MicroRNA-22 can reduce parathymosin expression in transdifferentiated hepatocytes

Pancreatic acinar cells AR42J-B13 can transdifferentiate into hepatocyte-like cells permissive for efficient hepatitis B virus (HBV) replication. Here, we profiled miRNAs differentially expressed in AR42J-B13 cells before and after transdifferentiation to hepatocytes, using chip-based microarray. Significant increase of miRNA expression, including miR-21, miR-22, and miR-122a, was confirmed by stem-loop real-time PCR and Northern blot analyses. In contrast, miR-93, miR-130b, and a number of other miRNAs, were significantly reduced after transdifferentiation. To investigate the potential significance of miR-22 in hepatocytes, we generated cell lines stably expressing miR-22. By 2D-DIGE, LC-MS/MS, and Western blot analyses, we identified several potential target genes of miR-22, including parathymosin. In transdifferentiated hepatocytes, miR-22 can inhibit both mRNA and protein expression of parathymosin, probably through a direct and an indirect mechanism. We tested two computer predicted miR-22 target sites at the 3' UTR of parathymosin, by the 3' UTR reporter gene assay. Treatment with anti-miR-22 resulted in significant elevation of the reporter activity. In addition, we observed an in vivo inverse correlation between miR-22 and parathymosin mRNA in their tissue distribution in a rat model. The phenomenon that miR-22 can reduce parathymosin protein was also observed in human hepatoma cell lines Huh7 and HepG2. So far, we detected no major effect on several transdifferentiation markers when AR42J-B13 cells were transfected with miR-22, or anti-miR-22, or a parathymosin expression vector, with or without dexamethasone treatment. Therefore, miR-22 appears to be neither necessary nor sufficient for transdifferentiation. We discussed the possibility that altered expression of some other microRNAs could induce cell cycle arrest leading to transdifferentiation.     

Upregulation of miR-760 and miR-186 Is Associated with Replicative Senescence in Human Lung Fibroblast Cells

We have previously shown that microRNAs (miRNAs) miR-760, miR-186, miR-337-3p, and miR-216b stimulate premature senescence through protein kinase CK2 (CK2) down-regulation in human colon cancer cells. Here, we examined whether these four miRNAs are involved in the replicative senescence of human lung fibroblast IMR-90 cells. miR-760 and miR-186 were significantly upregulated in replicatively senescent IMR-90 cells, and their joint action with both miR-337-3p and miR-216b was necessary for efficient downregulation of the α subunit of CK2 (CK2α) in IMR-90 cells. A mutation in any of the four miRNA-binding sequences within the CK2α 3′-untranslated region (UTR) indicated that all four miRNAs should simultaneously bind to the target sites for CK2α downregulation. The four miRNAs increased senescence-associated β-galactosidase (SA-β-gal) staining, p53 and p21^Cip1/WAF1 expression, and reactive oxygen species (ROS) production in proliferating IMR-90 cells. CK2α over-expression almost abolished this event. Taken together, the present results suggest that the upregulation of miR-760 and miR-186 is associated with replicative senescence in human lung fibroblast cells, and their cooperative action with miR-337-3p and miR-216b may induce replicative senescence through CK2α downregulation-dependent ROS generation.     

Dependence of Intracellular and Exosomal microRNAs on Viral E6/E7 Oncogene Expression in HPV-positive Tumor Cells

