staining. RESULTS: HGM was observed in 420 Chinese patients, yielding a prevalence of 0.4%. The majority of patients had a single patch (300/420; 71.4%), while the remainder had two (84/420; 20%) or multiple patches (36/420; 8.6%). The size of the patches and the distance from the patch to the frontal incisor teeth varied significantly. The large majority of HGM patches were flat (393/420; 93.6%), whereas the remaining patches were slightly elevated. The primary histological characteristic was fundic-type (216/420; 51.4%) within the HGM patch, and antral- (43/420; 10.2%) and transitional-type (65/420; 15.5%) mucosa were also observed. The prevalence of intestinal metaplasia was 3.1% (13/420) and the prevalence of dysplasia was 1.4% (6/420), indicating the necessity for endoscopic follow-up in patients with HGM. Esophageal and extraesophageal complaints were also observed in patients with HGM. Dysphagia and epigastric discomfort (odds ratios: 6.836 and 115.826, respectively; Ps < 0.05) were independent risk factors for HGM. CONCLUSION: Clinical complaints should be considered to improve the detection rate of HMG. The prevalence of intestinal metaplasia and dysplasia also indicates a need for endoscopic follow-up.