Результаты поиска

Alu-dependent RNA editing of GLI1 promotes malignant regeneration in multiple myeloma.

Despite novel therapies, relapse of multiple myeloma (MM) is virtually inevitable. Amplification of chromosome 1q, which harbors the inflammation-responsive RNA editase adenosine deaminase acting on RNA (ADAR)1 gene, occurs in 30-50% of MM patients and portends a poor prognosis. Since adenosine-to-inosine RNA editing has recently emerged as a driver of cancer progression, genomic amplification combined with inflammatory cytokine activation of ADAR1 could stimulate MM progression and therapeutic resistance. Here, we report that high ADAR1 RNA expression correlates with reduced patient survival

Совпадение в полном тексте источника
… . ** p < 0.01, by unpaired, two-tailed Student’s t -test. l Relative GLI1- Luciferase / Renilla reporter activity (mean ± S.E.M.). … Dual reporter assays HEK293T cells were plated in 96-well plates (0.3 × 10 5 cells per well) 24 h prior to transfection with Lipofectamine 2000 (Life Technologies). pCDH backbone/GLI1 WT/GLI1 R701G plasmids were co-transfected with a dual GLI reporter system (6X-GLI1- Luciferase reporter and Renilla …

Targeting of multiple myeloma-related angiogenesis by miR-199a-5p mimics: in vitro and in vivo anti-tumor activity.

Multiple myeloma (MM) cells induce relevant angiogenic effects within the human bone marrow milieu (huBMM) by the aberrant expression of angiogenic factors. Hypoxia triggers angiogenic events within the huBMM and the transcription factor hypoxia-inducible factor-1α (HIF-1α) is over-expressed by MM cells. Since synthetic miR-199a-5p mimics negatively regulates HIF-1α, we here investigated a miRNA-based therapeutic strategy against hypoxic MM cells. We indeed found that enforced expression of miR-199a-5p led to down-modulated expression of HIF-1α as well as of other pro-angiogenic factors such a

Совпадение в полном тексте источника
… The firefly luciferase activity was normalized to renilla luciferase activity. The data are shown as relative luciferase activity of miR-199a-5p-transfected cells as compared to the control (NC) of a total of six experiments from three independent transfections. P<0,05. … Firefly and Renilla luciferase activities were measured consecutively using the dual-luciferase assay kit (Promega Corporation, Madison, WI, USA) 24 hours after transfection. Data are expressed as luminescence from firefly luciferase divided by luminescence from renilla luciferase. …

DNA-demethylating and anti-tumor activity of synthetic miR-29b mimics in multiple myeloma.

Aberrant DNA methylation plays a relevant role in multiple myeloma (MM) pathogenesis. MicroRNAs (miRNAs) are a class of small non-coding RNAs that recently emerged as master regulator of gene expression by targeting protein-coding mRNAs. However, miRNAs involvement in the regulation of the epigenetic machinery and their potential use as therapeutics in MM remain to be investigated. Here, we provide evidence that the expression of de novo DNA methyltransferases (DNMTs) is deregulated in MM cells. Moreover, we show that miR-29b targets DNMT3A and DNMT3B mRNAs and reduces global DNA methylation i

Совпадение в полном тексте источника
… The firefly luciferase activity was normalized to renilla luciferase activity. The data are shown as relative luciferase activity of miR-29b-transfected cells as compared to the control (NC) of a total of six experiments from three independent transfections. °P<0,01. (C). … Firefly and Renilla luciferase activities were measured consecutively using the dual-luciferase assay kit (Promega) 24 hours after transfection. DNA extraction and measurement of global DNA methylation levels DNA was extracted by a commercial kit (Invitrogen). …

Recurrent Angiolymphoid Hyperplasia with Eosinophilia of the Parotid.

Mitochondrial fusion plays an essential role in mitochondrial calcium homeostasis, bioenergetics, autophagy and quality control. Fusion is q … ferase assay. Two human cell lines are targeted with either the amino or a carboxy terminal part of Renilla luciferase along with a leucine zipper to ensure dimerization upon mixing. Mitochondria are isolated from each cell line, and fused. The fusion reaction can occur without the cytosol under physiological conditions in the presence of energy, appropri …

Targeting HLA-E in Lung Cancer: The Therapeutic Potential of IRF5-Engineered M1-Macrophage-Derived Exosomes.

