scholarly journals Elucidation of the BMI1 interactome identifies novel regulatory roles in glioblastoma

NAR Cancer ◽  
2021 ◽  
Vol 3 (1) ◽  
Author(s):  
Verónica Freire-Benéitez ◽  
Nicola Pomella ◽  
Thomas O Millner ◽  
Anaëlle A Dumas ◽  
Maria Victoria Niklison-Chirou ◽  
...  
Keyword(s):  
Chromatin Immunoprecipitation ◽  
Protein Composition ◽  
Mrna Splicing ◽  
Cholesterol Transport ◽  
Normal Brain ◽  
Regulatory Functions ◽  
Polycomb Repressive Complex 1 ◽  
Normal Brain Development

Abstract Glioblastoma (GBM) is the most common and aggressive intrinsic brain tumour in adults. Epigenetic mechanisms controlling normal brain development are often dysregulated in GBM. Among these, BMI1, a structural component of the Polycomb Repressive Complex 1 (PRC1), which promotes the H2AK119ub catalytic activity of Ring1B, is upregulated in GBM and its tumorigenic role has been shown in vitro and in vivo. Here, we have used protein and chromatin immunoprecipitation followed by mass spectrometry (MS) analysis to elucidate the protein composition of PRC1 in GBM and transcriptional silencing of defining interactors in primary patient-derived GIC lines to assess their functional impact on GBM biology. We identify novel regulatory functions in mRNA splicing and cholesterol transport which could represent novel targetable mechanisms in GBM.

scholarly journals Pre-mRNA splicing alters mRNP composition: evidence for stable association of proteins at exon–exon junctions

2000 ◽  
Vol 14 (9) ◽  
pp. 1098-1108 ◽  
Author(s):  
Hervé Le Hir ◽  
Melissa J. Moore ◽  
Lynne E. Maquat
Keyword(s):  
Direct Evidence ◽  
Protein Composition ◽  
Mrna Splicing ◽  
Core Component ◽  
Nonsense Mediated Decay ◽  
Exon Junctions ◽  
Stable Association ◽  
Gradient Fractionation

We provide direct evidence that pre-mRNA splicing alters mRNP protein composition. Using a novel in vitro cross-linking approach, we detected several proteins that associate with mRNA exon–exon junctions only as a consequence of splicing. Immunoprecipitation experiments suggested that these proteins are part of a tight complex around the junction. Two were identified as SRm160, a nuclear matrix-associated splicing coactivator, and hPrp8p, a core component of U5 snRNP and spliceosomes. Glycerol gradient fractionation showed that a subset of these proteins remain associated with mRNA after its release from the spliceosome. These results demonstrate that the spliceosome can leave behind signature proteins at exon–exon junctions. Such proteins could influence downstream metabolic events in vivo such as mRNA transport, translation, and nonsense-mediated decay.

scholarly journals LPTO-06. A NOVEL BRAIN-PERMEANT CHEMOTHERAPEUTIC AGENT FOR THE TREATMENT OF BREAST CANCER LEPTOMENINGEAL METASTASIS

2019 ◽  
Vol 1 (Supplement_1) ◽  
pp. i7-i7
Author(s):  
Jiaojiao Deng ◽  
Sophia Chernikova ◽  
Wolf-Nicolas Fischer ◽  
Kerry Koller ◽  
Bernd Jandeleit ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Brain Tumors ◽  
Cell Lines ◽  
Chemotherapeutic Agent ◽  
Leptomeningeal Metastasis ◽  
Breast Cancer Cell Lines ◽  
Normal Brain ◽  
Breast Cancers

