scholarly journals MicroRNA-21 is upregulated during the proliferative phase of liver regeneration, targets Pellino-1, and inhibits NF-κB signaling

2010 ◽  
Vol 298 (4) ◽  
pp. G535-G541 ◽  
Author(s):  
Rebecca T. Marquez ◽  
Erik Wendlandt ◽  
Courtney Searcey Galle ◽  
Kathy Keck ◽  
Anton P. McCaffrey
Keyword(s):  
Liver Regeneration ◽  
Cultured Cells ◽  
Inverse Correlation ◽  
Luciferase Reporter ◽  
Mrna Levels ◽  
Messenger Rnas ◽  
Early Stages ◽  
Small Regulatory Rnas ◽  
Reporter Assays ◽  
Parenchymal Cells

During liver regeneration, normally quiescent liver cells reenter the cell cycle, nonparenchymal and parenchymal cells divide, and proper liver architecture is restored. The gene expression programs regulating these transitions are not completely understood. MicroRNAs are a newly discovered class of small regulatory RNAs that silence messenger RNAs by binding to their 3′-untranslated regions (UTRs). A number of microRNAs, including miR-21, have been shown to be involved in regulation of cell proliferation. We performed partial hepatectomies on mice and allowed the liver to regenerate for 1, 6, 12, 24, and 48 h and 4 and 7 days. We compared the expression of miR-21 in the posthepatectomy liver to the prehepatectomy liver by Northern blot and found that miR-21 was upregulated during the early stages of liver regeneration. NF-κB signaling is also activated very early during liver regeneration. It has been previously reported that NF-κB upregulates the miR-21 precursor transcript. The predicted miR-21 target, Pellino ( Peli1), is a ubiquitin ligase involved in activating NF-κB signaling. We observed an inverse correlation between miR-21 and Peli1 mRNA levels during liver regeneration. miR-21 overexpression in cultured cells inhibited a Peli1 3′-UTR luciferase reporter. Using NF-κB reporter assays, we determined that miR-21 overexpression inhibits NF-κB signaling. In conclusion, miR-21 expression was upregulated during early stages of liver regeneration. Targeting of Peli1 by miR-21 could potentially provide the basis for a negative feedback cycle regulating NF-κB signaling.

LINC00265 maintains hepatocyte proliferation during liver regeneration by targeting miRNA-28-5p

2021 ◽  
Vol 85 (3) ◽  
pp. 528-536
Author(s):  
Sheng Yu ◽  
Zhonglin Cui ◽  
Jie Zhou ◽  
Kai Wang ◽  
Qingping Li ◽  
...  
Keyword(s):  
Liver Resection ◽  
Liver Regeneration ◽  
Therapeutic Option ◽  
Weight Ratio ◽  
Hepatocyte Proliferation ◽  
Phase Cell ◽  
Luciferase Reporter ◽  
Hepatocyte Regeneration ◽  
M Phase ◽  
Reporter Assays

ABSTRACT Long noncoding RNAs have been implicated in many biological processes, but their roles in liver regeneration still need to be illustrated. Therefore, we aimed to investigate the role of LINC00265 as a pivotal regulator of hepatocyte proliferation during liver regeneration. It was found that LINC00265 is significantly upregulated in rat liver tissues at various time points after 2/3 liver resection. LINC00265 knockdown inhibited hepatocyte proliferation, induced cell apoptosis and led to G2/M phase cell cycle arrestment. In rats subjected to surgery, LINC00265 knockdown decreased liver/body weight ratio, attenuated improvement from liver damage and reduced Ki67 and PCNA expression. Luciferase reporter assays confirmed that miR-28-5p was a direct target of LINC00265, and inhibition of miR-28-5p abolished the effect of LINC00265 knockdown. In summary, LINC00265 might maintain hepatocyte proliferation by targeting miR-28-5p during liver regeneration and should be considered as a promising therapeutic option for hepatocyte regeneration after liver resection.

scholarly journals miR-301-3p directly regulates Cx43 to mediate the development of gastric cancer