Immunotherapy is a pivotal approach in the treatment of lung cancer. Although HLA-E is a potential target for tumor immunotherapy, its role in lung cancer remains unclear. Previous studies have identified the transcription factor IRF5 as a characteristic gene of M1-like macrophages, highlighting its crucial role in promoting antitumor immune responses. In this study, we developed an engineered M1-like macrophage exosomes expressing IRF5 (IRF5 M1-exos) and demonstrated their ability to inhibit proliferation, migration, and invasion of lung cancer cells. Moreover, our experiments using a nude mo

Совпадение в полном тексте источника
… Each DNA construct (2 μg) and the Renilla construct (0.3 μg, Promega) were combined with lipo3000 (10 μL, Qiagen, Germany). At room temperature, the mixture was incubated for 20 min. … Firefly and Renilla luciferase enzymatic activities were measured using the Dual‐Luciferase Reporter assay System (Promega) and a luminometer. All luciferase assays were conducted in duplicate or triplicate, and each experiment was replicated at least twice. …

LncRNA OIP5‑AS1 aggravates house dust mite‑induced inflammatory responses in human bronchial epithelial cells via the miR‑143‑3p/HMGB1 axis.

Bronchial asthma poses a serious threat to human health. Previous studies have documented the role of long non‑coding RNAs (lncRNAs) in asthma. However, the molecular mechanism underlying bronchial asthma remains unclear. The aim of the present study was to evaluate the role of the lncRNA Opa‑interacting protein 5 antisense RNA1 (OIP5‑AS1) in the house dust mite‑induced inflammatory response in human bronchial epithelial cells. BEAS‑2B cells were treated with Dermatophagoides pteronyssinus peptidase 1 (Der p1) to establish an in vitro model of asthma. OIP5‑AS1 expression levels increased in BE

Совпадение в полном тексте источника
… After 48-h incubation, firefly and Renilla luciferase activities were measured using a Bright-Glo™ Luciferase Assay system (Promega Corporation). Firefly luciferase activity was normalized to Renilla luciferase activity. …

Структурное и механистическое профилирование модулятуры Hurr1 vidofludimus позволяет проектировать лиганд на основе структуры.

В центре биомедицинских исследований находится фактор нейрозащиты, связанный с ядерным рецептором 1 (Nurr1, NR4A2) для его многообещающей нейрозащитной роли в болезни Паркинсона, болезни Альцгеймера и множественном склерозе. Его деятельность может контролироваться лигандами, предлагающими доступ к фармакологической модуляции Hurr1. Вместе с тем связующие эпитопы и механизмы молекулярной активации синтетических активаторов Hurr1 по-прежнему остаются труднодостижимыми, но имеют важное значение для продвижения к новым лекарствам лигандов Hurr1. Здесь мы охарактеризовали диссоциацию Hurr1 димера и

Совпадение в полном тексте источника
… The assays were conducted in a uniform format using a Gal4-responsive firefly luciferase reporter construct and a constitutively expressed (SV40 promoter) renilla luciferase as control gene for normalization. … Firefly luminescence was divided by Renilla luminescence and multiplied by 1000, resulting in relative light units (RLUs) to normalize for transfection efficiency and cell growth. Fold activation was obtained by dividing the mean RLU of a test sample by the mean RLU of the untreated control. …

A novel mechanism in regulating drug sensitivity, growth, and apoptosis of bortezomib-resistant multiple myeloma cells: the USP4/KLF2/HMGA2 cascade.

BackgroundMultiple myeloma (MM) is a malignant disorder originating from plasma cells. Bortezomib (BTZ) resistance has become a huge obstacle to MM treatment. Herein, we elucidated the action of Kruppel-like factor 2 (KLF2), a crucial transcription factor (TF), on BTZ resistance of MM.MethodsTwo BTZ-resistant cell lines (MM1.S/BTZ and NCI-H929/BTZ) were generated and used. KLF2 mRNA was quantified by quantitative PCR, and protein expression was analyzed by immunoblotting. MTT cell cytotoxicity assay was used to test BTZ sensitivity. Cell growth was detected by MTT and EdU assays. Flow cytometr

Совпадение в полном тексте источника
… Meantime, the pRL-TK Renilla vector (Promega, Paris, France) was co-transfected and used as the control for normalization. 48 h later, the Dual-Luciferase Assay Kit (Beyotime) was used for the measurement of luciferase activity (firefly/ Renilla ). …

Aberrant NSUN2-mediated m5C modification of exosomal LncRNA MALAT1 induced RANKL-mediated bone destruction in multiple myeloma.