Abstract Leptomeningeal metastasis (LM), a spread of cancer to the cerebrospinal fluid and meninges, is universally and rapidly fatal due to poor detection and no effective treatment. Breast cancers account for a majority of LMs from solid tumors, with triple-negative breast cancers (TNBCs) having the highest propensity to metastasize to LM. The treatment of LM is challenged by poor drug penetration into CNS and high neurotoxicity. Therefore, there is an urgent need for new modalities and targeted therapies able to overcome the limitations of current treatment options. Quadriga has discovered a novel, brain-permeant chemotherapeutic agent that is currently in development as a potential treatment for glioblastoma (GBM). The compound is active in suppressing the growth of GBM tumor cell lines implanted into the brain. Radiolabel distribution studies have shown significant tumor accumulation in intracranial brain tumors while sparing the adjacent normal brain tissue. Recently, we have demonstrated dose-dependent in vitro and in vivo anti-tumor activity with various breast cancer cell lines including the human TNBC cell line MDA-MB-231. To evaluate the in vivo antitumor activity of the compound on LM, we used the mouse model of LM based on the internal carotid injection of luciferase-expressing MDA-MB-231-BR3 cells. Once the bioluminescence signal intensity from the metastatic spread reached (0.2 - 0.5) x 106 photons/sec, mice were dosed i.p. twice a week with either 4 or 8 mg/kg for nine weeks. Tumor growth was monitored by bioluminescence. The compound was well tolerated and caused a significant delay in metastatic growth resulting in significant extension of survival. Tumors regressed completely in ~ 28 % of treated animals. Given that current treatments for LM are palliative with only few studies reporting a survival benefit, Quadriga’s new agent could be effective as a therapeutic for both primary and metastatic brain tumors such as LM. REF: https://onlinelibrary.wiley.com/doi/full/10.1002/pro6.43

scholarly journals MiR-4448 is involved in deltamethrin resistance by targeting CYP4H31 in Culex pipiens pallens

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Xixi Li ◽  
Shengli Hu ◽  
Haitao Yin ◽  
Hongbo Zhang ◽  
Dan Zhou ◽  
...  
Keyword(s):  
Culex Pipiens ◽  
Oral Feeding ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
Culex Pipiens Pallens ◽  
Deltamethrin Resistance ◽  
Regulatory Functions ◽  
Pipiens Pallens

Abstract Background Culex pipiens (Cx. pipiens) complex, which acts as a vector of viruses and is widespread and abundant worldwide, including West Nile virus, Japanese encephalitis virus, and Sindbis virus, can cause serious vector-borne diseases affecting human health. Unfortunately, mosquitoes have developed deltamethrin resistance because of its long-term overuse, representing a major challenge to mosquito control. Understanding the molecular regulatory mechanisms of resistance is vital to control mosquitoes. MicroRNAs (miRNAs) are short non-coding RNAs that have been demonstrated to be important regulators of gene expression across a wide variety of organisms, which might function in mosquito deltamethrin resistance. In the present study, we aimed to investigate the regulatory functions of miR-4448 and CYP4H31 in the formation of insecticidal resistance in mosquito Culex pipiens pallens. Methods We used quantitative real-time reverse transcription PCR to measure miR-4448 and CYP4H31 (encoding a cytochrome P450) expression levels. The regulatory functions of miR-4448 and CYP4H31 were assessed using dual-luciferase reporter assays. Then, oral feeding, RNA interference, and the American Centers for Disease Control and Prevention bottle bioassay were used to determine miR-4448’s association with deltamethrin resistance by targeting CYP4H31in vivo. Cell Counting Kit-8 (CCK-8) was also used to detect the viability of pIB/V5-His-CYP4H31-transfected C6/36 cells after deltamethrin treatment in vitro. Results MiR-4448 was downregulated in the deltamethrin-resistant strain (DR strain), whereas CYP4H31 was downregulated in deltamethrin-susceptible strain. CYP4H31 expression was downregulated by miR-4448 recognizing and binding to its 3′ untranslated region. Functional verification experiments showed that miR-4448 overexpression resulted in lower expression of CYP4H31. The mortality of miR-4448 mimic-injected DR strain mosquitoes was higher than that of the controls. CCK-8 assays showed that CYP4H31 decreased cellular resistance to deltamethrin in vitro and the mortality of the DR strain increased when CYP4H31 was knocked down in vivo. Conclusions In mosquitoes, miR-4448 participates in deltamethrin resistance by targeting CYP4H31. The results of the present study increase our understanding of deltamethrin resistance mechanisms.

