Role of ursolic acid chalcone,a synthetic analogue of ursolic acid,in inhibiting the properties of CD133+ sphere-forming cells in liver stem cells

The expression of CD133 decreases with differentiation of tumor cell, indicating that CD133 is a specific marker for isolation and identification of CSCs. In the present study the effect of Ursolic acid chalcone (UAC) on CD133⁺ hepatocellular carcinoma cell (HCC CSCs) differentiation, their self-renewal, tumorigenic capacity and sensitivity to chemotherapeutic drugs was studied. The results demonstrated that UAC inhibits the expression of CD133⁺ in a dose and time-dependent manner in PLC/PRF/5 and Huh7 HCC cells. The inhibition was significant at 50 μM and on day 8. The percentage of CD133⁺ cells decreased from an initial 59.3% in PLC/PRF/5 to 37.1% and 78.2% in Huh7 to 59.2% on treatment with UAC. There was inhibition of Oct4, Tert, Bmi1, β-catenin, ABCG2, and tumor sphere-related gene Ep300. In addition it also decreased number of CK19-positive cells and increased number of CK8/18-positive cells. UAC treatment caused a decrease in self-renewal capability and increase in sensitivity to doxorubicin and vincristine drugs in CD133⁺ HCC CSCs. Therefore, UAC can be a potent therapeutic agent to target differentiation of CSC in HCC.     

The expression profile of Oct4 and Sox2 in the carcinogenesis of oral mucosa

This study is to detect the co-expression of embryonic stem cell-related markers (Oct4 and Sox2) in the carcinogenesis of oral mucosa. The expression profile of these markers was studied by immunohistochemistry assay in rat and human samples. The normal oral mucosa (20 cases) and the transforming oral mucosa (20 cases) were performed in rat samples. The precancerous lesions (20 cases), OSCCs in primary site (116 cases), corresponding epithelial non-cancer tissues adjacent to the OSCC (20 cases) and 46 paired metastatic OSCCs in lymph nodes were performed in human samples. The co-expression of the two markers was defined as both of them are positively detected in the same site of one case under one selected field of microscope. The results indicated that Oct4 and Sox2 were individually detected in normal oral mucosa, but they cannot be co-expressed in the same site of one case. The co-expression of Oct4 and Sox2 (Oct4⁺Sox2⁺) was frequently detected in the transforming oral mucosa of rat (16/20), precancerous lesions of human (12/20) and epithelial non-cancer tissues adjacent to the OSCC (18/20). Also, Oct4⁺Sox2⁺ profile was remarkable noted in the primary sites of OSCCs (38/116). In the 46 paired OSCCs (primary sites with lymph node metastasis), Oct4⁺Sox2⁺ profile (8/46) was less frequently detected than Oct4^low/-Sox2^low/- (14/46) profile in the metastatic sites. To conclude, this study suggests Oct4 and Sox2 are expressed in normal oral mucosa, premalignant diseases, primary sites of OSCCs and metastasis sites of OSCCs. Oct4⁺Sox2⁺ profile may contribute to the malignant transformation of oral mucosa.     

MicroRNAs in metabolism

MicroRNAs (miRNAs) have within the past decade emerged as key regulators of metabolic homoeostasis. Major tissues in intermediary metabolism important during development of the metabolic syndrome, such as β‐cells, liver, skeletal and heart muscle as well as adipose tissue, have all been shown to be affected by miRNAs. In the pancreatic β‐cell, a number of miRNAs are important in maintaining the balance between differentiation and proliferation (miR‐200 and miR‐29 families) and insulin exocytosis in the differentiated state is controlled by miR‐7, miR‐375 and miR‐335. MiR‐33a and MiR‐33b play crucial roles in cholesterol and lipid metabolism, whereas miR‐103 and miR‐107 regulates hepatic insulin sensitivity. In muscle tissue, a defined number of miRNAs (miR‐1, miR‐133, miR‐206) control myofibre type switch and induce myogenic differentiation programmes. Similarly, in adipose tissue, a defined number of miRNAs control white to brown adipocyte conversion or differentiation (miR‐365, miR‐133, miR‐455). The discovery of circulating miRNAs in exosomes emphasizes their importance as both endocrine signalling molecules and potentially disease markers. Their dysregulation in metabolic diseases, such as obesity, type 2 diabetes and atherosclerosis stresses their potential as therapeutic targets. This review emphasizes current ideas and controversies within miRNA research in metabolism.     

