scholarly journals Gene-activated matrix harboring a miR20a-expressing plasmid promotes rat cranial bone augmentation

2021 ◽  
Vol 8 (2) ◽  
Author(s):  
Rena Shido ◽  
Yoshinori Sumita ◽  
Masahito Hara ◽  
Mayumi Iwatake ◽  
Shun Narahara ◽  
...  
Keyword(s):  
Target Genes ◽  
Osteoblastic Differentiation ◽  
Cranial Bone ◽  
Bone Marrow Mscs ◽  
Bone Augmentation ◽  
Bone Surface ◽  
Naked Plasmid ◽  
Rat Bone

Abstract Gene-activated matrix (GAM) has a potential usefulness in bone engineering as an alternate strategy for the lasting release of osteogenic proteins but efficient methods to generate non-viral GAM remain to be established. In this study, we investigated whether an atelocollagen-based GAM containing naked-plasmid (p) DNAs encoding microRNA (miR) 20a, which may promote osteogenesis in vivo via multiple pathways associated with the osteogenic differentiation of mesenchymal stem/progenitor cells (MSCs), facilitates rat cranial bone augmentation. First, we confirmed the osteoblastic differentiation functions of generated pDNA encoding miR20a (pmiR20a) in vitro, and its transfection regulated the expression of several of target genes, such as Bambi1 and PPARγ, in rat bone marrow MSCs and induced the increased expression of BMP4. Then, when GAMs fabricated by mixing 100 μl of 2% bovine atelocollagen, 20 mg β-TCP granules and 0.5 mg (3.3 μg/μl) AcGFP plasmid-vectors encoding miR20a were transplanted to rat cranial bone surface, the promoted vertical bone augmentation was clearly recognized up to 8 weeks after transplantation, as were upregulation of VEGFs and BMP4 expressions at the early stages of transplantation. Thus, GAM-based miR delivery may provide an alternative non-viral approach by improving transgene efficacy via a small sequence that can regulate the multiple pathways.

scholarly journals Gene-Activated Matrix with Self-Assembly Anionic Nano-Device Containing Plasmid DNAs for Rat Cranial Bone Augmentation

Materials ◽  
2021 ◽  
Vol 14 (22) ◽  
pp. 7097
Author(s):  
Masahito Hara ◽  
Yoshinori Sumita ◽  
Yukinobu Kodama ◽  
Mayumi Iwatake ◽  
Hideyuki Yamamoto ◽  
...  
Keyword(s):  
Self Assembly ◽  
Electrostatic Interactions ◽  
Cranial Bone ◽  
Bone Augmentation ◽  
Mesenchymal Progenitor Cells ◽  
Anionic Polymer ◽  
Morphogenetic Protein ◽  
Low Cytotoxicity

We have developed nanoballs, a biocompatible self-assembly nano-vector based on electrostatic interactions that arrange anionic macromolecules to polymeric nanomaterials to create nucleic acid carriers. Nanoballs exhibit low cytotoxicity and high transfection efficiently in vivo. This study investigated whether a gene-activated matrix (GAM) composed of nanoballs containing plasmid (p) DNAs encoding bone morphogenetic protein 4 (pBMP4) could promote bone augmentation with a small amount of DNA compared to that composed of naked pDNAs. We prepared nanoballs (BMP4-nanoballs) constructed with pBMP4 and dendrigraft poly-L-lysine (DGL, a cationic polymer) coated by γ-polyglutamic acid (γ-PGA; an anionic polymer), and determined their biological functions in vitro and in vivo. Next, GAMs were manufactured by mixing nanoballs with 2% atelocollagen and β-tricalcium phosphate (β-TCP) granules and lyophilizing them for bone augmentation. The GAMs were then transplanted to rat cranial bone surfaces under the periosteum. From the initial stage, infiltrated macrophages and mesenchymal progenitor cells took up the nanoballs, and their anti-inflammatory and osteoblastic differentiations were promoted over time. Subsequently, bone augmentation was clearly recognized for up to 8 weeks in transplanted GAMs containing BMP4-nanoballs. Notably, only 1 μg of BMP4-nanoballs induced a sufficient volume of new bone, while 1000 μg of naked pDNAs were required to induce the same level of bone augmentation. These data suggest that applying this anionic vector to the appropriate matrices can facilitate GAM-based bone engineering.

