scholarly journals Feeding of Enterococcus faecium NCIMB 10415 Leads to Intestinal miRNA-423-5p-Induced Regulation of Immune-Relevant Genes

2016 ◽  
Vol 82 (8) ◽  
pp. 2263-2269 ◽  
Author(s):  
Susanne Kreuzer-Redmer ◽  
Jennifer C. Bekurtz ◽  
Danny Arends ◽  
Ralf Bortfeldt ◽  
Barbara Kutz-Lohroff ◽  
...  
Keyword(s):  
Mode Of Action ◽  
Enterococcus Faecium ◽  
Target Genes ◽  
Immune Cell ◽  
Animal Health ◽  
Luciferase Reporter ◽  
Content Type ◽  
Immunoglobulin Kappa ◽  
Genes Expression ◽  
Reporter Assays

ABSTRACTProbiotics are widely used in human and animal health, but little is known about the mode of action of probiotics. One possible mechanism at the molecular level could be an influence on microRNAs (miRNAs) and the related immune-relevant target genes. Here, we analyzed differential expression of miRNA and potential target genes of ileal and jejunal lymphatic tissues fromEnterococcus faeciumNCIMB 10415-fed piglets versus untreated controls by using next-generation sequencing. We identified miR-423-5p as being greatly affected by the treatment group (2.32-fold;P= 0.014). Validation by reverse transcription-quantitative PCR (RT-qPCR) confirmed a significant upregulation of miR-423-5p (2.11-fold;P= 0.03) and, additionally, downregulation of the important immune-relevant immunoglobulin lambda light C region (IGLC) (0.61-fold;P= 0.03) and immunoglobulin kappa constant (IGKC) (0.69-fold;P= 0.04) target genes. Expression analysis of miR-423-5p and IGLC at different age points shows a clear anticorrelated relationship. Luciferase reporter assays with a HeLa cell line verified IGLC as a target of miR-423-5p. The results provided evidence for an effect of feeding ofE. faeciumon the expression of miR-423-5p and on the regulation of the IGLC gene through miR-423-5p. This might be a possible mode of action ofE. faeciumon immune cell regulation in the small intestine.

scholarly journals Ehrlichia chaffeensis TRP32 Is a Nucleomodulin That Directly Regulates Expression of Host Genes Governing Differentiation and Proliferation

2016 ◽  
Vol 84 (11) ◽  
pp. 3182-3194 ◽  
Author(s):  
Tierra R. Farris ◽  
Paige S. Dunphy ◽  
Bing Zhu ◽  
Clayton E. Kibler ◽  
Jere W. McBride
Keyword(s):  
Dna Binding ◽  
Host Cell ◽  
Gene Transcription ◽  
Noncoding Rna ◽  
Target Genes ◽  
Immune Cell ◽  
Luciferase Reporter ◽  
Ehrlichia Chaffeensis ◽  
Content Type

Ehrlichia chaffeensis is an obligately intracellular bacterium that reprograms the mononuclear phagocyte through diverse effector-host interactions to modulate numerous host cell processes, including transcription. In a previous study, we reported that E. chaffeensis TRP32, a type 1 secreted effector, interacts with multiple host nucleus-associated proteins and also autoactivates reporter gene expression in yeast. In this study, we demonstrate that TRP32 is a nucleomodulin that binds host DNA and alters host gene transcription. TRP32 enters the host cell nucleus via a noncanonical translocation mechanism that involves phosphorylation of Y179 located in a C-terminal trityrosine motif. Both genistein and mutation of Y179 inhibited TRP32 nuclear entry. An electromobility shift assay (EMSA) demonstrated TRP32 host DNA binding via its tandem repeat domain. TRP32 DNA-binding and motif preference were further confirmed by supershift assays, as well as competition and mutant probe analyses. Using chromatin immunoprecipitation with next-generation sequencing (ChIP-seq), we determined that TRP32 binds a G-rich motif primarily within ±500 bp of the gene transcription start site. An ontology analysis identified genes involved in processes such as immune cell differentiation, chromatin remodeling, and RNA transcription and processing as primary TRP32 targets. TRP32-bound genes ( n = 1,223) were distributed on all chromosomes and included several global regulators of proliferation and inflammation such as those encoding FOS, JUN, AKT3, and NRAS and noncoding RNA genes microRNA 21 (miRNA 21) and miRNA 142. TRP32 target genes were differentially regulated during infection, the majority of which were repressed, and direct repression/activation of these genes by TRP32 was confirmed in vitro with a cellular luciferase reporter assay.