Specific types of human papillomaviruses (HPVs) cause cervical cancer. Cervical cancers exhibit aberrant cellular microRNA (miRNA) expression patterns. By genome-wide analyses, we investigate whether the intracellular and exosomal miRNA compositions of HPV-positive cancer cells are dependent on endogenous E6/E7 oncogene expression. Deep sequencing studies combined with qRT-PCR analyses show that E6/E7 silencing significantly affects ten of the 52 most abundant intracellular miRNAs in HPV18-positive HeLa cells, downregulating miR-17-5p, miR-186-5p, miR-378a-3p, miR-378f, miR-629-5p and miR-7-5p, and upregulating miR-143-3p, miR-23a-3p, miR-23b-3p and miR-27b-3p. The effects of E6/E7 silencing on miRNA levels are mainly not dependent on p53 and similarly observed in HPV16-positive SiHa cells. The E6/E7-regulated miRNAs are enriched for species involved in the control of cell proliferation, senescence and apoptosis, suggesting that they contribute to the growth of HPV-positive cancer cells. Consistently, we show that sustained E6/E7 expression is required to maintain the intracellular levels of members of the miR-17~92 cluster, which reduce expression of the anti-proliferative p21 gene in HPV-positive cancer cells. In exosomes secreted by HeLa cells, a distinct seven-miRNA-signature was identified among the most abundant miRNAs, with significant downregulation of let-7d-5p, miR-20a-5p, miR-378a-3p, miR-423-3p, miR-7-5p, miR-92a-3p and upregulation of miR-21-5p, upon E6/E7 silencing. Several of the E6/E7-dependent exosomal miRNAs have also been linked to the control of cell proliferation and apoptosis. This study represents the first global analysis of intracellular and exosomal miRNAs and shows that viral oncogene expression affects the abundance of multiple miRNAs likely contributing to the E6/E7-dependent growth of HPV-positive cancer cells.     

Sox9-Regulated miRNA-574-3p Inhibits Chondrogenic Differentiation of Mesenchymal Stem Cells

The aim of this study was to identify new microRNAs (miRNAs) that are modulated during the differentiation of mesenchymal stem cells (MSCs) toward chondrocytes. Using large scale miRNA arrays, we compared the expression of miRNAs in MSCs (day 0) and at early time points (day 0.5 and 3) after chondrogenesis induction. Transfection of premiRNA or antagomiRNA was performed on MSCs before chondrogenesis induction and expression of miRNAs and chondrocyte markers was evaluated at different time points during differentiation by RT-qPCR. Among miRNAs that were modulated during chondrogenesis, we identified miR-574-3p as an early up-regulated miRNA. We found that miR-574-3p up-regulation is mediated via direct binding of Sox9 to its promoter region and demonstrated by reporter assay that retinoid X receptor (RXR)α is one gene specifically targeted by the miRNA. In vitro transfection of MSCs with premiR-574-3p resulted in the inhibition of chondrogenesis demonstrating its role during the commitment of MSCs towards chondrocytes. In vivo, however, both up- and down-regulation of miR-574-3p expression inhibited differentiation toward cartilage and bone in a model of heterotopic ossification. In conclusion, we demonstrated that Sox9-dependent up-regulation of miR-574-3p results in RXRα down-regulation. Manipulating miR-574-3p levels both in vitro and in vivo inhibited chondrogenesis suggesting that miR-574-3p might be required for chondrocyte lineage maintenance but also that of MSC multipotency.     

A Pituitary Homeobox 2 (Pitx2):microRNA-200a-3p:β-catenin Pathway Converts Mesenchymal Cells to Amelogenin-expressing Dental Epithelial Cells

Pitx2, Wnt/β-catenin signaling, and microRNAs (miRs) play a critical role in the regulation of dental stem cells during embryonic development. In this report, we have identified a Pitx2:β-catenin regulatory pathway involved in epithelial cell differentiation and conversion of mesenchymal cells to amelogenin expressing epithelial cells via miR-200a. Pitx2 and β-catenin are expressed in the labial incisor cervical loop or epithelial stem cell niche, with decreased expression in the differentiating ameloblast cells of the mouse lower incisor. Bioinformatics analyses reveal that miR-200a-3p expression is activated in the pre-ameloblast cells to enhance epithelial cell differentiation. We demonstrate that Pitx2 activates miR-200a-3p expression and miR-200a-3p reciprocally represses Pitx2 and β-catenin expression. Pitx2 and β-catenin interact to synergistically activate gene expression during odontogenesis and miR-200a-3p attenuates their expression and directs differentiation. To understand how this mechanism controls cell differentiation and cell fate, oral epithelial and odontoblast mesenchymal cells were reprogrammed by a two-step induction method using Pitx2 and miR-200a-3p. Conversion to amelogenin expressing dental epithelial cells involved an up-regulation of the stem cell marker Sox2 and proliferation genes and decreased expression of mesenchymal markers. E-cadherin expression was increased as well as ameloblast specific factors. The combination of Pitx2, a regulator of dental stem cells and miR-200a converts mesenchymal cells to a fully differentiated dental epithelial cell type. This pathway and reprogramming can be used to reprogram mesenchymal or oral epithelial cells to dental epithelial (ameloblast) cells, which can be used in tissue repair and regeneration studies.     

