scholarly journals Dendrimer-Coated Gold Nanoparticles for Efficient Folate-Targeted mRNA Delivery In Vitro

Pharmaceutics ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 900
Author(s):  
Londiwe Simphiwe Mbatha ◽  
Fiona Maiyo ◽  
Aliscia Daniels ◽  
Moganavelli Singh
Keyword(s):  
Gold Nanoparticles ◽  
Messenger Rna ◽  
Transgene Expression ◽  
Vaccine Development ◽  
Luciferase Reporter ◽  
Kb Cells ◽  
Luciferase Reporter Gene ◽  
Attractive Candidate ◽  
Mcf 7

Messenger RNA (mRNA) is not an attractive candidate for gene therapy due to its instability and has therefore received little attention. Recent studies show the advantage of mRNA over DNA, especially in cancer immunotherapy and vaccine development. This study aimed to formulate folic-acid-(FA)-modified, poly-amidoamine-generation-5 (PAMAM G5D)-grafted gold nanoparticles (AuNPs) and to evaluate their cytotoxicity and transgene expression using the luciferase reporter gene (FLuc-mRNA) in vitro. Nanocomplexes were spherical and of favorable size. Nanocomplexes at optimum nanoparticle:mRNA (w/w) binding ratios showed good protection of the bound mRNA against nucleases and were well tolerated in all cell lines. Transgene expression was significantly (p < 0.0001) higher with FA-targeted, dendrimer-grafted AuNPs (Au:G5D:FA) in FA receptors overexpressing MCF-7 and KB cells compared to the G5D and G5D:FA NPs, decreasing significantly (p < 0.01) in the presence of excess competing FA ligand, which confirmed nanocomplex uptake via receptor mediation. Overall, transgene expression of the Au:G5D and Au:G5D:FA nanocomplexes exceeded that of G5D and G5D:FA nanocomplexes, indicating the pivotal role played by the inclusion of the AuNP delivery system. The favorable properties imparted by the AuNPs potentiated an increased level of luciferase gene expression.

scholarly journals Dendrimer functionalized folate-targeted gold nanoparticles for luciferase gene silencing in vitro: A proof of principle study

2019 ◽  
Vol 69 (1) ◽  
pp. 49-61 ◽  
Author(s):  
Londiwe Simphiwe Mbatha ◽  
Fiona Chepkoech Maiyo ◽  
Moganavelli Singh
Keyword(s):  
Gold Nanoparticles ◽  
Gene Silencing ◽  
Folate Receptor ◽  
Sirna Delivery ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Band Shift ◽  
Delivery Vehicles ◽  
Interfering Rna

Abstract Use of exogenous small interfering RNA (siRNA) has shown potential in gene silencing. The need for target-specific siRNA delivery vehicles is crucial to successful gene silencing. This study is aimed at developing and evaluating the safety and efficiency of siRNA delivery using unmodified and folic acid (FA) modified poly(amidoamine) generation 5 (PAMAM G5D) functionalized gold nanoparticles (Au:G5D/Au:G5D:FA) in vitro. All formulations were physico--chemically characterized and nanocomplexes were evaluated using the band shift, dye displacement, nuclease protection, MTT cell viability, and luciferase reporter gene assays. Nanocomplexes bound and protected siRNA against degrading RNases, and were well tolerated by the cells. The Au:G5D:FA nanocomplexes elicited excellent gene silencing in folate receptor expressing HeLa-Tat-Luc cells, decreasing significantly in the presence of excess FA ligand, indicating nanocomplex uptake by the mechanism of receptor mediation. These results highlight the synergistic role played by Au and the dendrimer in enhancement of transgene silencing.

Benzisothiazolone Derivatives Exhibit Cytotoxicity in Hodgkin’s Lymphoma Cells through NF-κB Inhibition and are Synergistic with Doxorubicin and Etoposide

2020 ◽  
Vol 20 (6) ◽  
pp. 715-723
Author(s):  
Natarajan Nandakumar ◽  
Pushparathinam Gopinath ◽  
Jacob Gopas ◽  
Kannoth M. Muraleedharan
Keyword(s):  
Synergistic Effect ◽  
Hodgkin’S Lymphoma ◽  
A549 Cells ◽  
Hodgkin's Lymphoma ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Lymphoma Cells ◽  
Nuclear Extracts ◽  
Gene Assay