scholarly journals Nanoparticles Delivering Antagomir-483-5p to Bone Marrow Mesenchymal Stem Cells Prevent Osteoporosis by Increasing Bone Formation

2021 ◽  
Author(s):  
Yue Zhou ◽  
Hao Jia ◽  
Aihua Hu ◽  
Rangru Liu ◽  
Xiangzhou Zeng ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Formation ◽  
Small Molecules ◽  
Target Genes ◽  
Osteoblastic Differentiation ◽  
Mitogen Activated Protein Kinase ◽  
Osteoporosis Prevention ◽  
Bone Formation Markers

Abstract Background: Promoting bone marrow mesenchymal stem cell (BMSC) osteoblastic differentiation is a promising therapeutic strategy for osteoporosis (OP). The present study demonstrates that miR-483-5p inhibits the osteogenic differentiation of BMSCs. Therefore, selectively delivering the nanoparticles carrying antagomir-483-5p (miR-483-5p inhibitor) to BMSCs is expected to become an effective treatment drug for OP.Methods: Real-time PCR assays were used to analyse miR-483-5p, ALP and Bglap levels in BMSCs of ovariectomized and aged osteoporotic mice. To selectively and efficiently deliver antagomir-483-5p to BMSCs in vivo, immunoglobulin G and poloxamer-188 were used to encapsulate the functional small molecules, and BMSC-targeting aptamer was employed to confirm the direction of the nanoparticles. Luciferase assays were used to determine the target genes of miR-483-5p. Western blot assays and immunohistochemistry staining were used to detect the targets in vitro and vivo.Results: miR-483-5p levels were increased in BMSCs of ovariectomized and aged osteoporotic mice. Inhibition of miR-483-5p levels in BMSCs by antagomir-483-5p in vitro promoted the expression of bone formation markers, such as ALP and Bglap. The FAM-BMSC-aptamer-nanoparticles carrying antagomir-483-5p were taken up by BMSCs, resulting in stimulation of BMSC osteoblastic differentiation in vitro and osteoporosis prevention in vivo. Furthermore, our research demonstrated that mitogen-activated protein kinase 1 (MAPK1) and SMAD family member 5 (Smad5) were direct targets of miR-483-5p in regulating BMSC osteoblastic differentiation and osteoporosis pathological processes. Conclusions: The important therapeutic role of FAM-BMSC-aptamer-nanoparticles carrying antagomir-483-5p in osteoporosis was established in our study. These nanoparticles are novel candidate for the clinical prevention and treatment of osteoporosis. The optimized targeted drug delivery platform for small molecules will provide new ideas for the treatment of clinical diseases.

scholarly journals Osteoprotective Effects of Loganic Acid on Osteoblastic and Osteoclastic Cells and Osteoporosis-Induced Mice

2020 ◽  
Vol 22 (1) ◽  
pp. 233
Author(s):  
Eunkuk Park ◽  
Chang Gun Lee ◽  
Eunguk Lim ◽  
Seokjin Hwang ◽  
Seung Hee Yun ◽  
...  
Keyword(s):  
Osteoclast Differentiation ◽  
Osteoblastic Differentiation ◽  
Common Disease ◽  
Root Extract ◽  
Mouse Bone Marrow ◽  
Mineral Density ◽  
Bone Mineral Density Loss ◽  
In Vivo Experiments