MicroRNA signatures of iPSCs and endoderm-derived tissues

MicroRNAs (miRNAs), small non-coding RNAs that fine-tune gene expression, play multiple roles in the cell, including cell fate specification. We have analyzed the differential expression of miRNAs during fibroblast reprogramming into induced pluripotent stem cells (iPSCs) and endoderm induction from iPSCs upon treatment with high concentrations of Activin-A. The reprogrammed iPSCs assumed an embryonic stem cell (ESC)-like miRNA signature, marked by the induction of pluripotency clusters miR-290–295 and miR-302/367 and conversely the downregulation of the let-7 family. On the other hand, endoderm induction in iPSCs resulted in the upregulation of 13 miRNAs. Given that the liver and the pancreas are common derivatives of the endoderm, analysis of the expression of these 13 upregulated miRNAs in hepatocytes and pancreatic islets revealed a tendency for these miRNAs to be expressed more in pancreatic islets than in hepatocytes. These observations provide insights into how differentiation may be guided more efficiently towards the endoderm and further into the liver or pancreas. Moreover, we also report novel miRNAs enriched for each of the cell types analyzed.     

PSD-95 protects the pancreas against pathological damage through p38 MAPK signaling pathway in acute pancreatitis

Acute pancreatitis is one of the leading causes of gastrointestinal disorder-related hospitalizations, yet its pathogenesis remains to be fully elucidated. Postsynaptic density protein-95 (PSD-95) is closely associated with tissue inflammation and injury. We aimed to investigate the expression of PSD-95 in pancreatic acinar cells, and its function in regulating the inflammatory response and pancreatic pathological damage in acute pancreatitis. A mouse model of edematous acute pancreatitis was induced with caerulein and lipopolysaccharide in C57BL/6 mice. Tat-N-dimer was injected to inhibit the PSD-95 activity separately, or simultaneously with SB203580, inhibitor of p38 MAPK phosphorylation. Rat pancreatic acinar cells AR42J were cultured with 1 μM caerulein to build a cell model of acute pancreatitis. PSD-95-knockdown and negative control cell lines were constructed by lentiviral transfection of AR42J cells. Paraffin-embedded pancreatic tissue samples were processed for routine HE staining to evaluate the pathological changes of human and mouse pancreatic tissues. Serum amylase and inflammatory cytokine levels were detected with specific ELISA kits. Immunofluorescence, immunohistochemical, Western-blot, and qRT-PCR were used to detect the expression levels of PSD-95, p38, and phosphorylated p38. Our findings showed that PSD-95 is expressed in the pancreatic tissues of humans, C57BL/6 mice, and AR42J cells, primarily in the cytoplasm. PSD-95 expression increased at 2 h, reaching the peak at 6 h in mice and 12 h in AR42J cells. IL-6, IL-8, and TNF-α increased within 2 h of disease induction. The pancreatic histopathologic score was greater in the PSD-95 inhibition group compared with the control (P < 0.05), while it was lesser when phosphorylation of p38 MAPK was inhibited compared with the PSD-95 inhibition group (P < 0.05). Moreover, phosphorylation of p38 MAPK increased statistically after PSD-95 knocked-down. In conclusion, PSD-95 effectively influences the pathological damage of the pancreas in acute pancreatitis by affecting the phosphorylation of p38 MAPK.     