Aberrant expression of microRNAs in T cells from patients with ankylosing spondylitis contributes to the immunopathogenesis

Ankylosing spondylitis (AS) is a chronic inflammatory disorder characterized by dysregulated T cells. We hypothesized that the aberrant expression of microRNAs (miRNAs) in AS T cells involved in the pathogenesis of AS. The expression profile of 270 miRNAs in T cells from five AS patients and five healthy controls were analysed by real‐time polymerase chain reaction (PCR). Thirteen miRNAs were found potentially differential expression. After validation, we confirmed that miR‐16, miR‐221 and let‐7i were over‐expressed in AS T cells and the expression of miR‐221 and let‐7i were correlated positively with the Bath Ankylosing Spondylitis Radiology Index (BASRI) of lumbar spine in AS patients. The protein molecules regulated by miR‐16, miR‐221 and let‐7i were measured by Western blotting. We found that the protein levels of Toll‐like receptor‐4 (TLR‐4), a target of let‐7i, in T cells from AS patients were decreased. In addition, the mRNA expression of interferon (IFN)‐γ was elevated in AS T cells. Lipopolysaccharide (LPS), a TLR‐4 agonist, inhibited IFN‐γ secretion by anti‐CD3⁺anti‐CD28 antibodies‐stimulated normal T cells but not AS T cells. In the transfection studies, we found the increased expression of let‐7i enhanced IFN‐γ production by anti‐CD3⁺anti‐CD28⁺ lipopolysaccharide (LPS)‐stimulated normal T cells. In contrast, the decreased expression of let‐7i suppressed IFN‐γ production by anti‐CD3⁺anti‐CD28⁺ LPS‐stimulated AS T cells. In conclusion, we found that miR‐16, miR‐221 and let‐7i were over‐expressed in AS T cells, but only miR‐221 and let‐7i were associated with BASRI of lumbar spine. In the functional studies, the increased let‐7i expression facilitated the T helper type 1 (IFN‐γ) immune response in T cells.     

The circulating microRNome demonstrates distinct lymphocyte subset‐dependent signatures

Upon activation, lymphocytes release vesicles containing microRNAs (miRNAs). However, little is known as to whether this release results in modulation of circulating miRNAs (the miRNome) in the serum. The present work aims to identify lymphocyte subset‐specific signatures of miRNAs within the serum circulating miRNome. We therefore assessed serum miRNA expression profiles in wild‐type mice; in mice lacking either CD4⁺ T cells, CD8⁺ T cells, invariant natural killer T (iNKT) cells, or B cells; and, as a control, in mice in which Dicer has been ablated in T lymphocytes. We found that specific serum miRNAs are differentially modulated when different lymphocyte subsets are lacking. In particular, the serum level of miR‐181b‐5p, previously demonstrated to be fundamental for the development of iNKT cells, is specifically reduced in mice in which iNKT cells are absent. Interestingly, our results indicate a direct link between the biological role of a single miRNA in lymphocyte development and its serum level, and prove that even a population composed of relatively few cells in vivo, such as iNKT lymphocytes, has a measurable effect on the serum circulating miRNome.     

Systematic evaluation of the miRNA‐ome and its downstream effects on mRNA expression identifies gastric cancer progression