Osteoporosis is a common disease caused by an imbalance of processes between bone resorption by osteoclasts and bone formation by osteoblasts in postmenopausal women. The roots of Gentiana lutea L. (GL) are reported to have beneficial effects on various human diseases related to liver functions and gastrointestinal motility, as well as on arthritis. Here, we fractionated and isolated bioactive constituent(s) responsible for anti-osteoporotic effects of GL root extract. A single phytochemical compound, loganic acid, was identified as a candidate osteoprotective agent. Its anti-osteoporotic effects were examined in vitro and in vivo. Treatment with loganic acid significantly increased osteoblastic differentiation in preosteoblast MC3T3-E1 cells by promoting alkaline phosphatase activity and increasing mRNA expression levels of bone metabolic markers such as Alpl, Bglap, and Sp7. However, loganic acid inhibited osteoclast differentiation of primary-cultured monocytes derived from mouse bone marrow. For in vivo experiments, the effect of loganic acid on ovariectomized (OVX) mice was examined for 12 weeks. Loganic acid prevented OVX-induced bone mineral density loss and improved bone structural properties in osteoporotic model mice. These results suggest that loganic acid may be a potential therapeutic candidate for treatment of osteoporosis.

scholarly journals Biomimetic reduced graphene oxide coated collagen scaffold for in situ bone regeneration

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Sajad Bahrami ◽  
Nafiseh Baheiraei ◽  
Mostafa Shahrezaee
Keyword(s):  
Graphene Oxide ◽  
Reduced Graphene Oxide ◽  
Cranial Bone ◽  
Drying Methods ◽  
Porous Scaffolds ◽  
Alizarin Red ◽  
Reduced Graphene ◽  
Coated Collagen

AbstractA variety of bone-related diseases and injures and limitations of traditional regeneration methods require new tissue substitutes. Tissue engineering and regeneration combined with nanomedicine can provide different natural or synthetic and combined scaffolds with bone mimicking properties for implantation in the injured area. In this study, we synthesized collagen (Col) and reduced graphene oxide coated collagen (Col-rGO) scaffolds, and we evaluated their in vitro and in vivo effects on bone tissue repair. Col and Col-rGO scaffolds were synthesized by chemical crosslinking and freeze-drying methods. The surface topography, and the mechanical and chemical properties of scaffolds were characterized, showing three-dimensional (3D) porous scaffolds and successful coating of rGO on Col. The rGO coating enhanced the mechanical strength of Col-rGO scaffolds to a greater extent than Col scaffolds by 2.8 times. Furthermore, Col-rGO scaffolds confirmed that graphene addition induced no cytotoxic effects and enhanced the viability and proliferation of human bone marrow-derived mesenchymal stem cells (hBMSCs) with 3D adherence and expansion. Finally, scaffold implantation into rabbit cranial bone defects for 12 weeks showed increased bone formation, confirmed by Hematoxylin–Eosin (H&E) and alizarin red staining. Overall, the study showed that rGO coating improves Col scaffold properties and could be a promising implant for bone injuries.

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 TMOD-15. EFFICACY OF THE MDM2 INHIBITOR KRT-232 IN GLIOBLASTOMA PATIENT-DERIVED XENOGRAFT MODELS

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii231-ii231
Author(s):  
Rachael Vaubel ◽  
Ann Mladek ◽  
Yu Zhao ◽  
Shiv K Gupta ◽  
Minjee Kim ◽  
...  
Keyword(s):  
Target Genes ◽  
Xenograft Model ◽  
Culture Conditions ◽  
Patient Derived Xenograft ◽  
Fold Induction ◽  
Extended Survival ◽  
Mdm2 Amplification ◽  
Mdm2 Inhibitor