2021 ◽  
Vol 49 (9) ◽  
pp. 030006052110331
Author(s):  
Shasha Liu ◽  
Yang Zhao ◽  
Huan Liu ◽  
Xing Zhao ◽  
Xingbin Shen
Keyword(s):  
Gastric Cancer ◽  
Cell Lines ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Mrna Levels ◽  
Clinical Prognosis ◽  
Expression Levels ◽  
Reporter Assays ◽  
Cx43 Mrna ◽  
Development Of Gastric Cancer

Objective Identifying novel biomarkers involved in the development of gastric cancer (GC) can provide potential therapeutic strategies and improve clinical prognosis. miR-301-3p and Cx43 are reportedly dysregulated in GC. miR-301-3p and Cx43 interaction, and their functions in GC progression, are still poorly understood. Methods The expression levels of miR-301-3p and Cx43 in GC tissues and cell lines with various differentiation degrees were evaluated by RT-qPCR. The interaction between miR-301-3p and Cx43 was assessed by dual-luciferase reporter assays. CCK8 and Transwell assays were employed to assess the effects of the miR-301-3p- Cx43 axis on GC cell proliferation, migration, and invasion. Results Cx43 was significantly downregulated in GC tissues and cell lines, while miR-301-3p expression was negatively correlated with Cx43 mRNA levels. The expression levels of Cx43 and miR-301-3p were closely associated with the differentiation, TNM stage, vascular invasion, and lymph node metastasis status of GC patients. Cx43 overexpression could suppress the proliferation, migration, and invasion of GC cells. Cx43 mRNA is a direct target of miR-301-3p, and transfection of an miR-301-3p mimic could reverse the inhibitory effects of Cx43. Conclusion The miR-301-3p- Cx43 axis is involved in the development and progression of GC by affecting the proliferation, migration, and invasion of GC cells.

scholarly journals CRP Stimulates GDF15 Expression in Endothelial Cells through p53

2018 ◽  
Vol 2018 ◽  
pp. 1-9 ◽  
Author(s):  
Yoonseo Kim ◽  
Nicole Noren Hooten ◽  
Michele K. Evans
Keyword(s):  
Endothelial Cells ◽  
Luciferase Reporter ◽  
Mrna Levels ◽  
Aortic Endothelial Cells ◽  
Reactive Protein ◽  
Direct Target Gene ◽  
Reporter Assays ◽  
Direct Target ◽  
Human Aortic Endothelial Cells

Growth differentiation factor 15 (GDF15) is a multifunctional, secreted protein that is a direct target gene of p53. GDF15 is a prospective biomarker of cardiovascular disease (CVD). C-reactive protein (CRP), like GDF15, is implicated in inflammation and an independent biomarker of CVD. However, the molecular interactions between GDF15 and CRP remain unexplored. In women, we found a significant relationship between hsCRP and GDF15 serum and mRNA levels. In vitro treatment of cultured human aortic endothelial cells (HAECs) with purified CRP or transfection of a CRP plasmid into HAECs induced GDF15 expression. Dual-luciferase reporter assays confirmed that CRP significantly increased the levels of GDF15 promoter luciferase activity, indicating that CRP induces GDF15 transcription. Chromatin immunoprecipitation (ChIP) assays confirmed that p53 was recruited to both p53 binding sites 1 and 2 in the GDF15 promoter in response to CRP. We have uncovered a linkage between CRP and GDF15, a new clue that could be important in the pathogenesis of endothelial inflammation.

scholarly journals MicroRNA-34A inhibits the growth, invasion and metastasis of gastric cancer by targeting PDGFR and MET expression

2014 ◽  
Vol 34 (3) ◽  
Author(s):  
Yang Peng ◽  
Jin-Jun Guo ◽  
Yan-Min Liu ◽  
Xiao-Ling Wu
Keyword(s):  
Gastric Cancer ◽  
Tyrosine Kinases ◽  
Cancer Metastasis ◽  
Cell Signalling ◽  
Growth Factor Receptor ◽  
Luciferase Reporter ◽  
Messenger Rnas ◽  
Reporter Assays ◽  
Phosphoinositide 3 Kinase ◽  
Cancer Tissues