Screening candidate microR-15a- IRAK2 regulatory pairs for predicting the response to Staphylococcus aureus-induced mastitis in dairy cows

2019 ◽  
Vol 86 (4) ◽  
pp. 425-431 ◽  
Author(s):  
Zhi Chen ◽  
Jingpeng Zhou ◽  
Xiaolong Wang ◽  
Yang Zhang ◽  
Xubin Lu ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Differential Expression ◽  
High Throughput Sequencing ◽  
Target Genes ◽  
Interleukin 1 ◽  
Differentially Expressed ◽  
Luciferase Reporter ◽  
Expression Of Genes ◽  
Chinese Holstein Cows ◽  
Reporter Assays

AbstractWe established a mastitis model using exogenous infection of the mammary gland of Chinese Holstein cows with Staphylococcus aureus and extracted total RNA from S. aureus-infected and healthy mammary quarters. Differential expression of genes due to mastitis was evaluated using Affymetrix technology and results revealed a total of 1230 differentially expressed mRNAs. A subset of affected genes was verified via Q-PCR and pathway analysis. In addition, Solexa high-throughput sequencing technology was used to analyze profiles of miRNA in infected and healthy quarters. These analyses revealed a total of 52 differentially expressed miRNAs. A subset of those results was verified via Q-PCR. Bioinformatics techniques were used to predict and analyze the correlations among differentially expressed miRNA and mRNA. Results revealed a total of 329 pairs of negatively associated miRNA/mRNA, with 31 upregulated pairs of mRNA and 298 downregulated pairs of mRNA. Differential expression of miR-15a and interleukin-1 receptor-associated kinase-like 2 (IRAK2), were evaluated by western blot and luciferase reporter assays. We conclude that miR-15a and miR-15a target genes (IRAK2) constitute potential miRNA–mRNA regulatory pairs for use as biomarkers to predict a mastitis response.

scholarly journals LukS-PV-Regulated MicroRNA-125a-3p Promotes THP-1 Macrophages Differentiation and Apoptosis by Down-Regulating NF1 and Bcl-2

2017 ◽  
Vol 44 (3) ◽  
pp. 1093-1105 ◽  
Author(s):  
Xiao-Xi Sun ◽  
Shan-Shan Zhang ◽  
Chun-Yang Dai ◽  
Jing Peng ◽  
Qing Pan ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
Cellular Differentiation ◽  
Target Genes ◽  
B Cell Lymphoma ◽  
Neurofibromatosis Type ◽  
Luciferase Reporter ◽  
Loss Of Function ◽  
Leukaemia Cell Line ◽  
Reporter Assays

Background/Aims: LukS-PV is a component of Panton-Valentine leukocidin (PVL). We have previously demonstrated that LukS-PV potently promoted differentiation and induced apoptosis in THP-1 cells. However, the precise mechanisms of these actions remain unknown. MicroRNAs (miRs) play important roles in cellular differentiation and apoptosis. This study aimed to investigate the role of miR-125a-3p in LukS-PV-regulated differentiation and apoptosis and its underlying mechanism in THP-1 cells. Methods: MicroRNA profiling analyses were conducted to determine differential miRNA expression levels in THP-1 cells treated with LukS-PV. Cell differentiation and apoptosis were measured in THP-1 cells by gain-of-function and loss-of-function experiments. Bioinformatics analysis and luciferase reporter assays were used to confirm the targets of miR-125a-3p. The effects of the miR-125a-3p targets on cellular differentiation were determined by knocking them down. Results: MiR-125a-3p was up-regulated after treating the human monocytic leukaemia cell line THP-1 with LukS-PV. In addition, miR-125a-3p positively regulated apoptosis and differentiation in THP-1 cells treated with LukS-PV. Concordantly, luciferase reporter assays confirmed that neurofibromatosis type 1 (NF1) and B-cell lymphoma 2 (Bcl-2) were direct target genes of miR-125a-3p. Moreover, NF1 knockdown in THP-1 cells significantly promoted differentiation in vitro. Finally, the extracellular signal-regulated kinase (ERK) pathway, a downstream target of NF1, was activated after NF1 knockdown. Conclusions: These findings confirm that miR-125a-3p is involved in LukS-PV-mediated cell differentiation and apoptosis in THP-1 cells.