scholarly journals EXTH-70. ENHANCEMENT OF ANTITUMOR ACTIVITY BY USING 5-ALA–MEDIATED SONODYNAMIC THERAPY TO INDUCE APOPTOSIS AND NECROSIS IN A MOUSE GLIOMA MODEL

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi97-vi97
Author(s):  
Satoshi Suehiro ◽  
Takanori Ohnishi ◽  
Akihiro Inoue ◽  
Daisuke Yamashita ◽  
Masahiro Nishikawa ◽  
...  
Keyword(s):  
Tumor Cells ◽  
Malignant Gliomas ◽  
Glioma Cells ◽  
High Intensity Focused Ultrasound ◽  
Control Group ◽  
Normal Brain ◽  
Sonodynamic Therapy ◽  
Cytotoxic Effects

Abstract OBJECTIVE High invasiveness of malignant gliomas frequently causes local tumor recurrence. To control such recurrence, novel therapies targeted toward infiltrating glioma cells are required. Here, we examined cytotoxic effects of sonodynamic therapy (SDT) combined with a sonosensitizer, 5-aminolevulinic acid (5-ALA), on malignant gliomas both in vitro and in vivo. METHODS In vitro cytotoxicity of 5-ALA-SDT was evaluated in U87 and U251 glioma cells and in U251Oct-3/4 glioma stemlike cells. Treatment-related apoptosis was analyzed using flow cytometry. Intracellular reactive oxygen species (ROS) were measured and the role of ROS in treatment-related cytotoxicity was examined. Effects of 5-ALA-SDT with high-intensity focused ultrasound (HIFU) on tumor growth, survival of glioma-transplanted mice, and histological features of the mouse brains were investigated. RESULTS The 5-ALA-SDT inhibited cell growth and changed cell morphology. Flow cytometric analysis indicated that 5-ALA-SDT induced apoptotic cell death. The 5-ALA-SDT generated higher ROS than in the control group, and inhibition of ROS generation completely eliminated the cytotoxic effects of 5-ALA-SDT. In the in vivo study, 5-ALA-SDT with HIFU greatly prolonged survival of the tumor-bearing mice compared with that of the control group (p < 0.05). Histologically, 5-ALA-SDT produced mainly necrosis of the tumor tissue in the focus area and induced apoptosis of the tumor cells in the perifocus area around the target of the HIFU-irradiated field. Normal brain tissues around the ultrasonic irradiation field of HIFU remained intact. CONCLUSIONS The 5-ALA-SDT was cytotoxic toward malignant gliomas. Generation of ROS by the SDT was thought to promote apoptosis of glioma cells. The 5-ALA-SDT with HIFU induced tumor necrosis in the focus area and apoptosis in the perifocus area of the HIFU-irradiated field. These results suggest that 5-ALA-SDT with HIFU may present a less invasive and tumor-specific therapy, not only for a tumor mass but also for infiltrating tumor cells in malignant gliomas.

scholarly journals PDTM-17. MiR-126, miR-369-5p AND miR-487b DRIVE PEDIATRIC GLIOBLASTOMA INVASION VIA KCNA1

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi190-vi191
Author(s):  
Yulun Huang ◽  
Lin Qi ◽  
Mari Kogiso ◽  
Yuchen Du ◽  
Frank Braun ◽  
...  
Keyword(s):  
Cell Migration ◽  
Neurological Deficits ◽  
Integrated Analysis ◽  
Normal Brain ◽  
Tumor Cell Migration ◽  
Monolayer Cultures ◽  
Invasive Capacity ◽  
Resected Primary Tumor