We investigated the differential expression of Dicer and Drosha, as well as that of microRNA (miRNA), in adjacent normal and tumour samples of patients with gastric cancer. The expression of Dicer and Drosha was studied by immunohistochemistry in 332 gastric cancers and correlated with clinico‐pathological patient characteristics. Differential expression of miRNAs was studied using the Invitrogen NCode^? Multi‐Species miRNA Microarray Probe Set containing 857 mammalian probes in a test set of six primary gastric cancers (three with and three without lymph node metastases). Differential expression was validated by RT‐PCR on an independent validation set of 20 patients with gastric cancer. Dicer and Drosha were differentially expressed in non‐neoplastic and neoplastic gastric tissue. The expression of Drosha correlated with local tumour growth and was a significant independent prognosticator of patient survival. Twenty miRNAs were up‐ and two down‐regulated in gastric carcinoma compared with non‐neoplastic tissue. Six of these miRNAs separated node‐positive from node‐negative gastric cancers, ie miR‐103, miR‐21, miR‐145, miR‐106b, miR‐146a, and miR‐148a. Five miRNAs expressed differentially in node‐positive cancers had conserved binding sites for mRNAs differentially expressed in the same set of tumour samples. Gastric cancer shows a complex derangement of the miRNA‐ome, including Dicer and Drosha. These changes correlate independently with patient prognosis and probably influence local tumour growth and nodal spread. Copyright © 2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

T cell-expressed microRNAs critically regulate germinal center T follicular helper cell function and maintenance in acute viral infection in mice

Constitutive T cell-intrinsic miRNA expression is required for the differentiation of naïve CD4⁺ T cells into Tfh cells, thus making it difficult to study the role of miRNAs in the maintenance of already established Tfh cells and ongoing germinal center (GC) responses. To overcome this problem, we here used temporally controlled ablation of mature miRNAs specifically in CD4⁺ T cells during acute LCMV infection in mice. T cell-intrinsic miRNA expression was not only critical at early stages of Tfh cell differentiation, but also important for the maintenance of already established Tfh cells. In addition, CD4⁺ T cell-specific ablation of miRNAs resulted in impaired GC B cell responses. Notably, miRNA deficiency also compromised the antigen-specific CD4⁺ T cell compartment, Th1 cells, Treg cells, and Tfr cells. In conclusion, our results highlight miRNAs as important regulators of Tfh cells, thus providing novel insights into the molecular events that govern T cell–B cell interactions and Th cell identity.     

CD133<Superscript>+</Superscript> single cell-derived progenies of colorectal cancer cell line SW480 with different invasive and metastatic potential

Single cell progenies (SCPs) inherit biological properties from their isogenetic mother cells. If a single cancer cell can give rise to progenies, which can be passaged sustainably in vitro and produce tumor in xenotransplantation, the cell should be cancer initiating cell. CD133 (Prominin-1, Prom1) is the marker of human colorectal cancer (CRC) stem cells and probably a marker of metastatic cancer stem cells (CSCs). Thirty-three SCPs of CRC cell line SW480 were isolated by limited dilution methods, thirty of which are CD133 positive and three negative. All of the CD133⁺ SCPs are tumorigenic, and the subcutaneous tumors expanded rapidly, while only 1 of 3 CD133⁻ SCPs developed a minimal tumor in nude mice. Orthotopic transplantation experiments showed that CD133⁺ SCPs possessed heterogeneity in intestinal wall invasion, lymph node and liver metastases. CD133⁺ SCPs varied in cell growth, invasive ability, epithelial-mesenchymal-transition and expression of CSCs markers (CD133, CD44, and CXCR4) associated with metastatic potential. CD133⁻ SCPs did not produce secondary transplanted tumor, intestinal invasion and metastasis. The results indicated CD133⁺ subpopulation of SW480 SCPs bear heterogeneous invasive and metastatic ability, and CRC-CSCs might be a heterogeous subpopulation.     

Clinical and biological implications of CD133-positive and CD133-negative cells in glioblastomas

A number of recent reports have demonstrated that only CD133-positive cancer cells of glioblastoma multiforme (GBM) have tumor-initiating potential. These findings raise an attractive hypothesis that GBMs can be cured by eradicating CD133-positive cancer stem cells (CSCs), which are a small portion of GBM cells. However, as GBMs are known to possess various genetic alterations, GBMs might harbor heterogeneous CSCs with different genetic alterations. Here, we compared the clinical characteristics of two GBM patient groups divided according to CD133-positive cell ratios. The CD133-low GBMs showed more invasive growth and gene expression profiles characteristic of mesenchymal or proliferative subtypes, whereas the CD133-high GBMs showed features of cortical and well-demarcated tumors and gene expressions typical of proneuronal subtype. Both CD133-positive and CD133-negative cells purified from four out of six GBM patients produced typical GBM tumor masses in NOD-SCID brains, whereas brain mass from CD133-negative cells showed more proliferative and angiogenic features compared to that from CD133-positive cells. Our results suggest, in contrast to previous reports that only CD133-positive cells of GBMs can initiate tumor formation in vivo CD133-negative cells also possess tumor-initiating potential, which is indicative of complexity in the identification of cancer cells for therapeutic targeting.     