The impact of exosome-mediated crosstalk between multiple myeloma (MM) cells and osteoclasts (OCs) on bone lesions remains to be investigated. Here, we identified NSUN2 and YBX1-mediated m5C modifications upregulated LncRNA MALAT1 expression in MM cells, which could be transported to OCs via exosomes and promote bone lesions. Methodologically, RNA-seq was carried out to detect the cargoes of exosomes. TRAP staining and WB were used to evaluate osteoclastogenesis in vitro. Micro-CT and bone histomorphometric analyses were performed to identify bone destruction in vivo. RNA pull-down, RIP, MeRIP

Совпадение в полном тексте источника
… Wild-type sequences of RANKL mRNA and MALAT1 that bind to miR-17 were subcloned into a 3’ untranslated region (UTR) of the Renilla luciferase gene within the psiCHECK2 plasmid (Promega, USA). … Firefly luciferase activity was normalized to Renilla luciferase activity to control for transfection efficiency. The relative luciferase activity (luciferase ratio) was calculated by comparing the normalized luciferase activity of the experimental group to that of the control group. …

CPUY201112, a novel synthetic small-molecule compound and inhibitor of heat shock protein Hsp90, induces p53-mediated apoptosis in MCF-7 cells.

Heat-shock protein 90 (Hsp90) is highly expressed in many tumor cells and is associated with the maintenance of malignant phenotypes. Targeting Hsp90 has had therapeutic success in both solid and hematological malignancies, which has inspired more studies to identify new Hsp90 inhibitors with improved clinical efficacy. Using a fragment-based approach and subsequent structural optimization guided by medicinal chemistry principles, we identified the novel compound CPUY201112 as a potent Hsp90 inhibitor. It binds to the ATP-binding pocket of Hsp90 with a kinetic dissociation (Kd) constant of 27

Совпадение в полном тексте источника
… The cells were co-transfected with pHspA1A-Luc and pRL-SV40 (a plasmid encoding Renilla luciferase) using Lipofectamine 2000. After 6 h, the cells were treated with the indicated concentrations of the various compounds for 24 h. … The level of Hsp70 activation for a sample was calculated as the ratio between firefly and Renilla luciferase activity for the same sample. Typically, each sample was run in triplicate. Results were obtained from at least three separate experiments. …

Ginsenoside Rg3 inhibits melanoma cell proliferation through down-regulation of histone deacetylase 3 (HDAC3) and increase of p53 acetylation.

Malignant melanoma is an aggressive and deadly form of skin cancer, and despite recent advances in available therapies, is still lacking in completely effective treatments. Rg3, a monomer extracted from ginseng roots, has been attempted for the treatment of many cancers. It is reported that the expressions of histone deacetylase 3 (HDAC3) and p53 acetylation correlate with tumor cell growth. However, the antitumor effect of Rg3 on melanoma and the mechanism by which it regulates HDAC3 expression and p53 acetylation remain unknown. We found high expression of HDAC3 in human melanoma tissues to

Совпадение в полном тексте источника
… Luciferase reporter assay p53 luciferase activity was determined in A375 and C8161 cells cotransfected, using Lipofectamine 2000 Reagent, with 2 µg of p53 luciferase plasmids and 0.2 µg of pGL3, which constitutively expressed renilla luciferase. … Firefly luciferase activity was measured and the reading was normalized to renilla luciferase activity, which served as an internal control for transfection efficiency. …

Endothelial TFEB signaling-mediated autophagic disturbance initiates microglial activation and cognitive dysfunction.

