scholarly journals Construction and Experimental Validation of a Petri net Model of Wnt/β-catenin Signaling

2016 ◽  
Author(s):  
Annika Jacobsen ◽  
Nika Heijmans ◽  
Folkert Verkaar ◽  
Martine J. Smit ◽  
Jaap Heringa ◽  
...  
Keyword(s):  
Wnt Signaling ◽  
Negative Feedback ◽  
Petri Net ◽  
Wnt Pathway ◽  
Target Genes ◽  
Luciferase Reporter ◽  
Developmental Defects ◽  
Wnt Ligands ◽  
Reporter Assays

The Wnt/β-catenin signaling pathway is important for multiple developmental processes and tissue maintenance in adults. Consequently, deregulated signaling is involved in a range of human diseases including cancer and developmental defects. A better understanding of the intricate regulatory mechanism and effect of physiological (active) and pathophysiological (hyperactive) WNT signaling is important for predicting treatment response and developing novel therapies. The constitutively expressed CTNNB1 (commonly and hereafter referred to as β-catenin) is degraded by a destruction complex, composed of amongst other AXIN1 and GSK3. The destruction complex is inhibited during active signaling leading to β-catenin stabilization and induction of β-catenin/TCF target genes. In this study we investigated the mechanism and effect of β-catenin stabilization during active and hyperactive WNT signaling in a combined in silico and in vitro approach. We constructed a Petri net model of Wnt/β-catenin signaling including main players from the plasma membrane (WNT ligands and receptors), cytoplasmic effectors and the downstream negative feedback target gene AXIN2. We simulated the model with active (i.e. WNT stimulation) and hyperactive (i.e. GSK3 inhibition) signaling, which led to the following observations: 1) A dose- and time-dependent response was observed for both WNT stimulation and GSK3 inhibition. 2) The Wnt-pathway activity was 2-fold higher for GSK3 inhibition compared to WNT stimulation. Both of these observations were corroborated by TCF/LEF luciferase reporter assays. Using this experimentally validated model we simulated the effect of the negative feedback regulator AXIN2 upon WNT stimulation and observed an attenuated β-catenin stabilization. We furthermore simulated the effect of APC inactivating mutations, yielding a stabilization of β-catenin levels comparable to the Wnt-pathway activities observed in colorectal and breast cancer. Our model can be used for further investigation and viable predictions of the role of Wnt/β-catenin signaling in oncogenesis and development.

The BCL9 Oncogene Promotes Tumor Progression by Conferring Enhanced Proliferative, Metastatic and Angiogenic Properties to Myeloma Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 648-648
Author(s):  
Mala Mani ◽  
Jui Dutta ◽  
Yunyu Zhang ◽  
Daniel E Carrasco ◽  
Yiming Zhou ◽  
...  
Keyword(s):  
Wnt Signaling ◽  
Tumor Progression ◽  
Cyclin D1 ◽  
Wnt Pathway ◽  
Target Genes ◽  
Metastatic Potential ◽  
Luciferase Reporter ◽  
Aberrant Expression

Abstract Wnt signaling plays an important role in tissue development and maintenance during embryogenesis, cell differentiation, and stem cell growth. Several components of the Wnt signaling cascade have been shown to function as either tumor suppressor proteins or as oncogenes in multiple human cancers, underscoring the relevance of this pathway in oncogenesis. Deregulation of the canonical Wnt/b-catenin pathway has been implicated in numerous human epithelial malignancies as well as hematologic malignancies including multiple myeloma (MM), generating immense interest in these molecules as targets for cancer therapy. Activation of Wnt/b-catenin in cancer has been associated with mutations that enable b-catenin to escape degradation by the proteasome, thereby allowing its accumulation in the nucleus where it functions as a transcriptional regulator in conjunction with coactivators by constitutively activating target genes such as c-Myc and Cyclin D1. To date, however, no mutations in Wnt pathway have been documented in MM, suggesting that mechanisms other than gene mutation may contribute to Wnt pathway deregulation. BCL9, a key component of the Wnt pathway, is required for b-catenin transcriptional activity and resides on chromosome 1q21, a region frequently involved in secondary chromosomal aberrations associated with MM tumor progression. Here we provide evidence that dysregulation of BCL9 expression is a novel oncogenic mechanism of Wnt pathway activation in MM. Using in vitro and in vivo functional analyses, we demonstrate that BCL9 is a bonafide oncogene that is aberrantly expressed in MM and associated with survival. Using the TCF- specific luciferase reporter, we show that enforced expression of BCL9 in MM cells enhanced b-catenin mediated transcription by >12 fold, suggesting a possible role of BCL9 overexpression in the pathogenesis of MM. BCL9 enhanced proliferation (1.5 fold, P<0.02), migration (3.5 fold, P<0.0001) and the metastatic potential of MM cells. We also showed that BCL9 plays an important role in tumor progression by regulating Cyclin D1 and c-Myc mediated cell proliferation, CD44 mediated tumor metastasis, as well as VEGF mediated host angiogenesis. Importantly, BCL9 knockdown significantly increased the survival in a xenograft mouse model of human MM (P=0.001), associated with decreased tumor burden and host angiogenesis. In summary, we have demonstrated that BCL9 is a novel and potent oncogene of the Wnt pathway in MM, playing fundamental roles in tumor progression by regulating proliferation, migration, invasion, angiogenesis and the metastatic potential of tumor cells. The pleiotropic roles of BCL9 and its aberrant expression highlight its importance as an attractive and novel therapeutic target in the treatment of MM.