Within the family of RTKs (receptor tyrosine kinases), PDGFR (platelet-derived growth factor receptor) has been implicated in carcinogenesis and tumour development. miRNAs (microRNAs), which can target the mRNAs (messenger RNAs) of cancer-associated genes, are abnormally expressed in various cancers. In this study, our aim was to identify the miRNAs that target PDGFR-α/β and to study the functions of these miRNAs. miR-34a was predicted to target PDGFR, and luciferase reporter assays showed that miR-34a could directly target PDGFR. Meanwhile, we found that miR-34a was down-regulated in gastric cancer tissues and was associated with metastasis. Our findings showed that miR-34a could inhibit gastric cancer cell migration, invasion and proliferation, but these tumourigenic properties were only partially restored when PDGFR-α/β was overexpressed. In subsequent experiments, we found that the overexpression of both PDGFR and MET could completely restore the gastric cancer tumourigenic properties. Moreover, the cancer-associated cell signalling pathway was studied, and we found that miR-34a could inhibit Akt [PKB (protein kinase B)] phosphorylation, which was restored by the overexpression of both PDGFR and MET. In conclusion, miR-34a may act as a potential tumour suppressor in gastric cancer and is associated with the mechanisms of gastric cancer metastasis; miR-34a can inhibit gastric cancer tumourigenesis by targeting PDGFR and MET through the PI3K (phosphoinositide 3-kinase)/Akt pathway.

scholarly journals MiR-155 promotes anaplastic thyroid cancer progression by directly targeting SOCS1

BMC Cancer ◽  
2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Wei Zhang ◽  
Wenyue Ji ◽  
Xudong Zhao
Keyword(s):  
Thyroid Cancer ◽  
Cancer Progression ◽  
Inverse Correlation ◽  
Anaplastic Thyroid Cancer ◽  
Luciferase Reporter ◽  
Rare Type ◽  
Invasion And Migration ◽  
Extrathyroidal Invasion ◽  
Reporter Assays ◽  
And Migration

Abstract Background Anaplastic thyroid cancer (ATC) is considered to be a rare type of thyroid cancer but takes up the most important proportion of thyroid cancer-related deaths. Therefore, the development of molecular targeted therapy is an exciting strategy in the management of ATC. Methods miR-155 and SOCS1 expression were measured by qRT-PCR as well as western blot analysis. 8305c and FRO cells were transfected and cultured for apoptosis assays, transwell, MTT on miR-155 or SOCS1 suppression and overexpression. Dual-luciferase reporter assays and SOCS1 restoration experimentswas implemented for define the relation between SOCS1 and miR-155. In addition, the correlation between miR-155 expression and patients’ clinicopathological features were also explored. Results Aberrant miR-155 and SOCS1 expression and inverse correlation were found in ATC samples. In addition, it indicated that miR-155 expression correlated with cervical metastasis as well as extrathyroidal invasion. Moreover, we demonstrated that miR-155 inhibited 8305c and FRO cells apoptosis, promoted proliferation, invasion and migration. Furthermore, miR-155 inhibition was associated with a significant overexpression of SOCS1. Additionally, luciferase reporter assays presented that miR-155 could bind to SOCS1 3′-UTR, influencing its stability negatively and finally lowering SOCS1 levels. Moreover, it was illustrated that the impacts of miR-155 suppression were reversed by the inhibition of SOCS1 on cell proliferation, apoptosis as well as invasion. Conclusions Aberrant miR-155/SOCS1 expression has been included in ATC progression: miR-155 overexpression leads to SOCS1 suppression and develops ATC progression. Thus, miR-155 has been considered to be an underlying therapeutic target for ATC.

scholarly journals γ-Protocadherins, Presenilin-mediated Release of C-terminal Fragment Promotes Locus Expression