Effect of microRNA-27a-5p on apoptosis and inflammatory response of pancreatic acinar cells in acute pancreatitis by targeting PTEN

To investigate the apoptosis and inflammatory response of microRNA-27a-5p (miR-27a-5p) in pancreatic acinar cells of acute pancreatitis (AP) and its related mechanisms. Rat pancreatic acinar cell line AR42J was treated with caerulein (10nmol/L) to construct an acute pancreatitis cell model. Quantitative real-time polymerase chain reaction was performed to measure the expression of miR-27a-5p; The miR-27a-5p mimic was transfected into cell, and the apoptosis rate of the cells was detected by flow cytometry; The levels of TNF-α, IL-1, and IL-6 in the culture supernatant were determined by enzyme-linked immunosorbent assay; TargetScans database predicted and dual luciferase reporter gene assay verified the relationship between miR-27a-5p and the phosphatase and tensin homolog deleted on chromosome 10 (PTEN); The recovery experiment explored the apoptosis and the effects of inflammatory responses. The expression of miR-27a-5p decreased gradually (P < 0.05) and the expression of PTEN increased gradually (P < 0.05) with the prolongation of acting time. Upregulation of miR-27a-5p significantly promoted cell apoptosis (P < 0.05) and inhibited inflammatory response (P < 0.05); The TargetScans database predicted that the 3'UTR of PTEN contains a base complementary to the miR-27a-5p seed region. Cotransfection of wild-type vector (PTEN-WT) with miR-27a-5p mimic or miR-27a-5p inhibitor significantly affected the relative activity of luciferase (P < 0.05), and no significant impact was observed in mutant PTEN-MUT. Compared with miR-27a-5p + pcDNA group, transfection of miR-27a-5p mimic and pcDNA-PTEN significantly increased the expression of PTEN (P < 0.05), decreased the apoptotic rate (P < 0.05), and increased the inflammatory response (P < 0.05). miR-27a-5p induced apoptosis and inhibited the inflammatory response of pancreatic acinar cells in AP by targeting PTEN.     

Pancreatic acinar cells utilize tyrosine to synthesize L-dihydroxyphenylalanine

The pancreatic β cells can synthesize dopamine by taking L-dihydroxyphenylalanine, but whether pancreatic acinar cells synthesize dopamine has not been confirmed. By means of immunofluorescence, the tyrosine hydroxylase -immunoreactivity and aromatic amino acid decarboxylase (AADC)- immunoreactivity were respectively observed in pancreatic acinar cells and islet β cells. Treatment with L-dihydroxyphenylalanine, not tyrosine, caused the production of dopamine in the incubation of INS-1 cells (rat islet β cell line) and primary isolated islets, which was blocked by AADC inhibitor NSD-1015. However, only L-dihydroxyphenylalanine, but not dopamine, was detected when AR42J cells (rat pancreatic acinar cell line) were treated with tyrosine, which was blocked by tyrosine hydroxylase inhibitor AMPT. Dopamine was detected in the coculture of INS-1 cells with AR42J cells after treatment with tyrosine. In an in vivo study, pancreatic juice contained high levels of L-dihydroxyphenylalanine and dopamine. Both L-dihydroxyphenylalanine and dopamine accompanied with pancreatic enzymes and insulin in the pancreatic juice were all significantly increased after intraperitoneal injection of bethanechol chloride and their increases were all blocked by atropine. Inhibiting TH with AMPT blocked bethanechol chloride-induced increases in L-dihydroxyphenylalanine and dopamine, while inhibiting AADC with NSD-1015 only blocked the dopamine increase. Bilateral subdiaphragmatic vagotomy of rats leads to significant decreases of L-dihydroxyphenylalanine and dopamine in pancreatic juice. These results suggested that pancreatic acinar cells could utilize tyrosine to synthesize L-dihydroxyphenylalanine, not dopamine. Islet β cells only used L-dihydroxyphenylalanine, not tyrosine, to synthesize dopamine. Both L-dihydroxyphenylalanine and dopamine were respectively released into the pancreatic duct, which was regulated by the vagal cholinergic pathway. The present study provides important evidences for the source of L-dihydroxyphenylalanine and dopamine in the pancreas.     