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 Differentiation of Human Cardiac Atrial Appendage Stem Cells into Adult Cardiomyocytes: A Role for the Wnt Pathway?

2020 ◽  
Vol 21 (11) ◽  
pp. 3931
Author(s):  
Leen Willems ◽  
Annick Daniëls ◽  
Yanick Fanton ◽  
Loes Linsen ◽  
Lize Evens ◽  
...  
Keyword(s):  
Stem Cells ◽  
Wnt Signaling ◽  
Wnt Pathway ◽  
Target Genes ◽  
Cardiac Differentiation ◽  
Differentiation Markers ◽  
Proliferation And Differentiation ◽  
Frizzled Receptors ◽  
Myocardial Differentiation

Human cardiac stem cells isolated from atrial appendages based on aldehyde dehydrogenase activity (CASCs) can be expanded in vitro and differentiate into mature cardiomyocytes. In this study, we assess whether Wnt activation stimulates human CASC proliferation, whereas Wnt inhibition induces cardiac maturation. CASCs were cultured as described before. Conventional PCR confirmed the presence of the Frizzled receptors. Small-molecule inhibitors (IWP2, C59, XAV939, and IWR1-endo) and activator (CHIR99021) of the Wnt/β -catenin signaling pathway were applied, and the effect on β-catenin and target genes for proliferation and differentiation was assessed by Western blot and RT-qPCR. CASCs express multiple early cardiac differentiation markers and are committed toward myocardial differentiation. They express several Frizzled receptors, suggesting a role for Wnt signaling in clonogenicity, proliferation, and differentiation. Wnt activation increases total and active β-catenin levels. However, this does not affect CASC proliferation or clonogenicity. Wnt inhibition upregulated early cardiac markers but could not induce mature myocardial differentiation. When CASCs are committed toward myocardial differentiation, the Wnt pathway is active and can be modulated. However, despite its role in cardiogenesis and myocardial differentiation of pluripotent stem-cell populations, our data indicate that Wnt signaling has limited effects on CASC clonogenicity, proliferation, and differentiation.

scholarly journals OIP5-AS1 contributes to tumorigenesis in hepatocellular carcinoma by miR-300/YY1-activated WNT pathway

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Yu Wang ◽  
Lei Dou ◽  
Yun Qin ◽  
Huiyuan Yang ◽  
Peng Yan
Keyword(s):  
Cell Growth ◽  
Wnt Pathway ◽  
Luciferase Reporter ◽  
Cell Cycle Distribution ◽  
Reporter Assays ◽  
Tumor Types ◽  
Regulatory Functions ◽  
Hcc Cells