scholarly journals MicroRNA 322 Aggravates Dexamethasone-Induced Muscle Atrophy by Targeting IGF1R and INSR

2020 ◽  
Vol 21 (3) ◽  
pp. 1111 ◽  
Author(s):  
Hongwei Geng ◽  
Qinglong Song ◽  
Yunyun Cheng ◽  
Haoyang Li ◽  
Rui Yang ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Muscle Atrophy ◽  
Target Genes ◽  
Immunosuppressive Agent ◽  
Skeletal Muscle Atrophy ◽  
Luciferase Reporter ◽  
C2c12 Myotubes ◽  
High Dose ◽  
Reporter Assays ◽  
Underlying Mechanisms

Dexamethasone (Dex) has been widely used as a potent anti-inflammatory, antishock, and immunosuppressive agent. However, high dose or long-term use of Dex is accompanied by side effects including skeletal muscle atrophy, whose underlying mechanisms remain incompletely understood. A number of microRNAs (miRNAs) have been shown to play key roles in skeletal muscle atrophy. Previous studies showed significantly increased miR-322 expression in Dex-treated C2C12 myotubes. In our study, the glucocorticoid receptor (GR) was required for Dex to increase miR-322 expression in C2C12 myotubes. miR-322 mimic or miR-322 inhibitor was used for regulating the expression of miR-322. Insulin-like growth factor 1 receptor (IGF1R) and insulin receptor (INSR) were identified as target genes of miR-322 using luciferase reporter assays and played key roles in Dex-induced muscle atrophy. miR-322 overexpression promoted atrophy in Dex-treated C2C12 myotubes and the gastrocnemius muscles of mice. Conversely, miR-322 inhibition showed the opposite effects. These data suggested that miR-322 contributes to Dex-induced muscle atrophy via targeting of IGF1R and INSR. Furthermore, miR-322 might be a potential target to counter Dex-induced muscle atrophy. miR-322 inhibition might also represent a therapeutic approach for Dex-induced muscle atrophy.

scholarly journals LncRNA SNHG6 promotes chemoresistance through ULK1-induced autophagy by sponging miR-26a-5p in colorectal cancer cells

2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Xinke Wang ◽  
Zhixian Lan ◽  
Juan He ◽  
Qiuhua Lai ◽  
Xiang Yao ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Western Blotting ◽  
Target Genes ◽  
Bioinformatics Analysis ◽  
Chemotherapy Resistance ◽  
Micro Rna ◽  
Luciferase Reporter ◽  
Qrt Pcr ◽  
Reporter Assays

Abstract Background Chemotherapy resistance is one of the main causes of recurrence in colorectal cancer (CRC) patients and leads to poor prognosis. Long noncoding RNAs (lncRNAs) have been reported to regulate chemoresistance. We aimed to determine the role of the lncRNA small nucleolar RNA host gene 6 (SNHG6) in CRC cell chemoresistance. Methods Cell drug sensitivity tests and flow cytometry were performed to analyze CRC cell chemoresistance. Animal models were used to determine chemoresistance in vivo, and micro RNA (miRNA) binding sites were detected by dual-luciferase reporter assays. Bioinformatics analysis was performed to predict miRNAs binding to SNHG6 and target genes of miR-26a-5p. SNHG6/miR-26a-5p/ULK1 axis and autophagy-related proteins were detected by qRT-PCR and western blotting. Furthermore, immunofluorescence was employed to confirm the presence of autophagosomes. Results SNHG6 enhanced CRC cell resistance to 5-fluorouracil (5-FU), promoted autophagy, inhibited 5-FU-induced apoptosis, and increased 5-FU resistance in vivo. Bioinformatics analysis showed that miR-26a-5p might bind to SNHG6 and target ULK1, and dual-luciferase reporter assays confirmed this activity. qRT-PCR and western blotting showed that SNHG6 was able to negatively regulate miR-26a-5p but correlated positively with ULK1. Conclusion SNHG6 may promote chemoresistance through ULK1-induced autophagy by sponging miR-26a-5p in CRC cells.