MicroRNAs Regulate Human Adipocyte Lipolysis: Effects of miR-145 Are Linked to TNF-α

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression and have multiple effects in various tissues including adipose inflammation, a condition characterized by increased local release of the pro-lipolytic cytokine tumor necrosis factor-alpha (TNF-α). Whether miRNAs regulate adipocyte lipolysis is unknown. We set out to determine whether miRNAs affect adipocyte lipolysis in human fat cells. To this end, eleven miRNAs known to be present in human adipose tissue were over-expressed in human in vitro differentiated adipocytes followed by assessments of TNF-α and glycerol levels in conditioned media after 48 h. Three miRNAs (miR-145, -26a and let-7d) modulated both parameters in parallel. However, while miR-26a and let-7d decreased, miR-145 increased both glycerol release and TNF-α secretion. Further studies were focused therefore on miR-145 since this was the only stimulator of lipolysis and TNF-α secretion. Time-course analysis demonstrated that miR-145 over-expression up-regulated TNF-α expression/secretion followed by increased glycerol release. Increase in TNF-α production by miR-145 was mediated via activation of p65, a member of the NF-κB complex. In addition, miR-145 down-regulated the expression of the protease ADAM17, resulting in an increased fraction of membrane bound TNF-α, which is the more biologically active form of TNF-α. MiR-145 overexpression also increased the phosphorylation of activating serine residues in hormone sensitive lipase and decreased the mRNA expression of phosphodiesterase 3B, effects which are also observed upon TNF-α treatment in human adipocytes. We conclude that miR-145 regulates adipocyte lipolysis via multiple mechanisms involving increased production and processing of TNF-α in fat cells.     

DNA Methylation-mediated Repression of miR-941 Enhances Lysine (K)-specific Demethylase 6B Expression in Hepatoma Cells

MicroRNAs (miRNAs) have been shown to play important roles in carcinogenesis. However, their underlying mechanisms of action in hepatocellular carcinoma (HCC) are poorly understood. Recent evidence suggests that epigenetic silencing of miRNAs through tumor suppression by CpG island hypermethylation may be a common hallmark of human tumors. Here, we demonstrated that miR-941 was significantly down-regulated in HCC tissues and cell lines and was generally hypermethylated in HCC. The overexpression of miR-941 suppressed in vitro cell proliferation, migration, and invasion and inhibited the metastasis of HCC cells in vivo. Furthermore, the histone demethylase KDM6B (lysine (K)-specific demethylase 6B) was identified as a direct target of miR-941 and was negatively regulated by miR-941. The ectopic expression of KDM6B abrogated the phenotypic changes induced by miR-941 in HCC cells. We demonstrated that miR-941 and KDM6B regulated the epithelial-mesenchymal transition process and affected cell migratory/invasive properties.     

Regulation of NF-κB signaling by oxidized glycerophospholipid and IL-1β induced miRs-21-3p and -27a-5p in human aortic endothelial cells