Abstract Background It has reported that long non-coding RNAs (lncRNAs) exerted regulatory functions by targeting specific genes through a competing endogenous RNA (ceRNA) pathway. LncRNA OIP5-AS1 has been identified as a tumor-enhancer in several tumor types. Nonetheless, its molecular mechanism in HCC remains to be masked. Aim of the study This study was aimed at exploring whether and how OIP5-AS1 exert functions in HCC. Methods qRT-PCR and western blot were employed for detecting gene expression. CCK-8, colony formation and EdU assays were implemented to evaluate the proliferative ability of HCC cells. Caspase-3 activity and flow cytometry analyses were implemented to determine cell apoptosis and cell cycle distribution. RNA pull down, ChIP, RIP and luciferase reporter assays explored the interplays between molecules. Results YY1 was upregulated in HCC cells, and silenced YY1 restrained HCC cell proliferation in vitro and hampered tumor growth in vivo. Later, we discovered that miR-300 could regulate WNT pathway via targeting YY1. Furthermore, OIP5-AS1 was identified as the sponge of miR-300 and promoted cell growth in HCC. Importantly, YY1 transcriptionally activate OIP5-AS1 in turn. Rescue experiments indicated that miR-300 inhibition or YY1 overexpression abrogated the inhibitive effect of OIP5-AS1 silencing on the malignant growth of HCC cells. Conclusions OIP5-AS1/miR-300/YY1 feedback loop facilitates cell growth in HCC by activating WNT pathway.

scholarly journals Alternative isoforms of KDM2A and KDM2B lysine demethylases negatively regulate canonical Wnt signaling

2020 ◽  
Author(s):  
Dijana Lađinović ◽  
Daniel Pinkas ◽  
Otakar Raška ◽  
František Liška ◽  
Ivan Raška ◽  
...  
Keyword(s):  
Wnt Signaling ◽  
Transcriptional Repression ◽  
Target Genes ◽  
Luciferase Reporter ◽  
Dna Binding Domain ◽  
Biological Processes ◽  
Canonical Wnt Signaling ◽  
Developmental Defects ◽  
Canonical Wnt ◽  
Alternative Isoforms

AbstractA precisely balanced activity of canonical Wnt signaling is essential for a number of biological processes and its perturbation leads to developmental defects or diseases. Here, we demonstrate that alternative isoforms of the KDM2A and KDM2B lysine demethylases have the ability to negatively regulate canonical Wnt signaling. These KDM2A and KDM2B isoforms (KDM2A-SF and KDM2B-SF) lack the N-terminal demethylase domain, but they are able to bind to activated promoters in order to repress them. We have observed that KDM2A-SF and KDM2B-SF bind to and repress the promoters of AXIN2 and CYCLIN D1, two canonical Wnt signaling target genes. Moreover, KDM2A-SF and KDM2B-SF can repress a Wnt-responsive luciferase reporter. The transcriptional repression mediated by KDM2A-SF and KDM2B-SF, but also by KDM2A-LF, is dependent on their DNA binding domain, while the N-terminal demethylase domain is dispensable for this process. Surprisingly, KDM2B-LF is unable to repress both the endogenous promoters and the luciferase reporter. Finally, we show that both KDM2A-SF and KDM2B-SF are able to interact with TCF7L1, one of the transcriptional mediators of canonical Wnt signaling. KDM2A-SF and KDM2B-SF are thus likely to affect the transcription of the TCF7L1 target genes also through this interaction.

scholarly journals MiR-193a-3p is an Important Tumour Suppressor in Lung Cancer and Directly Targets KRAS

2017 ◽  
Vol 44 (4) ◽  
pp. 1311-1324 ◽  
Author(s):  
Qian Fan ◽  
Xiuting Hu ◽  
Haiyang Zhang ◽  
Shengguang Wang ◽  
Huilai Zhang ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Cell Migration ◽  
Target Genes ◽  
Biological Effects ◽  
Tumour Suppressor ◽  
Luciferase Reporter ◽  
Cellular Functions ◽  
Reporter Assays

Background/Aims: MicroRNAs (miRNAs) have emerged as major regulators of tumour development and progression in non-small cell lung cancer (NSCLC). However, the role of miR-193a-3p in NSCLC is still unclear. Methods: Quantitative RT-PCR was used to detect miR-193a-3p expression levels in NSCLC tumour tissues. CCK8, EdU and cell migration assays were performed to analyse the biological functions of miR-193a-3p in NSCLC cells. Luciferase reporter assays were used to validate the bioinformatics-predicted target genes of miR-193a-3p. Western blotting and RNA/DNA interference carried out to evaluate the association between miR-193a-3p and KRAS. Results: miR-193a-3p expression was decreased in the NSCLC tumour tissues. We investigated the biological effects of miR-193a-3p both in vivo and in vitro and found that enforced expression of miR-193a-3p inhibited tumour formation and suppressed cell proliferation and cell migration. KRAS was found to be a potential target of miR-193a-3p, and dual luciferase reporter assays showed that miR-193a-3p directly binds to the 3’-untranslated region (3’-UTR) of KRAS mRNA. In addition, we found that changing the expression of KRAS had the opposite results to those induced by miR-193a-3p in the NSCLC cells. Importantly, simultaneous overexpression of miR-193a-3p and KRAS could counteract the effects of both on cellular functions. Conclusion: These findings highlight an important role for miR-193a-3p as a tumour suppressor in NSCLC pathogenesis via the regulation of KRAS expression.