Progenitor cell niches in the human pancreatic duct system and associated pancreatic duct glands: an anatomical and immunophenotyping study

Pancreatic duct glands (PDGs) are tubule‐alveolar glands associated with the pancreatic duct system and can be considered the anatomical counterpart of peribiliary glands (PBGs) found within the biliary tree. Recently, we demonstrated that endodermal precursor niches exist fetally and postnatally and are composed functionally of stem cells and progenitors within PBGs and of committed progenitors within PDGs. Here we have characterized more extensively the anatomy of human PDGs as novel niches containing cells with multiple phenotypes of committed progenitors. Human pancreata (n = 15) were obtained from cadaveric adult donors. Specimens were processed for histology, immunohistochemistry and immunofluorescence. PDGs were found in the walls of larger pancreatic ducts (diameters > 300 μm) and constituted nearly 4% of the duct wall area. All of the cells identified were negative for nuclear expression of Oct4, a pluripotency gene, and so are presumably committed progenitors and not stem cells. In the main pancreatic duct and in large interlobular ducts, Sox9⁺ cells represented 5–30% of the cells within PDGs and were located primarily at the bottom of PDGs, whereas rare and scattered Sox9⁺ cells were present within the surface epithelium. The expression of PCNA, a marker of cell proliferation, paralleled the distribution of Sox9 expression. Sox9⁺ PDG cells proved to be Pdx1⁺/Ngn3^+/–/Oct4A^?. Nearly 10% of PDG cells were positive for insulin or glucagon. Intercalated ducts contained Sox9⁺/Pdx1⁺/Ngn3⁺ cells, a phenotype that is presumptive of committed endocrine progenitors. Some intercalated ducts appeared in continuity with clusters of insulin‐positive cells organized in small pancreatic islet‐like structures. In summary, PDGs represent niches of a population of Sox9⁺ cells exhibiting a pattern of phenotypic traits implicating a radial axis of maturation from the bottoms of the PDGs to the surface of pancreatic ducts. Our results complete the anatomical background that links biliary and pancreatic tracts and could have important implications for the common patho‐physiology of biliary tract and pancreas.     

Genetic Variants in MicroRNAs and Their Binding Sites Are Associated with the Risk of Parkinson Disease

MicroRNAs (miRNAs) are small noncoding RNAs that serve as key regulators of gene expression. They have been shown to be involved in a wide range of biological processes including neurodegenerative diseases. Genetic variants in miRNAs or miRNA‐binding sites on their target genes could affect miRNA function and contribute to disease risk. Here, we investigated the association of miRNA‐related genetic variants with Parkinson disease (PD) using data from the largest GWAS on PD. Of 243 miRNA variants, we identified rs897984:T>C in miR‐4519 (P value = 1.3×10^?5 and OR = 0.93) and rs11651671:A>G in miR‐548at‐5p (P value = 1.1×10^?6 and OR = 1.09) to be associated with PD. We showed that the variant's mutant alleles change the secondary structure and decrease expression level of their related miRNAs. Subsequently, we highlighted target genes that might mediate the effects of miR‐4519 and miR‐548at‐5p on PD. Among them, we experimentally showed that NSF is a direct target of miR‐4519. Furthermore, among 48,844 miRNA‐binding site variants, we found 32 variants (within 13 genes) that are associated with PD. Four of the host genes, CTSB, STX1B, IGSF9B, and HSD3B7, had not previously been reported to be associated with PD. We provide evidence supporting the potential impact of the identified miRNA‐binding site variants on miRNA‐mediated regulation of their host genes.