Background: The authors investigated the NF-κB inhibitory role of three Benzisothiazolone (BIT) derivatives (1, 2 and 3) in Hodgkin’s Lymphoma cells (L428) which constitutively express activated NF-κB. All three compounds showed dose-dependent NF-κB inhibition (78.3, 70.7 and 34.6%) in the luciferase reporter gene assay and were found cytotoxic at IC50 values of 3.3μg/ml, 4.35μg/ml and 13.8μg/ml, respectively by the XTT assay. BIT 1and BIT 2 (but not BIT 3) suppressed both NF-κB subunits p50 and p65 in cytoplasmic and nuclear extracts in a concentration-dependent manner. Furthermore, BIT 1 showed a moderate synergistic effect with the standard chemotherapy drugs etoposide and doxorubicin, whereas BIT 2 and 3 showed a moderate additive effect to antagonistic effect. Cisplatin exhibited an antagonist effect on all the compounds tested under various concentrations, except in the case of 1.56μg/ml of BIT 3 with 0.156μg/ml of cisplatin. The compounds also inhibited the migration of adherent human lung adenocarcinoma cells (A549) in vitro. We conclude that especially BIT 1 and BIT 2 have in vitro anti-inflammatory and anti-cancer activities, which can be further investigated for future potential therapeutic use. Methods: Inspired by the electrophilic sulfur in Nuphar alkaloids, monomeric and dimeric benzisothiazolones were synthesized from dithiodibenzoic acid and their NF-κB inhibitory role was explored. NF-κB inhibition and cytotoxicity of the synthesized derivatives were studied using luciferase reporter gene assay and XTTassay. Immunocytochemistry studies were performed using L428 cells. Cell migration assay was conducted using the A549 cell line. L428 cells were used to conduct combination studies and the results were plotted using CompuSyn software. Results: Benzisothiazolone derivatives exhibited cytotoxicity in Hodgkin’s Lymphoma cells through NF-κB inhibition. Potent compounds showed suppression of both NF-κB subunits p50 and p65 in a concentrationdependent manner, both in cytoplasmic and nuclear extracts. Combination studies suggest that benzisothiazolone derivatives possess a synergistic effect with etoposide and doxorubicin. Furthermore, the compounds also inhibited the migration of A549 cells. Conclusion: Benzisothiazolones bearing one or two electrophilic sulfur atoms as part of the heterocyclic framework exhibited cytotoxicity in Hodgkin’s Lymphoma cells through NF-κB inhibition. In addition, these derivatives also exhibited a synergistic effect with etoposide and doxorubicin along with the ability to inhibit the migration of A549 cells. Our study suggests that BIT-based new chemical entities could lead to potential anticancer agents.

scholarly journals Impact of RARα and miR-138 on retinoblastoma etoposide resistance

Tumor Biology ◽  
2021 ◽  
Vol 43 (1) ◽  
pp. 11-26
Author(s):  
Maike Busch ◽  
Natalia Miroschnikov ◽  
Jaroslaw Thomas Dankert ◽  
Marc Wiesehöfer ◽  
Klaus Metz ◽  
...  
Keyword(s):  
Cell Viability ◽  
Cell Lines ◽  
Cancer Progression ◽  
Treated Patient ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Gene Assay ◽  
The Impact

BACKGROUND: Retinoblastoma (RB) is the most common childhood eye cancer. Chemotherapeutic drugs such as etoposide used in RB treatment often cause massive side effects and acquired drug resistances. Dysregulated genes and miRNAs have a large impact on cancer progression and development of chemotherapy resistances. OBJECTIVE: This study was designed to investigate the involvement of retinoic acid receptor alpha (RARα) in RB progression and chemoresistance as well as the impact of miR-138, a potential RARα regulating miRNA. METHODS: RARα and miR-138 expression in etoposide resistant RB cell lines and chemotherapy treated patient tumors compared to non-treated tumors was revealed by Real-Time PCR. Overexpression approaches were performed to analyze the effects of RARα on RB cell viability, apoptosis, proliferation and tumorigenesis. Besides, we addressed the effect of miR-138 overexpression on RB cell chemotherapy resistance. RESULTS: A binding between miR-138 and RARα was shown by dual luciferase reporter gene assay. The study presented revealed that RARα is downregulated in etoposide resistant RB cells, while miR-138 is endogenously upregulated. Opposing RARα and miR-138 expression levels were detectable in chemotherapy pre-treated compared to non-treated RB tumor specimen. Overexpression of RARα increases apoptosis levels and reduces tumor cell growth of aggressive etoposide resistant RB cells in vitro and in vivo. Overexpression of miR-138 in chemo-sensitive RB cell lines partly enhances cell viability after etoposide treatment. CONCLUSIONS: Our findings show that RARα acts as a tumor suppressor in retinoblastoma and is downregulated upon etoposide resistance in RB cells. Thus, RARα may contribute to the development and progression of RB chemo-resistance.