scholarly journals Circular RNA circSDHC serves as a sponge for miR-127-3p to promote the proliferation and metastasis of renal cell carcinoma via the CDKN3/E2F1 axis

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Junjie Cen ◽  
Yanping Liang ◽  
Yong Huang ◽  
Yihui Pan ◽  
Guannan Shu ◽  
...  
Keyword(s):  
Renal Cell Carcinoma ◽  
Cell Carcinoma ◽  
Renal Cell ◽  
Target Genes ◽  
Expression Patterns ◽  
Luciferase Reporter ◽  
Circular Rnas ◽  
Proliferation And Metastasis

Abstract Background There is increasing evidence that circular RNAs (circRNAs) have significant regulatory roles in cancer development and progression; however, the expression patterns and biological functions of circRNAs in renal cell carcinoma (RCC) remain largely elusive. Method Bioinformatics methods were applied to screen for circRNAs differentially expressed in RCC. Analysis of online circRNAs microarray datasets and our own patient cohort indicated that circSDHC (hsa_circ_0015004) had a potential oncogenic role in RCC. Subsequently, circSDHC expression was measured in RCC tissues and cell lines by qPCR assay, and the prognostic value of circSDHC evaluated. Further, a series of functional in vitro and in vivo experiments were conducted to assess the effects of circSDHC on RCC proliferation and metastasis. RNA pull-down assay, luciferase reporter and fluorescent in situ hybridization assays were used to confirm the interactions between circSDHC, miR-127-3p and its target genes. Results Clinically, high circSDHC expression was correlated with advanced TNM stage and poor survival in patients with RCC. Further, circSDHC promoted tumor cell proliferation and invasion, both in vivo and in vitro. Analysis of the mechanism underlying the effects of circSDHC in RCC demonstrated that it binds competitively to miR-127-3p and prevents its suppression of a downstream gene, CDKN3, and the E2F1 pathway, thereby leading to RCC malignant progression. Furthermore, knockdown of circSDHC caused decreased CDKN3 expression and E2F1 pathway inhibition, which could be rescued by treatment with an miR-127-3p inhibitor. Conclusion Our data indicates, for the first time, an essential role for the circSDHC/miR-127-3p/CDKN3/E2F1 axis in RCC progression. Thus, circSDHC has potential to be a new therapeutic target in patients with RCC.

scholarly journals The nuclear factor κB inhibitor (E)-2-fluoro-4′-methoxystilbene inhibits firefly luciferase

2012 ◽  
Vol 32 (6) ◽  
pp. 531-537 ◽  
Author(s):  
Albert Braeuning ◽  
Silvia Vetter
Keyword(s):  
Photon Emission ◽  
Gene Promoter ◽  
Firefly Luciferase ◽  
Nuclear Factor Κb ◽  
Signalling Pathway ◽  
Luciferase Reporter ◽  
Nuclear Factor ◽  
Reporter System ◽  
Reporter Assays

Photinus pyralis (firefly) luciferase is widely used as a reporter system to monitor alterations in gene promoter and/or signalling pathway activities in vitro. The enzyme catalyses the formation of oxyluciferin from D-luciferin in an ATP-consuming reaction involving photon emission. The purpose of the present study was to characterize the luciferase-inhibiting potential of (E)-2-fluoro-4′-methoxystilbene, which is known as a potent inhibitor of the NF-κB (nuclear factor κB) signalling pathway that is used to modulate the NF-κB signalling pathway in vitro. Results show that (E)-2-fluoro-4′-methoxystilbene effectively inhibits firefly luciferase activity in cell lysates and living cells in a non-competitive manner with respect to the luciferase substrates D-luciferin and ATP. By contrast, the compound has no effect on Renilla and Gaussia luciferases. The mechanism of firefly luciferase inhibition by (E)-2-fluoro-4′-methoxystilbene, as well as its potency is comparable to its structure analogue resveratrol. The in vitro use of trans-stilbenes such as (E)-2-fluoro-4′-methoxystilbene or resveratrol compromises firefly luciferase reporter assays as well as ATP/luciferase-based cell viability assays.