Abstract Diffuse invasion is one of the key features that make GBM particularly difficult to treat. We hypothesize that direct comparison of matched invasive (GBMINV) and tumor core GBM cells (GBMTC) would facilitate the discovery of drivers of pediatric GBM (pGBM) invasion. However, GBMINV cells are extremely difficult to obtain from normal brain tissues because aggressive surgical resection of normal tissue carries the risk of serious neurological deficits. Most past and current studies on GBM invasion were and are forced to utilize the resected primary tumor masses. To overcome this barrier, we utilized a panel of 6 pediatric patient tumor-derived orthotopic xenograft (PDOX) mouse models to isolate matching pairs of GBMTC cells and GBMINV cells and confirmed a significantly elevated invasive capacity in GBMINV cells both in vitro and in vivo. Global profiling of 768 human microRNA using a real-time PCR-based Taqman system identified 23 microRNAs were upregulated in the GBMINV cells in at least 4 of the 6 pGBM models as compared with the matching GBMTC cells. We subsequently showed that silencing the top three miRNAINV, miR-126, miR-369-5p, and miR-487b, suppressed tumor cell migration in vitro (both as neurospheres and monolayer cultures) without affecting cell proliferation, and blocked pGBM invasion in mouse brains. Integrated analysis of the mRNA profiling of the same set of GBMTC and GBMINV cells revealed the affected signaling pathways and identified KCNA1 as the sole common computational target gene of the three miRNAINV. Treatment of three pairs of GBMTC and GBMINV cells with two KCNA1 inhibitors, ADWX1 and Agitoxin 2, caused significant suppression of pGBM cell migration in vitro. In conclusion, this study revealed an intrinsically elevated invasive phenotype in GBMINV cells, identified miR-126, -369-5p, and -487b as novel drivers of pGBM invasion, and characterized KCNA1 as a potential therapeutic target for arresting pGBM invasion.

scholarly journals Functional Domains of the TOL Plasmid Transcription Factor XylS

2000 ◽  
Vol 182 (4) ◽  
pp. 1118-1126 ◽  
Author(s):  
Niilo Kaldalu ◽  
Urve Toots ◽  
Victor de Lorenzo ◽  
Mart Ustav
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Dependent Manner ◽  
Tol Plasmid ◽  
Terminal Fragment ◽  
Central Regions ◽  
Basic Features ◽  
Regulatory Functions

ABSTRACT The alkylbenzoate degradation genes of Pseudomonas putida TOL plasmid are positively regulated by XylS, an AraC family protein, in a benzoate-dependent manner. In this study, we used deletion mutants and hybrid proteins to identify which parts of XylS are responsible for the DNA binding, transcriptional activation, and benzoate inducibility. We found that a 112-residue C-terminal fragment of XylS binds specifically to the Pm operator in vitro, protects this sequence from DNase I digestion identically to the wild-type (wt) protein, and activates the Pm promoter in vivo. When overexpressed, that C-terminal fragment could activate transcription as efficiently as wt XylS. All the truncations, which incorporated these 112 C-terminal residues, were able to activate transcription at least to some extent when overproduced. Intactness of the 210-residue N-terminal portion was found to be necessary for benzoate responsiveness of XylS. Deletions in the N-terminal and central regions seriously reduced the activity of XylS and caused the loss of effector control, whereas insertions into the putative interdomain region did not change the basic features of the XylS protein. Our results confirm that XylS consists of two parts which probably interact with each other. The C-terminal domain carries DNA-binding and transcriptional activation abilities, while the N-terminal region carries effector-binding and regulatory functions.