2005 ◽  
Vol 280 (16) ◽  
pp. 15888-15897 ◽  
Author(s):  
Boris Hambsch ◽  
Valery Grinevich ◽  
Peter H. Seeburg ◽  
Martin K. Schwarz
Keyword(s):  
Cortical Neurons ◽  
Intracellular Signaling ◽  
Luciferase Reporter ◽  
Type I ◽  
Mrna Levels ◽  
Signaling Mechanism ◽  
Neonatal Lethality ◽  
Reporter Assays ◽  
Complex Locus ◽  
Membrane Spanning

γ-Protocadherins (γ-pcdhs) are type I membrane-spanning glycoproteins, widely expressed in the mammal and required for survival. These cell adhesion molecules are expressed from a complex locus comprising 22 functional variable exons arranged in tandem, each encoding extracellular, transmembrane and intracellular sequence, and three exons for an invariant C-terminal domain (γ-ICD). However, the signaling mechanisms that lie downstream of γ-pcdhs have not been elucidated. Here we report that γ-pcdhs are subject to presenilin-dependent intramembrane cleavage (PS-IP), accompanied by shedding of the extracellular domain. The cleaved intracellular domain (γ-ICD) translocates to the cell nucleus and was detected in subsets of cortical neurons. Notably, gene-targeted mice lacking functional γ-ICD sequence showed severely reduced γ-pcdh mRNA levels and neonatal lethality. Most importantly, inhibition of γ-secretase decreased γ-pcdh locus expression. Luciferase reporter assays demonstrated that γ-pcdh promoter activity is increased by γ-ICD. These results reveal an intracellular signaling mechanism for γ-pcdhs and identify a novel vital target for the γ-secretase complex.

C-Myb Regulates Mir-101 Expression During the Early Stages of Erythropoiesis

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3876-3876
Author(s):  
Giselle Brito ◽  
Ningkun Li ◽  
Susan E. Shetzline
Keyword(s):  
Feedback Loop ◽  
Hematopoietic Cells ◽  
Luciferase Reporter ◽  
Negative Feedback Loop ◽  
Start Site ◽  
Transcription Start ◽  
Early Stages ◽  
Cd34 Cells ◽  
Reporter Assays ◽  
Cells Cultured

Abstract Abstract 3876 The proto-oncogene c-myb encodes the transcription factor c-Myb, which is predominantly expressed in immature hematopoietic cells where it plays an obligate role in definitive hematopoiesis. Given the critical functions of c-Myb in lineage commitment, proliferation, and differentiation, c-Myb regulatory factors are of great interest but remain incompletely defined. In recent years, c-Myb has been shown to regulate the expression of microRNA (miRNA) molecules in hematopoietic cells. MiRNA molecules are noncoding RNA molecules that are 21–23 nucleotides in length and function to hybridize to the 3′UTR region of its target mRNA to stimulate/repress translation or induce mRNA degradation. For example, in hematopoietic cells, miR-15a and c-Myb form an autoregulatory negative feedback loop in that over-expression of miR-15a in hematopoietic cells was determined to block erythroid and myeloid colony formation. In megakaryocytes, the hormone thrombopoietin induced miR-150 expression which subsequently functioned to degrade c-myb mRNA through direct interaction with c-myb's 3′-UTR. Our studies have focused on determining the physiologic function of the neuroendocrine Neuromedin U (NmU) during the early stages of erythropoiesis, because we recently determined that silencing NmU in primary human CD34+ cells impairs burst-forming units-erythroid and colony-forming unit-erythroid formation. In subsequent studies, we determined that c-Myb directly interacts with the NmU promoter at Myb Response Elements (MREs) distal to its transcription start site. Also, the expression profiles of NmU and c-myb are similar in CD34+ cells cultured under erythroid inducing conditions for 10 days, and silencing c-myb expression in hematopoietic cells inhibits NmU expression. To gain insight into the regulatory mechanism involved in NmU expression during the early stages of erythropoiesis, we hypothesized that miRNA molecules regulated by c-Myb would inhibit NmU expression through a negative feedback loop. To address this hypothesis, we first scanned the 3′-UTR of NmU and identified 24 different miRNA molecules predicted to interact with NmU's 3′-UTR. Second, we used luciferase reporter assays to determine which of the miRNA molecules interacted with NmU's 3′-UTR. Of the three miRNA molecules we tested, miR-101 directly interacted with NmU's 3′-UTR in a dose-dependent manner. Third, we determined the expression profile of miR-101 in primary CD34+ cells cultured under erythroid inducing conditions. The gene expression of miR-101 was inversely correlated with NmU and c-myb. Finally, because miR-101 contained 6 MREs, we determined the ability of c-Myb to directly interact with the promoter of miR-101 using chromatin immunoprecipitation (ChIP) assays. Using primers that flank the MREs proximal to miR-101's transcription start site, we observed a greater than 2-fold increase in the amplification of DNA recovered from ChIP assays completed with c-Myb antibody compared to ChIP assays completed with irrelevant antibody. Studies are underway to confirm by luciferase-reporter assays that c-Myb directly binds to and transactivates the miR-101 promoter. Collectively, these data identify a regulatory loop comprised of c-Myb, NmU, and miR-101 that could be of potential importance during human erythropoiesis. Disclosures: No relevant conflicts of interest to declare.