scholarly journals Let-7a regulates EV secretion and mitochondrial oxidative phosphorylation by targeting SNAP23 in colorectal cancer

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
You Dong Liu ◽  
Xiao Peng Zhuang ◽  
Dong Lan Cai ◽  
Can Cao ◽  
Qi Sheng Gu ◽  
...  
Keyword(s):  
Oxidative Phosphorylation ◽  
Metabolic Reprogramming ◽  
Luciferase Reporter ◽  
Mitochondrial Oxidative Phosphorylation ◽  
In Vivo Assays ◽  
Reporter Assays ◽  
Extracellular Mirna

Abstract Background MicroRNAs (miRNAs) are abundant in tumor-derived extracellular vesicles (EVs) and the functions of extracellular miRNA to recipient cells have been extensively studied with tumorigenesis. However, the role of miRNA in EV secretion from cancer cells remains unknown. Methods qPCR and bioinformatics analysis were applied for determining extracellular let-7a expression from CRC patient serum and cells. Nanosight particle tracking analysis was performed for investigating the effect of let-7a on EV secretion. Luciferase reporter assays was used for identifying targeted genes synaptosome-associated protein 23 (SNAP23). In vitro and in vivo assays were used for exploring the function of let-7a/SNAP23 axis in CRC progression. Bioenergetic assays were performed for investigating the role of let-7a/SNAP23 in cellular metabolic reprogramming. Results let-7a miRNA was elevated in serum EVs from CRC patients and was enriched in CRC cell-derived EVs. We determined that let-7a could suppress EV secretion directly targeting SNAP23. In turn, SNAP23 promotes EV secretion of let-7a to downregulate the intracellular let-7a expression. In addition, we found a novel mechanism of let-7a/SNAP23 axis by regulating mitochondrial oxidative phosphorylation (OXPHOS) through Lin28a/SDHA signaling pathway. Conclusions Let-7a plays an essential role in not only inhibiting EV secretion, but also suppressing OXPHOS through SNAP23, resulting in the suppression of CRC progression, suggesting that let-7a/SNAP23 axis could provide not only effective tumor biomarkers but also novel targets for tumor therapeutic strategies.

BMP4 and WNT signaling interact to promote mouse tracheal mesenchyme morphogenesis

2021 ◽  
Author(s):  
Natalia Bottasso Arias ◽  
Lauren Leesman ◽  
Kaulini Burra ◽  
John Snowball ◽  
Ronak M Shah ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Wnt Signaling ◽  
Rna Sequencing ◽  
Muscle Development ◽  
Target Genes ◽  
Bmp Signaling ◽  
Embryonic Mouse ◽  
Tracheal Cartilage ◽  
Cartilage Formation

Tracheobronchomalacia and Complete Tracheal Rings are congenital malformations of the trachea associated with morbidity and mortality for which the etiology remains poorly understood. Epithelial expression of Wls (a cargo receptor mediating Wnt ligand secretion) by tracheal cells is essential for patterning the embryonic mouse trachea's cartilage and muscle. RNA sequencing indicated that Wls differentially modulated the expression of BMP signaling molecules. We tested whether BMP signaling, induced by epithelial Wnt ligands, mediates cartilage formation. Deletion of Bmp4 from respiratory tract mesenchyme impaired tracheal cartilage formation that was replaced by ectopic smooth muscle, recapitulating the phenotype observed after epithelial deletion of Wls in the embryonic trachea. Ectopic muscle was caused in part by anomalous differentiation and proliferation of smooth muscle progenitors rather than tracheal cartilage progenitors. Mesenchymal deletion of Bmp4 impaired expression of Wnt/β-catenin target genes, including targets of WNTsignaling: Notum, and Axin2. In vitro, rBMP4 rescued the expression of Notum in Bmp4 deficient tracheal mesenchymal cells and induced Notum promoter activity via SMAD1/5. RNA sequencing of Bmp4 deficient tracheas identified genes essential for chondrogenesis and muscle development co-regulated by BMP and WNT signaling. During tracheal morphogenesis, WNT signaling induces Bmp4 in mesenchymal progenitors to promote cartilage differentiation and restrict trachealis muscle. In turn, Bmp4 differentially regulates the expression of Wnt/β-catenin targets to attenuate mesenchymal WNT signaling and to further support chondrogenesis.

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