Abstract WP127: Intravenous Administration of Microrna-122 Mimic for Treatment of Ischemic and Hemorrhagic Strokes in Rats

Stroke ◽  
2020 ◽  
Vol 51 (Suppl_1) ◽  
Author(s):  
DaZhi Liu
Keyword(s):  
Ischemic Stroke ◽  
Drug Targets ◽  
Target Genes ◽  
Time Window ◽  
Artery Occlusion ◽  
Luciferase Reporter ◽  
Mirna Expression Data ◽  
Reporter Assays ◽  
Target Binding

MicroRNAs(miRs)are very promising next generation drug targets due to their unique miR-target binding that is different from the traditional ligand-receptor. A single miR binds to complementary bases in the 3’ untranslated regions(3’UTR) of hundreds of target genes and down-regulates these genes. MiR therapeutics have been developed for treatment of various diseases, with several miR drugs(e.g., miR-122/miR-155 inhibitors, miR-16/29/34 mimics) being advanced into human trials in the last decade. Our previous studies showed that intravenous (i.v.) miR-122 mimic (2.4mg/kg, wrapped in PEG-liposomes) improves outcomes after suture middle cerebral artery occlusion(MCAO)-induced ischemic stroke (IS) with a 6 hour time window. In pilot whole genome miR expression studies, we demonstrated that microRNA-122 (miR-122) is the most significantly decreased miR in blood after intraventricularautologousfresh blood-induced intracerebral hemorrhage (ICH) in rats. As compared to sham controls, miR-122 decreased 28 fold at 3 hrs, 34 fold at 24 hrs, 31 fold at 7 days and 19 fold at 14 days in blood after ICH in rats. These miRNA expression data suggest that elevating miR-122 in blood has great potential to treat ICH in addition to IS. Therefore, we hypothesized that i.v. miR-122 mimic improves outcomes after ICH, in addition to IS. Our miR-122 targetome studies show that a set of miR-122 target genes (Pla2g2a, Vcam1, Nos2, Rhbdf1, Olig1, Nrep) are responsible for the therapeutic efficacy of miR-122 mimic on ischemic stroke, while another set of genes (Ywhaq, Klrk1, Tpst1, Vars2)account for efficacy in ICH. Using3’UTR luciferase reporter assays and anti-sense Morpholino Oligos (MOs), we show that miR-122 binds to 3’UTR of its target genes Pla2g2a and Vcam1, rather than Nos2. In addition, in vivo MO-miR-122-Pla2g2a blocks miR-122 mimic treatment-induced decrease of Pla2g2a in blood cells after ischemic stroke. In summary, our data suggest miR-122 mimic can treat IS and ICH. Therefore, the miR-122 mimic treatment for IS and ICH could likely be performed without brain imaging (e.g. in the ambulance, and/or emergency room) since it is broadly efficacious for both IS and ICH.

scholarly journals MicroRNA-185 induces potent autophagy via AKT signaling in hepatocellular carcinoma

Tumor Biology ◽  
2017 ◽  
Vol 39 (2) ◽  
pp. 101042831769431 ◽  
Author(s):  
Li Zhou ◽  
Shunai Liu ◽  
Ming Han ◽  
Shenghu Feng ◽  
Jinqiu Liang ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Hepg2 Cells ◽  
Target Genes ◽  
Untranslated Regions ◽  
Luciferase Reporter ◽  
Promising Therapeutic Target ◽  
Reporter Assays ◽  
Induced Apoptosis ◽  
First Time