scholarly journals MiR-206 regulates the progression of osteoporosis via targeting HDAC4

2021 ◽  
Vol 26 (1) ◽  
Author(s):  
Zhiyuan Lu ◽  
Dawei Wang ◽  
Xuming Wang ◽  
Jilong Zou ◽  
Jiabing Sun ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Apoptosis ◽  
Diagnostic Value ◽  
Expression Level ◽  
Luciferase Reporter ◽  
Control Group ◽  
Luciferase Reporter Gene ◽  
Mineral Density ◽  
Gene Assay

Abstract Background More and more studies have confirmed that miRNAs play an important role in maintaining bone remodeling and bone metabolism. This study investigated the expression level of miR-206 in the serum of osteoporosis (OP) patients and explored the effect and mechanism of miR-206 on the occurrence and development of osteoporosis. Methods 120 postmenopausal women were recruited, including 63 cases with OP and 57 women without OP. The levels of miR-206 were determined by qRT-PCR technology. Spearman correlation coefficient was used to evaluate the correlation of miR-206 with bone mineral density (BMD). An ROC curve was used to evaluate the diagnostic value of miR-206 in osteoporosis. The effects of miR-206 on cell proliferation and cell apoptosis of hFOBs were measured by CCK-8 assay and flow cytometry, respectively. Luciferase reporter gene assay was used to confirm the interaction of miR-206 and the 3′UTR of HDAC4. Results Serum miR-206 had low expression level in osteoporosis patient group compared with control group. The expression level of serum miR-206 had diagnostic value for osteoporosis, and the serum miR-206 levels were positively correlated with BMD. The down-regulated miR-206 could inhibit cell proliferation and promote cell apoptosis. Luciferase analysis indicated that HDAC4 was the target gene of miR-206. Conclusions MiR-206 could be used as a new potential diagnostic biomarker for osteoporosis, and in in vitro cell experiments, miR-206 may regulate osteoblast cell proliferation and apoptosis by targeting HDAC4.

scholarly journals Mechanism of MicroRNA-375 Promoter Methylation in Promoting Ovarian Cancer Cell Malignancy

2021 ◽  
Vol 20 ◽  
pp. 153303382098011
Author(s):  
Junjun Shu ◽  
Ling Xiao ◽  
Sanhua Yan ◽  
Boqun Fan ◽  
Xia Zou ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Protein Expression ◽  
Cell Lines ◽  
Cell Invasion ◽  
Promoter Methylation ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Gene Assay ◽  
Cell Malignancy

Objective: Ovarian cancer (OC) ranks one of the most prevalent fatal tumors of female genital organs. Aberrant promoter methylation triggers changes of microRNA (miR)-375 in OC. Our study aimed to evaluate the mechanism of methylated miR-375 promoter region in OC cell malignancy and to seek the possible treatment for OC. Methods: miR-375 promoter methylation level in OC tissues and cells was detected. miR-375 expression in OC tissues and cell lines was compared with that in demethylated cells. Role of miR-375 in OC progression was measured. Dual-luciferase reporter gene assay was utilized to verify the targeting relationship between miR-375 and Yes-associated protein 1 (YAP1). Then, Wnt/β-catenin pathway-related protein expression was tested. Moreover, xenograft transplantation was applied to confirm the in vitro experiments. Results: Highly methylated miR-375 was seen in OC tissues and cell lines, while its expression was decreased as the promoter methylation increased. Demethylation in OC cells brought miR-375 back to normal level, with obviously declined cell invasion, migration and viability and improved apoptosis. Additionally, miR-375 targeted YAP1 to regulate the Wnt/β-catenin pathway protein expression. Overexpressed YAP1 reversed the protein expression, promoted cell invasion, migration and viability while reduced cell apoptosis. Overexpressed miR-375 in vivo inhibited OC progression. Conclusion: Our study demonstrated that demethylated miR-375 inhibited OC growth by targeting YAP1 and downregulating the Wnt/β-catenin pathway. This investigation may offer novel insight for OC treatment.

scholarly journals MicroRNA-124 Regulates the Proliferation of Colorectal Cancer Cells by TargetingiASPP