Abstract Non-genotoxic reactivation of p53 by MDM2 inhibitors represents a promising therapeutic strategy for tumors with wild-type TP53, particularly tumors harboring MDM2 amplification. MDM2 controls p53 levels by targeting it for degradation, while disruption of the MDM2-p53 interaction causes rapid accumulation of p53 and activation of the p53 pathway. We examined the efficacy of the small molecule MDM2 inhibitor KRT-232, alone and in combination with radiation therapy (RT), in MDM2-amplified and/or p53 wildtype patient-derived xenograft (PDX) models of glioblastoma in vitro and in vivo. In vitro, glioblastoma PDX explant cultures showed sensitivity to KRT-232, both tumors with MDM2 amplification (GBM108 and G148) and non-amplified but TP53-wildtype lines (GBM10, GBM14, and GBM39), with IC50s ranging from 300-800 nM in FBS culture conditions. A TP53 p.F270C mutant PDX (GBM43) was inherently resistant, with IC50 >3000 nM. In the MDM2-amplified GBM108 line, KRT-232 led to a robust (5-6 fold) induction of p53-target genes p21, PUMA, and NOXA, with initiation of both apoptosis and senescence. Expression of p21 and PUMA was greater with KRT-232 in combination with RT (25-35 fold induction), while stable knock-down of p53 in GBM108 led to complete resistance to KRT-232. In contrast, GBM10 showed lower induction of p21 and PUMA (2-3 fold) and was more resistant to KRT-232. In an orthotopic GBM108 xenograft model, treatment with KRT-232 +/- RT for one week extended survival from 22 days (placebo) to 46 days (KRT-232 alone); combination KRT-232 + RT further extended survival (77 days) over RT alone (31 days). KRT-232 is an effective treatment in a subset of glioblastoma pre-clinical models alone and in combination with RT. Further studies are underway to understand the mechanisms conferring innate sensitivity or resistance to KRT-232.

scholarly journals Ways into Understanding HIF Inhibition

Cancers ◽  
2021 ◽  
Vol 13 (1) ◽  
pp. 159
Author(s):  
Tina Schönberger ◽  
Joachim Fandrey ◽  
Katrin Prost-Fingerle
Keyword(s):  
Protein Interactions ◽  
Target Genes ◽  
Protein Protein Interactions ◽  
Low Oxygen ◽  
Drug Candidates ◽  
Open Questions ◽  
Wide Range ◽  
Hif Pathway

Hypoxia is a key characteristic of tumor tissue. Cancer cells adapt to low oxygen by activating hypoxia-inducible factors (HIFs), ensuring their survival and continued growth despite this hostile environment. Therefore, the inhibition of HIFs and their target genes is a promising and emerging field of cancer research. Several drug candidates target protein–protein interactions or transcription mechanisms of the HIF pathway in order to interfere with activation of this pathway, which is deregulated in a wide range of solid and liquid cancers. Although some inhibitors are already in clinical trials, open questions remain with respect to their modes of action. New imaging technologies using luminescent and fluorescent methods or nanobodies to complement widely used approaches such as chromatin immunoprecipitation may help to answer some of these questions. In this review, we aim to summarize current inhibitor classes targeting the HIF pathway and to provide an overview of in vitro and in vivo techniques that could improve the understanding of inhibitor mechanisms. Unravelling the distinct principles regarding how inhibitors work is an indispensable step for efficient clinical applications and safety of anticancer compounds.

scholarly journals Genome-Wide Binding Analyses of HOXB1 Revealed a Novel DNA Binding Motif Associated with Gene Repression

2021 ◽  
Vol 9 (1) ◽  
pp. 6
Author(s):  
Narendra Pratap Singh ◽  
Bony De Kumar ◽  
Ariel Paulson ◽  
Mark E. Parrish ◽  
Carrie Scott ◽  
...  
Keyword(s):  
Dna Binding ◽  
Target Genes ◽  
Embryonic Stem ◽  
Binding Motif ◽  
Binding Assays ◽  
Histone Marks ◽  
Genome Wide ◽  
Hox Cofactors

Knowledge of the diverse DNA binding specificities of transcription factors is important for understanding their specific regulatory functions in animal development and evolution. We have examined the genome-wide binding properties of the mouse HOXB1 protein in embryonic stem cells differentiated into neural fates. Unexpectedly, only a small number of HOXB1 bound regions (7%) correlate with binding of the known HOX cofactors PBX and MEIS. In contrast, 22% of the HOXB1 binding peaks display co-occupancy with the transcriptional repressor REST. Analyses revealed that co-binding of HOXB1 with PBX correlates with active histone marks and high levels of expression, while co-occupancy with REST correlates with repressive histone marks and repression of the target genes. Analysis of HOXB1 bound regions uncovered enrichment of a novel 15 base pair HOXB1 binding motif HB1RE (HOXB1 response element). In vitro template binding assays showed that HOXB1, PBX1, and MEIS can bind to this motif. In vivo, this motif is sufficient for direct expression of a reporter gene and over-expression of HOXB1 selectively represses this activity. Our analyses suggest that HOXB1 has evolved an association with REST in gene regulation and the novel HB1RE motif contributes to HOXB1 function in part through a repressive role in gene expression.