Cognitive impairment caused by systemic chemotherapy is a critical question that perplexes the effective implementation of clinical treatment, but related molecular events are poorly understood. Herein, we show that bortezomib exposure leads to microglia activation and cognitive impairment, this occurs along with decreased nuclear translocation of TFEB (transcription factor EB), which is linked to macroautophagy/autophagy disorder, STAT3 (signal transducer and activator of transcription 3) phosphorylation and IL23A (interleukin 23 subunit alpha) expression. Pharmacological enhancement of TFEB

Совпадение в полном тексте источника
… Then, 500 ng Firefly luciferase reporter vectors driven by normal or mutant IL23A promoter region and 100 ng Renilla luciferase reporter vector pRL-TK (Beyotime, D2760) were transfected into HEK293 cells using Lipofectamine 3000 (Invitrogen, L3000008) according to the manufacturer. … Finally, the data was calculated by normalizing Firefly/Renilla relative light unit values. …

MiR-328-5p/BCL7A axis is involved in fracture healing by modulating osteoblast function.

BackgroundDelayed fracture healing severely impacts patients' quality of life and imposes economic burdens.ObjectiveThis study aimed to investigate the role of miR-328-5p in fracture healing and its underlying molecular mechanisms, providing novel insights for the management of delayed fracture healing.MethodsSerum miR-328-5p levels were measured by RT-qPCR in 125 tibial fracture patients. The diagnostic potential of miR-328-5p for delayed healing was assessed through ROC curve analysis. Osteogenic differentiation of hFOB 1.19 cells was induced using osteogenic medium followed by evaluation of

Совпадение в полном тексте источника
… Firefly luciferase activity was normalized to Renilla luciferase activity (mean ± SD, n = 3). D Serum BCL7A mRNA levels in patients with normal and delayed healing, quantified by RT-qPCR. … Firefly luciferase activity was normalized to Renilla luciferase activity (mean ± SD, n = 3). D Serum BCL7A mRNA levels in patients with normal and delayed healing, quantified by RT-qPCR. …

Wnt inhibitors reduce the unfolded protein response and enhance bortezomib-induced cell death in multiple myeloma.

Аннотация не предоставлена источником. Откройте карточку для перехода к публикации.

Совпадение в полном тексте источника
… Data is normalized to internal Renilla and FOP negative control, and shown as fold change to untreated control. Bars show the mean of 3 individual experiments, error bars represent the standard error of the mean and statistical significance was determined by unpaired t -test. …

MicroRNA-195 reverses the resistance to temozolomide through targeting cyclin E1 in glioma cells.

Glioma is the most common malignant tumor of the central nervous system with poor survival. Temozolomide (TMZ) is the first-line chemotherapy drug for initial and recurrent glioma treatment with a relatively good efficacy, which exerts its antitumor effects mainly through cell death induced by DNA double-strand breaks in the G1 and S phases. However, endogenous or acquired resistance to TMZ limits glioma patients' clinical outcome and is also an important cause of glioma replase. MicroRNA-195 (miR-195) plays an important role in the regulation of G1-phase/S-phase transition, DNA damage repair,

Совпадение в полном тексте источника
… Next, 100 μl of Stop & Glo reagent (Promega, Madison, Wisconsin, USA) was added to each well to initiate the renilla luciferase. The psiCHECK-2 vector, which provides constitutive expression of firefly luciferase, was co-transfected as an internal control. …

Связанный с K27 ROR__t Уничтожение Nedd4 потенциалами Th17-посредованная автонепроницаемость.

СПРАВОЧНАЯ ИНФОРМАЦИЯ: Было продемонстрировано, что THCT E3 Ubriquitin ligase Nedd4 позитивно регулирует реакцию клеток Т, однако его роль в дифференциации клеток и автономном иммунитете Т неизвестна. Считается, что 17 клеток играют ключевую роль в развитии и патогенезе аутоиммунных заболеваний. Тем не менее, правила, регулирующие активацию РОРТ в ходе дифференциации клеток TH17 с помощью сигнала TCR, пока еще не уточнены. Эти неизведанные аспекты вдохновляют нас на изучение потенциальной роли Nedd4 в автоматическом иммунитете Th17.МЕТОДЫ: Мы провели оценку воздействия дефицита Nedd4 на развит

Совпадение в полном тексте источника
… At ~ 80% confluence, cells were transfected with RORγt reporter plasmid (100 ng), pSV40-Renilla luciferase vector (50 ng), and expression vectors (Flag-tagged Nedd4 or Nedd4 C867A, together with HA-tagged ubiquitin, or Flag-tagged Nedd4 together with HA-tagged K27 ubiquitin or K27R ubiquitin; 0.5 μg) … Cells were collected after 24 h and lysed in 20 μl Passive Lysis Buffer; activities were measured by the Dual Luciferase system, according to the manufacturer’s instructions (Promega, WI, USA), and normalized against Renilla luciferase activities. …

MicroRNA-150-5p inhibits the proliferation and invasion of human larynx epidermiod cancer cells though regulating peptidyl-prolyl cis/trans isomerase.