2013 ◽  
Vol 2013 ◽  
pp. 1-10 ◽  
Author(s):  
Kuijie Liu ◽  
Hua Zhao ◽  
Hongliang Yao ◽  
Sanlin Lei ◽  
Zhendong Lei ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Cancer Cells ◽  
Target Prediction ◽  
Inhibitory Effect ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Small Noncoding Rnas ◽  
Colorectal Cancer Cells ◽  
Microrna 124

MicroRNAs are a class of small, noncoding RNAs that function as critical regulators of gene expression by targeting mRNAs for translational repression or degradation. In this study, we demonstrate that expression of microRNA-124 (miR-124) is significantly downregulated in colorectal cancer tissues and cell lines, compared to the matched adjacent tissues. We identified and confirmed inhibitor of apoptosis-stimulating protein of p53 (iASPP) as a novel, direct target of miR-124 using target prediction algorithms and luciferase reporter gene assays. Overexpression of miR-124 suppressed iASPP protein expression, upregulated expression of the downstream signaling molecule nuclear factor-kappa B (NF-κB), and attenuated cell viability, proliferation, and colony formation in SW480 and HT-29 colorectal cancer cells in vitro. Forced overexpression ofiASPPpartly rescued the inhibitory effect of miR-124 on SW480 and HT29 cell proliferation. Taken together, these findings shed light on the role and mechanism of action of miR-124, indicate that the miR-124/iASPP axis can regulate the proliferation of colorectal cancer cells, and suggest that miR-124 may serve as a potential therapeutic target for colorectal cancer.

scholarly journals Anti-TAR Polyamide Nucleotide Analog Conjugated with a Membrane-Permeating Peptide Inhibits Human Immunodeficiency Virus Type 1 Production

2002 ◽  
Vol 76 (8) ◽  
pp. 3881-3891 ◽  
Author(s):  
Neerja Kaushik ◽  
Amartya Basu ◽  
Paul Palumbo ◽  
Rene L. Myers ◽  
Virendra N. Pandey
Keyword(s):  
Human Immunodeficiency Virus ◽  
Antiviral Treatment ◽  
Significant Inhibition ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Nucleotide Analog ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT The emergence of drug-resistant variants has posed a significant setback against effective antiviral treatment for human immunodeficiency virus (HIV) infections. The choice of a nonmutable region of the viral genome such as the conserved transactivation response element (TAR element) in the 5′ long terminal repeat (LTR) may potentially be an effective target for drug development. We have earlier demonstrated that a polyamide nucleotide analog (PNA) targeted to the TAR hairpin element, when transfected into cells, can effectively inhibit Tat-mediated transactivation of HIV type 1 (HIV-1) LTR (T. Mayhood et al., Biochemistry 39:11532-11539, 2000). Here we show that this anti-TAR PNA (PNATAR), upon conjugation with a membrane-permeating peptide vector (transportan) retained its affinity for TAR in vitro similar to the unconjugated analog. The conjugate was efficiently internalized into the cells when added to the culture medium. Examination of the functional efficacy of the PNATAR-transportan conjugate in cell culture using luciferase reporter gene constructs resulted in a significant inhibition of Tat-mediated transactivation of HIV-1 LTR. Furthermore, PNATAR-transportan conjugate substantially inhibited HIV-1 production in chronically HIV-1-infected H9 cells. The mechanism of this inhibition appeared to be regulated at the level of transcription. These results demonstrate the efficacy of PNATAR-transportan as a potential anti-HIV agent.

scholarly journals LINC00839 Knockdown Restrains The Metastatic Behaviors of Nasopharyngeal Carcinoma By Sponging miR-454-3p

2021 ◽  
Author(s):  
Feng Ying Zhang ◽  
Xia Li ◽  
Ting Ting Huang ◽  
Mei Ling Xiang ◽  
Lin Lin Sun ◽  
...  
Keyword(s):  
Nasopharyngeal Carcinoma ◽  
Underlying Mechanism ◽  
Luciferase Reporter ◽  
Regulatory Function ◽  
Migration And Invasion ◽  
Luciferase Reporter Gene ◽  
Non Coding Rna ◽  
Invasive Capacity