Studies have demonstrated that microRNA 185 may be a promising therapeutic target in liver cancer. However, its role in hepatocellular carcinoma is largely unknown. In this study, the proliferation of human HepG2 cells was inhibited by transfection of microRNA 185 mimics. Cell-cycle analysis revealed arrest at the G0/G1 phase. Transfection of HepG2 cells with microRNA 185 mimics significantly induced apoptosis. These data confirmed microRNA 185 as a potent cancer suppressor. We demonstrated that microRNA 185 was a compelling inducer of autophagy, for the first time. When cell autophagy was inhibited by chloroquine or 3-methyladenine, microRNA 185 induced more cell apoptosis. MicroRNA 185 acted as a cancer suppressor by regulating AKT1 expression and phosphorylation. Dual-luciferase reporter assays indicated that microRNA 185 suppressed the expression of target genes including RHEB, RICTOR, and AKT1 by directly interacting with their 3′-untranslated regions. Binding site mutations eliminated microRNA 185 responsiveness. Our findings demonstrate a new role of microRNA 185 as a key regulator of hepatocellular carcinoma via autophagy by dysregulation of AKT1 pathway.

scholarly journals Mir-573 regulates cell proliferation and apoptosis by targeting Bax in human degenerative disc cells following hyperbaric oxygen treatment

2020 ◽  
Author(s):  
Song-Shu Lin ◽  
Chi-Chien Niu ◽  
Li-Jen Yuan ◽  
Tsung-Ting Tsai ◽  
Po-Liang Lai ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Hyperbaric Oxygen ◽  
Caspase 3 ◽  
Target Genes ◽  
Vital Role ◽  
Luciferase Reporter ◽  
Nucleus Pulposus Cells ◽  
Caspase 9 ◽  
Proliferation And Apoptosis ◽  
Reporter Assays

Abstract Background: MicroRNA (miRNA) plays a vital role in the intervertebral disc (IVD) degeneration. The expression level of miR-573 was downregulated whereas Bax was upregulated notably in human degenerative nucleus pulposus cells (NPCs). In this study, we aimed to investigate the role of miR-573 in human degenerative NPCs following hyperbaric oxygen (HBO) treatment. Methods: NPCs were separated from human degenerated IVD tissues. The control cells were maintained in 5% CO2/95% air and the hyperoxic cells were exposed to 100% O2 at 2.5 atmospheres absolute. MiRNA expression profiling was performed via microarray and confirmed by real-time PCR, and miRNA target genes were identified using bioinformatics and luciferase reporter assays. The mRNA and protein levels of Bax were measured. The proliferation of NPCs were detected using MTT assay. The protein expression levels of Bax, cleaved caspase 9, cleaved caspase 3, pro-caspase 9 and pro-caspase 3 were examined.Results: Bioinformatics analysis indicated that the 3′ untranslated region (UTR) of the Bax mRNA contained the “seed-matched-sequence” for hsa-miR-573, which was validated via reporter assays. MiR-573 was induced by HBO and simultaneous suppression of Bax was observed in NPCs. Knockdown of miR-573 resulted in upregulation of Bax expression in HBO-treated cells. In addition, overexpression of miR-573 by HBO increased cell proliferation and coupled with inhibition of cell apoptosis. The cleavage of pro‑caspase 9 and pro‑caspase 3 was suppressed while the levels of cleaved caspase 9 and caspase 3 were decreased in HBO-treated cells. Transfection with anti-miR-573 partly suppressed the effects of HBO. Conclusion: Mir-573 regulates cell proliferation and apoptosis by targeting Bax in human degenerative NPCs following HBO treatment.