scholarly journals MicroRNA-29b Contributes to Collagens Imbalance in Human Osteoarthritic and Dedifferentiated Articular Chondrocytes

2017 ◽  
Vol 2017 ◽  
pp. 1-12 ◽  
Author(s):  
David Moulin ◽  
Véronique Salone ◽  
Meriem Koufany ◽  
Thomas Clément ◽  
Isabelle Behm-Ansmant ◽  
...  
Keyword(s):  
Collagen Type ◽  
Articular Chondrocytes ◽  
Fold Increase ◽  
Luciferase Reporter ◽  
Type I ◽  
Mrna Levels ◽  
Chondrocyte Phenotype ◽  
Reporter Assays ◽  
The Impact

Objective. Decreased expression of collagen type II in favour of collagen type I or X is one hallmark of chondrocyte phenotype changes in osteoarthritic (OA) cartilage. MicroRNA- (miR-) 29b was previously shown to target collagens in several tissues. We studied whether it could contribute to collagen imbalance in chondrocytes with an impaired phenotype.Methods. After preliminary microarrays screening, miR-29b levels were measured by RT- quantitative PCR inin vitromodels of chondrocyte phenotype changes (IL-1βchallenge or serial subculturing) and in chondrocytes from OA and non-OA patients. Potential miR-29b targets identifiedin silicoin 3′-UTRs of collagens mRNAs were tested with luciferase reporter assays. The impact of premiR-29b overexpression in ATDC5 cells was studied on collagen mRNA levels and synthesis (Sirius red staining) during chondrogenesis.Results. MiR-29b level increased significantly in IL-1β-stimulated and weakly in subcultured chondrocytes. A 5.8-fold increase was observed in chondrocytes from OA versus non-OA patients. Reporter assays showed that miR-29b targeted COL2A1 and COL1A2 3′-UTRs although with a variable recovery upon mutation. In ATDC5 cells overexpressing premiR-29b, collagen production was reduced while mRNA levels increased.Conclusions. By acting probably as a posttranscriptional regulator with a different efficacy on COL2A1 and COL1A2 expression, miR-29b can contribute to the collagens imbalance associated with an abnormal chondrocyte phenotype.

scholarly journals Identification of microRNAs involved in NOD-dependent induction of pro-inflammatory genes in pulmonary endothelial cells

2020 ◽  
Author(s):  
Ann-Kathrin Vlacil ◽  
Evelyn Vollmeister ◽  
Wilhelm Bertrams ◽  
Florian Schoesser ◽  
Raghav Oberoi ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Direct Interaction ◽  
Luciferase Reporter ◽  
Mrna Levels ◽  
Pulmonary Endothelial Cells ◽  
Tnf Α ◽  
Reporter Assays ◽  
Mirna Expression Profiling ◽  
Bacterial Peptidoglycan