The phylogenetically invariant ACAGAGA and AGC sequences of U6 small nuclear RNA are more tolerant of mutation in human cells than in Saccharomyces cerevisiae

1993 ◽  
Vol 13 (9) ◽  
pp. 5377-5382
Author(s):  
B Datta ◽  
A M Weiner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transient Expression ◽  
Human Cells ◽  
Mrna Splicing ◽  
Small Nuclear Rna ◽  
Transient Expression Assay ◽  
U6 Snrna ◽  
Nuclear Rna

U6 small nuclear RNA (snRNA) is the most highly conserved of the five spliceosomal snRNAs that participate in nuclear mRNA splicing. The proposal that U6 snRNA plays a key catalytic role in splicing [D. Brow and C. Guthrie, Nature (London) 337:14-15, 1989] is supported by the phylogenetic conservation of U6, the sensitivity of U6 to mutation, cross-linking of U6 to the vicinity of the 5' splice site, and genetic evidence for extensive base pairing between U2 and U6 snRNAs. We chose to mutate the phylogenetically invariant 41-ACAGAGA-47 and 53-AGC-55 sequences of human U6 because certain point mutations within the homologous regions of Saccharomyces cerevisiae U6 selectively block the first or second step of mRNA splicing. We found that both sequences are more tolerant to mutation in human cells (assayed by transient expression in vivo) than in S. cerevisiae (assayed by effects on growth or in vitro splicing). These differences may reflect different rate-limiting steps in the particular assays used or differential reliance on redundant RNA-RNA or RNA-protein interactions. The ability of mutations in U6 nucleotides A-45 and A-53 to selectively block step 2 of splicing in S. cerevisiae had previously been construed as evidence that these residues might participate directly in the second chemical step of splicing; an indirect, structural role seems more likely because the equivalent mutations have no obvious phenotype in the human transient expression assay.

scholarly journals Tenovin-6 impairs autophagy by inhibiting autophagic flux

2017 ◽  
Vol 8 (2) ◽  
pp. e2608-e2608 ◽  
Author(s):  
Hongfeng Yuan ◽  
Brandon Tan ◽  
Shou-Jiang Gao
Keyword(s):  
Cell Types ◽  
Inhibitory Effect ◽  
Lymphocytic Leukemia ◽  
Autophagic Flux ◽  
Dependent Manner ◽  
Autophagy Pathway ◽  
Sarcoma Cells ◽  
Regulatory Functions

Abstract Tenovin-6 has attracted significant interest because it activates p53 and inhibits sirtuins. It has anti-neoplastic effects on multiple hematopoietic malignancies and solid tumors in both in vitro and in vivo studies. Tenovin-6 was recently shown to impair the autophagy pathway in chronic lymphocytic leukemia cells and pediatric soft tissue sarcoma cells. However, whether tenovin-6 has a general inhibitory effect on autophagy and whether there is any involvement with SIRT1 and p53, both of which are regulators of the autophagy pathway, remain unclear. In this study, we have demonstrated that tenovin-6 increases microtubule-associated protein 1 light chain 3 (LC3-II) level in diverse cell types in a time- and dose-dependent manner. Mechanistically, the increase of LC3-II by tenovin-6 is caused by inhibition of the classical autophagy pathway via impairing lysosomal function without affecting the fusion between autophagosomes and lysosomes. Furthermore, we have revealed that tenovin-6 activation of p53 is cell type dependent, and tenovin-6 inhibition of autophagy is not dependent on its regulatory functions on p53 and SIRT1. Our results have shown that tenovin-6 is a potent autophagy inhibitor, and raised the precaution in interpreting results where tenovin-6 is used as an inhibitor of SIRT1.

scholarly journals Smad9 is a key player of follicular selection in goose via keeping the balance of LHR transcription

2017 ◽  
Author(s):  
Daolun Yu ◽  
Fanghui Chen ◽  
Li Zhang ◽  
Hejian Wang ◽  
Jie Chen ◽  
...  
Keyword(s):  
Chromatin Immunoprecipitation ◽  
Hormone Receptor ◽  
Egg Production ◽  
Follicular Development ◽  
Luteinizing Hormone Receptor ◽  
Summary Statement ◽  
Follicular Selection ◽  
New Strategies