Exposure of endothelial cells (ECs) to agents such as oxidized glycerophospholipids (oxGPs) and cytokines, known to accumulate in atherosclerotic lesions, perturbs the expression of hundreds of genes in ECs involved in inflammatory and other biological processes. We hypothesized that microRNAs (miRNAs) are involved in regulating the inflammatory response in human aortic endothelial cells (HAECs) in response to oxGPs and interleukin 1β (IL-1β). Using next-generation sequencing and RT-quantitative PCR, we characterized the profile of expressed miRNAs in HAECs pre- and postexposure to oxGPs. Using this data, we identified miR-21-3p and miR-27a-5p to be induced 3- to 4-fold in response to oxGP and IL-1β treatment compared with control treatment. Transient overexpression of miR-21-3p and miR-27a-5p resulted in the downregulation of 1,253 genes with 922 genes overlapping between the two miRNAs. Gene Ontology functional enrichment analysis predicted that the two miRNAs were involved in the regulation of nuclear factor κB (NF-κB) signaling. Overexpression of these two miRNAs leads to changes in p65 nuclear translocation. Using 3′ untranslated region luciferase assay, we identified 20 genes within the NF-κB signaling cascade as putative targets of miRs-21-3p and -27a-5p, implicating these two miRNAs as modulators of NF-κB signaling in ECs.     

Reciprocal inhibition between miR-26a and NF-κB regulates obesity-related chronic inflammation in chondrocytes

Obesity is causally linked to osteoarthritis (OA), with the mechanism being not fully elucidated. miRNAs (miRs) are pivotal regulators of various diseases in multiple tissues, including inflammation in the chondrocytes. In the present study, we for the first time identified the expression of miR-26a in mouse chondrocytes. Decreased level of miR-26a was correlated to increased chronic inflammation in the chondrocytes and circulation in obese mouse model. Mechanistically, we demonstrated that miR-26a attenuated saturated free fatty acid-induced activation of NF-κB (p65) and production of proinflammatory cytokines in chondrocytes. Meanwhile, NF-κB (p65) also suppressed miR-26a production by directly binding to a predicted NF-κB binding element in the promoter region of miR-26a. Finally, we observed a negative correlation between NF-κB and miR-26a in human patients with osteoarthritis. Thus, we identified a reciprocal inhibition between miR-26a and NF-κB downstream of non-esterified fatty acid (NEFA) signalling in obesity-related chondrocytes. Our findings provide a potential mechanism linking obesity to cartilage inflammation.     

Osteogenic Differentiation Capacity of Human Skeletal Muscle-Derived Progenitor Cells

Heterotopic ossification (HO) is defined as the formation of ectopic bone in soft tissue outside the skeletal tissue. HO is thought to result from aberrant differentiation of osteogenic progenitors within skeletal muscle. However, the precise origin of HO is still unclear. Skeletal muscle contains two kinds of progenitor cells, myogenic progenitors and mesenchymal progenitors. Myogenic and mesenchymal progenitors in human skeletal muscle can be identified as CD56⁺ and PDGFRα⁺ cells, respectively. The purpose of this study was to investigate the osteogenic differentiation potential of human skeletal muscle-derived progenitors. Both CD56⁺ cells and PDGFRα⁺ cells showed comparable osteogenic differentiation potential in vitro. However, in an in vivo ectopic bone formation model, PDGFRα⁺ cells formed bone-like tissue and showed successful engraftment, while CD56⁺ cells did not form bone-like tissue and did not adapt to an osteogenic environment. Immunohistological analysis of human HO sample revealed that many PDGFRα⁺ cells were localized in proximity to ectopic bone formed in skeletal muscle. MicroRNAs (miRNAs) are known to regulate many biological processes including osteogenic differentiation. We investigated the participation of miRNAs in the osteogenic differentiation of PDGFRα⁺ cells by using microarray. We identified miRNAs that had not been known to be involved in osteogenesis but showed dramatic changes during osteogenic differentiation of PDGFRα⁺ cells. Upregulation of miR-146b-5p and -424 and downregulation of miR-7 during osteogenic differentiation of PDGFRα⁺ cells were confirmed by quantitative real-time RT-PCR. Inhibition of upregulated miRNAs, miR-146b-5p and -424, resulted in the suppression of osteocyte maturation, suggesting that these two miRNAs have the positive role in the osteogenesis of PDGFRα⁺ cells. Our results suggest that PDGFRα⁺ cells may be the major source of HO and that the newly identified miRNAs may regulate osteogenic differentiation process of PDGFRα⁺ cells.