Abstract Background Long intergenic non-coding RNA 00839 (LINC00839) has been verified as a cancer-promoting gene in malignancies. However, the significance of LINC00839 in nasopharyngeal carcinoma (NPC) has yet to be elaborated, as well as its underlying mechanism.Methods LINC00839 and miR-454-3p relative expression levels in NPC cells were examined by qRT-PCR. The growth of cells was examined by CCK-8 and colony formation assays. Cell migration and invasion were examined by wound healing and Transwell experiment, respectively. The binding sequence of LINC00839 and miR-454-3p was confirmed by the luciferase reporter gene experiment. The regulatory function of LINC00839 and miR-454-3p on c-Met was investigated by western blot.Results Here, we revealed that LINC00839 was elevated in NPC. Both LINC00839 knockdown and upregulation of miR-454-3p suppressed NPC cells proliferation, invasive capacity and EMT in vitro. Besides, LINC00839 was validated as a miR-454-3p “sponge”, and upregulation of LINC00839 could reverse miR-454-3p-mediated functions in NPC C666-1 and SUNE-1 cells. Furthermore, c-Met was determined to be targeted by miR-454-3p. Notably, c-Met was downregulated by LINC00839 knockdown through sponging miR-454-3p. In vivo, LINC00839 knockdown resulted in a slower tumor growth.Conclusions Altogether, knockdown of LINC00839 inhibits the aggressive properties of NPC cells via sponging miR-454-3p and regulating c-Met.

scholarly journals Circular RNA CDR1as Inhibits the Metastasis of Gastric Cancer through Targeting miR-876-5p/GNG7 Axis

2021 ◽  
Vol 2021 ◽  
pp. 1-13
Author(s):  
Jiajia Jiang ◽  
Rong Li ◽  
Junyi Wang ◽  
Jie Hou ◽  
Hui Qian ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Epithelial Mesenchymal Transition ◽  
Circular Rna ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Luciferase Reporter Gene ◽  
Mesenchymal Transition ◽  
Underlying Mechanisms

Circular RNA CDR1as has been demonstrated to participate in various cancer progressions as miRNA sponges. The exact underlying mechanisms of CDR1as on gastric cancer (GC) metastasis remain unknown. Here, we found that CDR1as knockdown facilitated GC cell migration and invasion while its overexpression inhibited the migration and invasion abilities of GC cells in vitro and in vivo. Moreover, epithelial-mesenchymal transition- (EMT-) associated proteins and MMP2 and MMP9 were downregulated by CDR1as. Bioinformatics analysis combined with dual-luciferase reporter gene assays, western blot, RT-qPCR analysis, and functional rescue experiments demonstrated that CDR1as served as a miR-876-5p sponge and upregulated the target gene GNG7 expression to suppress GC metastasis. In summary, our findings indicate that CDR1as suppresses GC metastasis through the CDR1as/miR-876-5p/GNG7 axis.

Bone Marrow Stromal Cells (BMSCs) Inhibit Retinal Ganglion Cell Apoptosis and Alleviate Inflammation Through Up-Regulation of MicroRNA-43 and Brain-Derived Neurotrophic Factor in Glaucoma

2021 ◽  
Vol 11 (12) ◽  
pp. 2478-2483
Author(s):  
Xiang Ji ◽  
Kai-Wen Zhou
Keyword(s):  
Neurotrophic Factor ◽  
Vision Loss ◽  
Luciferase Reporter ◽  
Retinal Ganglion ◽  
Luciferase Reporter Gene ◽  
Down Regulation ◽  
Related Factors ◽  
Gene Assay

Glaucoma is a leading cause of vision loss mainly due to retinal ganglion cells (RGC) loss. MicroRNAs (miRNAs) are highlighted as potential biomarkers in diseases. This study aims to investigate the role of miR-43 and BMSCs in the RGC apoptosis and glaucoma.RGCs were transfected with miR-43 inhibitors and mimics, and then co-cultured with BMSCs. RT-qPCR analysis was conducted to determine miR-43 expression, whilst Western blot, and flow cytometry were carried out to assess the role of miR-43 in apoptosis and inflammation. The interaction between miR-43 and BDNF, a neurotrophic factor, was detected by dual-luciferase reporter gene assay. Overexpression of miR-43 promoted RGC proliferation and decreased apoptosis. Furthermore, miR-43 overexpression diminished the contents of apoptosis- and inflammatory-related factors, and elevated the expression of BDNF. Down-regulation of BDNF exerted similar effect as down-regulation of miR-43, enhancing apoptosis and aggravating inflammation. Importantly, BMSC treatment reversed the in vitro inhibitory effect of si-BDNF on RGC with enhancement of miR-43 expression. Mechanically, miR-43 was indicated to target BDNF in glaucoma. Collectively, miR-43 delivered by BMSCs plays an important role in the inflammatory injury and abnormal apoptosis of RGC by regulating the expression of BDNF. These findings might help development of new treatment for glaucoma and provide a promising biomarker for diagnosis and treatment.

Sign in / Sign up

Export Citation Format

Share Document