scholarly journals MODL-16. ABEMACICLIB, A SELECTIVE CDK4/6 INHIBITOR, RESTRICTS GROWTH OF PEDIATRIC GLIAL-LINEAGE TUMORS IN VITRO AND IN VIVO

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii414-iii414
Author(s):  
Muh-Lii Liang ◽  
Tsung-Han Hsieh ◽  
Tai-Tong Wong
Keyword(s):  
Dna Repair ◽  
Therapeutic Strategy ◽  
Target Genes ◽  
Rna Seq ◽  
Downstream Target ◽  
Rb Phosphorylation ◽  
Glial Lineage

Abstract BACKGROUND Glial-lineage tumors constitute a heterogeneous group of neoplasms, comprising gliomas, oligodendrogliomas, and ependymomas, which account for 40%–50% of all pediatric central nervous system tumors. Advances in modern neuro-oncological therapeutics are aimed at improving neoadjuvant chemotherapy and deferring radiotherapy because radiation exposure may cause long-term side effects on the developing brain in young children. Despite aggressive treatment, more than half the high-grade gliomas (pHGGs) and one-third of ependymomas exhibit recurrence within 2 years of initial treatment. METHODS By using integrated bioinformatics and through experimental validation, we found that at least one gene among CCND1, CDK4, and CDK6 was overexpressed in pHGGs and ependymomas. RESULTS The use of abemaciclib, a highly selective CDK4/6 inhibitor, effectively inhibited cell proliferation and reduced the expression of cell cycle–related and DNA repair–related gene expression, which was determined through RNA-seq analysis. The efficiency of abemaciclib was validated in vitro in pHGGs and ependymoma cells and in vivo by using subcutaneously implanted ependymoma cells from patient-derived xenograft (PDX) in mouse models. Abemaciclib demonstrated the suppression of RB phosphorylation, downstream target genes of E2F, G2M checkpoint, and DNA repair, resulting in tumor suppression. CONCLUSION Abemaciclib showed encouraging results in preclinical pediatric glial-lineage tumors models and represented a potential therapeutic strategy for treating challenging tumors in children.

scholarly journals The Novel Role of PGC1α in Bone Metabolism

2021 ◽  
Vol 22 (9) ◽  
pp. 4670
Author(s):  
Cinzia Buccoliero ◽  
Manuela Dicarlo ◽  
Patrizia Pignataro ◽  
Francesco Gaccione ◽  
Silvia Colucci ◽  
...  
Keyword(s):  
Bone Metabolism ◽  
Osteoblastic Differentiation ◽  
In Vivo Studies ◽  
Peroxisome Proliferator ◽  
Sirtuin 3 ◽  
Peroxisome Proliferator Activated Receptor ◽  
Increased Risk

Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) is a protein that promotes transcription of numerous genes, particularly those responsible for the regulation of mitochondrial biogenesis. Evidence for a key role of PGC1α in bone metabolism is very recent. In vivo studies showed that PGC1α deletion negatively affects cortical thickness, trabecular organization and resistance to flexion, resulting in increased risk of fracture. Furthermore, in a mouse model of bone disease, PGC1α activation stimulates osteoblastic gene expression and inhibits atrogene transcription. PGC1α overexpression positively affects the activity of Sirtuin 3, a mitochondrial nicotinammide adenina dinucleotide (NAD)-dependent deacetylase, on osteoblastic differentiation. In vitro, PGC1α overexpression prevents the reduction of mitochondrial density, membrane potential and alkaline phosphatase activity caused by Sirtuin 3 knockdown in osteoblasts. Moreover, PGC1α influences the commitment of skeletal stem cells towards an osteogenic lineage, while negatively affects marrow adipose tissue accumulation. In this review, we will focus on recent findings about PGC1α action on bone metabolism, in vivo and in vitro, and in pathologies that cause bone loss, such as osteoporosis and type 2 diabetes.

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