ABSTRACTThe egg production of poultry depends on follicular development and selection. However, the mechanism of selecting the priority of hierarchical follicles is completely unknown. Smad9 is one of the important transcription factors in BMP/Smads pathway and involved in goose follicular initiation. To explore its potential role in goose follicle hierarchy determination, we first blocked Smad9 expression using BMP typeⅠreceptor inhibitor LDN–193189 both in vivo and in vitro. Unexpectedly, LDN–193189 administration could dramatically suppress Smad9 level and elevate egg production (7.08 eggs / bird, P< 0.05) of animals, and the estradiol (E2) and luteinizing hormone receptor (LHR) level were significantly increased (P< 0.05), but the progesterone (P4) and follicle stimulating hormone receptor (FSHR) mRNA remain unchanged. Surprisingly, Smad9 knockdown notably attenuated (P< 0.05) in E2, P4, FSHR and LHR level in goose granulosa cells (gGCs). Further chromatin immunoprecipitation (ChIP) assay of gGCs revealed that Smad9, served as a sensor of balance, bound to the LHR promoter regulating its transcription. These findings demonstrated that Smad9 is differentially expressed in goose follicles, and acts as a key player in controlling goose follicular selection.SUMMARY STATEMENTTo study the hierarchical development mechanism of avian follicle, new strategies can be found to improve the egg production of low-yielding poultry, such as geese.

scholarly journals lncRNA MIR22HG-Derived miR-22-5p Enhances the Radiosensitivity of Hepatocellular Carcinoma by Increasing Histone Acetylation Through the Inhibition of HDAC2 Activity

2021 ◽  
Vol 11 ◽  
Author(s):  
Qiao Jin ◽  
Hao Hu ◽  
Siqi Yan ◽  
Long Jin ◽  
Yuliang Pan ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Histone Acetylation ◽  
Chromatin Immunoprecipitation ◽  
Promoter Region ◽  
Animal Experiments ◽  
Primary Hepatocellular Carcinoma ◽  
Acetylation Level ◽  
Expression Levels

BackgroundWith the development of radiotherapy technology, radiotherapy has been increasingly used to treat primary hepatocellular carcinoma (HCC). However, due to radioresistance and the intolerance of the adjacent organs to radiation, the effects of radiotherapy are often unsatisfactory. Therefore, it is necessary to study radiosensitization in HCC.MethodA microarray was used to analyze the genes that were significantly associated with radiosensitivity. HCC cells, HepG2 and MHCC97H, were subjected to radiation in vitro. Real-time PCR was performed to determine MIR22HG (microRNA22 host gene) and miR-22-5p expression levels. Western blotting was performed to determine histone expression levels. A histone deacetylase (HDAC) whole cell assay was used to determine the activity of HDAC2. MTT, colony formation, 5-ethynyl-2′-deoxyuridine, and wound healing assays were performed to examine the function of MIR22HG and miR-22-5p in cellular radiosensitivity. Chromatin immunoprecipitation-PCR was used to confirm that HDAC2 affects the acetylation level of the MIR22HG promoter region. Finally, animal experiments were performed to demonstrate the in vivo effect of MIR22HG on the radiosensitivity of hepatoma.ResultsIrradiation can up-regulate MIR22HG expression and down-regulate HDAC2 expression. Inhibition of HDAC2 expression promotes histone acetylation in the MIR22HG promoter region and up-regulates MIR22HG expression. MIR22HG can increase radiosensitivity via miR-22-5p in HCC.ConclusionInhibition of HDAC2 expression promotes histone acetylation in the MIR22HG promoter region, thereby up-regulating the expression of MIR22HG and promoting the production of miR-22-5p, and ultimately increasing the sensitivity of liver cancer radiotherapy.

Sign in / Sign up

Export Citation Format

Share Document