AbstractThe nucleotide-binding oligomerization domain-containing proteins (NOD) 1 and 2 are mammalian cytosolic pattern recognition receptors sensing bacterial peptidoglycan fragments in order to initiate cytokine expression and pathogen host defence. Since endothelial cells are relevant cells for pathogen recognition at the blood/tissue interface, we here analysed the role of NOD1- and NOD2-dependently expressed microRNAs (miRNAs, miR) for cytokine regulation in murine pulmonary endothelial cells. The induction of inflammatory cytokines in response to NOD1 and NOD2 was confirmed by increased expression of tumour necrosis factor (Tnf)-α and interleukin (Il)-6. MiRNA expression profiling revealed NOD1- and NOD2-dependently regulated miRNA candidates, of which miR-147-3p, miR-200a-3p, and miR-298-5p were subsequently validated in pulmonary endothelial cells isolated from Nod1/2-deficient mice. In-silico analysis of the two down-regulated candidates miR-147-3p and miR-298-5p revealed predicted binding sites in the 3’ untranslated region (UTR) of the murine Tnf-α and Il-6 mRNA. Consequently, transfection of endothelial cells with miRNA mimics decreased Tnf-α and Il-6 mRNA levels. Finally, a novel direct interaction of miR-298-5p with the 3’UTR of the Il-6 mRNA was uncovered by luciferase reporter assays. We here identified a mechanism of miRNA-down-regulation by NOD stimulation thereby enabling the induction of inflammatory gene expression in endothelial cells.

scholarly journals The advanced lipoxidation end product precursor malondialdehyde induces IL-17E expression and skews lymphocytes to the Th17 subset

2015 ◽  
Vol 20 (4) ◽  
Author(s):  
Kartiga Natarajan ◽  
Gokila Devi Mathialagan ◽  
Somasundaram Raghavan ◽  
Narkunaraja Shanmugam
Keyword(s):  
T Cells ◽  
Molecular Mechanisms ◽  
Human Peripheral Blood ◽  
Peripheral Blood Lymphocytes ◽  
Luciferase Reporter ◽  
Diabetic Patients ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Jurkat T Cells ◽  
Reporter Assays

AbstractMalondialdehyde (MDA) is a highly reactive endogenous product of thromboxane synthesis in the prostagland and lipid peroxidation by reactive oxygen species. Elevated MDA levels occur in diabetes and atherosclerotic plaques. The aim of this study was to examine the molecular mechanisms of MDA-induced IL-17E cytokine expression and its effect on T-cell differentiation. Real-time PCR, RT-PCR and ELISA were used to assess the expression of IL-17 family cytokines in Jurkat T-cells and human peripheral blood lymphocytes (PBLCs) from diabetic subjects. Luciferase reporter assays were used for the promoter activation study. Pharmacological inhibitors were used for signaling pathway experiments. FACS analyses were used to measure the Th1, Th2 and Th17 subset levels. MDA induced significant (2- to 3-fold; p < 0.01) generation of IL-17E mRNA in a dose- and time-dependent manner in Jurkat T-cells and PBLCs. Elevated IL-17E mRNA levels were found in the lymphocytes from diabetic subjects. The increased IL-17E protein and mRNA levels correlate well with serum MDA levels from diabetic patients. Transient transfection of plasmid containing the minimum IL-17E promoter region (pIL-17E-Luc) showed a significant (2-fold; p < 0.01) increase in luciferase activity. Pretreatment of lymphocytes with pharmacological inhibitors showed the involvement of antioxidant, NF-ƙB, p38MAPK, PKC and ERK signaling pathways. Quantification of the Th1, Th2 and Th17 cell population in PBLCs via FACS analyses revealed an increase in the Th17 subset. These results show that MDA transcriptionally upregulates the expression of IL-17E in lymphocytes and alters lymphocyte differentiation towards the pathogenic Th17 subset.

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