PLAGL2 Cooperates in Leukemia Development by Direcly Inducing c-Mpl Expression.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 744-744
Author(s):  
Sean F. Landrette ◽  
Lucio H. Castilla
Keyword(s):  
Target Genes ◽  
Receptor Expression ◽  
Proximal Promoter ◽  
Luciferase Reporter ◽  
Pcr Analysis ◽  
Donor Cells ◽  
Human Acute Myeloid Leukemia ◽  
Reporter Assays ◽  
Leukemia Development ◽  
Upstream Regulators

Abstract Inversion of chromosome 16 is found in 12% of human acute myeloid leukemia (AML). This inversion creates the CBFΒ-SMMHC fusion protein, which hinders myeloid differentiation and triggers AML in collaboration with additional mutations. We have previously shown that the zinc finger transcription factor PLAGL2 efficiently cooperates with CbfΒ-SMMHC in murine AML, and that PLAGL2 transcript levels are significantly increased in inversion 16 human AML. The mechanism of PLAGL2 action in AML, however, is not understood. In order to identify candidate PLAGL2 target genes in AML, we performed gene expression profiling of primary hematopoietic progenitors expressing CbfΒ-SMMHC and PLAGL2, as well as in AML samples induced by CbfΒ-SMMHC and PLAGL2. The expression of c-Mpl transcript was increased 8 to 32 fold in progenitors and AML samples when compared to controls not expressing PLAGL2. This increase was confirmed by quantitative PCR analysis and correlated with increased c-Mpl receptor in membrane by flow cytometry. Analysis of the c-Mpl proximal promoter identified three conserved Plag binding sites. PLAGL2 binding to these sites was confirmed by chromatin immunoprecipitation and luciferase reporter assays. Furthermore, PLAGL2/CbfΒ-SMMHC AML cells show a significant increase in sensitivity to the c-Mpl-ligand thrombopoietin when compared to non-PLAGL2 CbfΒ-SMMHC-AML samples as measured by Jak2 phosphorylation. To test whether PLAGL2 induces AML by increasing c-Mpl levels, AML development was analyzed in bone-marrow transplantation assays using CbfΒ-SMMHC-expressing donor cells transduced with c-Mpl expressing retrovirus. All transplanted mice (8/8) succumbed to leukemia with a median latency of 8 weeks, while control groups remained unaffected for 16 weeks. The pathology of Mpl/CbfΒ-SMMHC AML was immuno-phenotypically and morphologically identical to that of PLAGL2/CbfΒ-SMMHC AML. This study demonstrates that PLAGL2 directly induces c-Mpl receptor expression, and that such increase cooperates with CbfΒ-SMMHC in AML development. These results suggest that AML can result from the alteration of upstream regulators of Mpl/Jak2 signaling.

scholarly journals Exosomal miR-21 regulates the TETs/PTENp1/PTEN pathway to promote hepatocellular carcinoma growth

2019 ◽  
Vol 18 (1) ◽  
Author(s):  
Liang-qi Cao ◽  
Xue-wei Yang ◽  
Yu-bin Chen ◽  
Da-wei Zhang ◽  
Xiao-Feng Jiang ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Target Genes ◽  
Underlying Mechanism ◽  
Luciferase Reporter ◽  
Pcr Analysis ◽  
Reporter Assays ◽  
Important Means ◽  
And Migration ◽  
Insight Into ◽  
Hcc Cells

Abstract Background As an important means of communication, exosomes play an important role in the development of hepatocellular carcinoma (HCC). Methods Bioinformatics analysis, dual-luciferase reporter assays, methylation-specific quantitative PCR, and ChIP-PCR analysis were used to gain insight into the underlying mechanism of miR-21 in HCC. Results The detection of miRNAs in exosomes of HCC showed that miR-21 expression in exosomes was positively correlated with the expression level of miR-21 in cells and negatively correlated with the expression of its target genes PTEN, PTENp1 and TETs. HCC cell-derived exosomes could increase miR-21 and p-Akt expression in HCC cells and downregulate the expression of PTEN, PTENp1 and TETs. MiR-21 inhibitors or PTENp1 overexpression vectors could weaken the effect of the abovementioned exosomes and simultaneously weaken their role in promoting cell proliferation and migration and inhibiting apoptosis. Further studies showed that miR-21 not only directly regulated the expression of PTEN, PTENp1 and TETs but also increased the methylation level of the PTENp1 promoter by regulating the expression of TETs, thereby inhibiting the expression of PTENp1 and further downregulating the expression of PTEN. Conclusions Exosomal miR-21 can regulate the expression of the tumor suppressor genes PTEN and PTENp1 in various ways and affect the growth of HCC cells.

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