ObjectiveThis study aimed to investigate the molecular mechanism of miR-150-5p regulating the malignant biological behavior of Human Epidermoid cancer cell (HEp-2) by targeting peptidyl-prolyl cis/trans isomerase NIMA-Interacting-1 (PIN1).MethodsFirstly, qRT-PCR and Western blot were adopted to detect the expression levels of miR-150-5p and PIN1 in cancer tissue and paracancerous tissues of patients with LSCC, and those in human bronchial epithelial cells (16 HBE) and HEp-2. Next, the targeted relationship between miR-150-5p and PIN1 was assessed by bioinformatics website and dual-luciferase r

Совпадение в полном тексте источника
… Later, relative firefly luciferase activity was calculated with Renilla luciferase activity serving as an internal control. Western blot The tissues and cells of each group were lysed 30 min with RIPA lysis buffer, and then crushed by sonication in ice bath. …

miR‑140‑5p inhibits the proliferation of multiple myeloma cells by targeting VEGFA.

MicroRNA (miR)‑140‑5p is associated with the growth and metastasis of various tumor cell types, yet its role in multiple myeloma (MM) remains unclear. Therefore, the present study aimed to investigate the regulatory effect of miR‑140‑5p on MM. Reverse transcription‑quantitative PCR analysis demonstrated that miR‑140‑5p was downregulated in MM cell lines, particularly in U266 and RPMI 8226 cells. A Cell Counting Kit‑8, wound healing and Transwell assays, as well as flow cytometry indicated that a miR‑140‑5p mimic could suppress cell viability, migration and invasion. In addition, the mimic prom

Совпадение в полном тексте источника
… The Renilla luciferase activity was used for reference. Reverse transcription-quantitative PCR (RT-qPCR) Total RNA was extracted from cells using TRIzol ® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). …

Exosomal miR-328 originated from pulmonary adenocarcinoma cells enhances osteoclastogenesis via downregulating Nrp-2 expression.

Osseous metastases of pulmonary carcinoma and the detailed mechanisms remain unclear, and the effects of exosomes (Exos) originated from pulmonary adenocarcinoma cells in this process have received a lot of attentions. Our study revealed that the Exos secreted from A549 cells (A549-Exos) enhanced osteoclastogenesis and osseous resorption in vitro. In addition, A549-Exos showed a targeted effect on bones to enhance osseous resorption in vivo. A549-exosomal miR-328 enhanced osseous resorption via downregulating neuropilin 2 (Nrp-2) expression, and A549-Exos miR-328 inhibitors suppressed osseous

Совпадение в полном тексте источника
… RAW 264.7 cells firstly received transfection of WT or mutant pGL3 construct and then received transfection of 40 ng pRL-TK Renilla-luciferase plasmid, miR-328 mimics or inhibitors respectively for 48 h. Luciferase activity was detected according to the method in the literature [ 30 ]. …

Interference of long non-coding RNA HAGLROS inhibits the proliferation and promotes the apoptosis of ovarian cancer cells by targeting miR-26b-5p.

Ovarian cancer (OV) is the fifth most common type of cancer affecting women worldwide. Long non-coding RNAs (lncRNAs) serve essential roles in the progression of OV. As such, the present study aimed to investigate the specific role of HAGLR opposite strand lncRNA (HAGLROS) in OV and the underlying mechanism of action through which HAGLROS exerts its effects on OV cells. In the present study, the expression of HAGLROS in several OV cell lines was first detected using reverse transcription-quantitative PCR. HAGLROS was then silenced to evaluate cell viability, proliferation and apoptosis, which

Совпадение в полном тексте источника
… After incubation for 48 h at 37˚C, firefly and Renilla luciferase activities were assessed. Statistical analysis GraphPad Prism version 8.0 (GraphPad Software, Inc.) was used to analyze the experimental data. All experiments were performed three times and the data were presented as mean ± SD. …