ABSTRACT
Abstract Micro-RNA-221(miR-221) is one of oncogenic miRNAs that plays a vital role in the development and progression of oral cancers. The aim of this study is to introduce a new gene therapy for oral squamous cell carcinoma by blocking the expression of oncogenic miR-221 by its inhibitor. The present work was performed on squamous cell carcinoma cell line SCC-25 and anti-miR-221 was delivered to the cells using an ultrasound micro bubbles. Assessment of the effect of miR-221 inhibitor on SCC-25 cells was done using MTT assay, cell cycle analysis and apoptosis detection. In addition, reverse transcription-polymerase chain reaction was also used to detect the expression -miR-221 and its target genes. Using ANOVA, statistical analysis of the results showed significant inhibition of cell viability with and induction of cell apoptosis of SCC-25 cell line after transfection. Moreover, the expression of miR-221, Epidermal growth factor receptor (EGFR) and CDKNIB/p27 were downregulated without significant difference. Transfection of SCC-25 by inhibitor of miR-221 resulting in blockage of its expression leading to arresting of tumor growth. These results proved the effective role of micro-RNA inhibitors as novel therapeutic agent for oral cancers.
Resumo Micro-RNA-221 (miR-221) é um dos miRNAs oncogênicos que desempenham um papel vital no desenvolvimento e progressão de carcinomas orais. O objetivo deste estudo é apresentar uma nova terapia gênica para o carcinoma epidermóide oral por meio do bloqueio da expressão do miR-221 oncogênico por seu inibidor. O presente trabalho foi realizado na linhagem de células de carcinoma de células escamosas SCC-25 e o anti-miR-221 foi administrado às células usando micro-bolhas de ultrassom. A avaliação do efeito do inibidor miR-221 em células SCC-25 foi feita usando ensaio de MTT, análise do ciclo celular e detecção de apoptose. Além disso, a reação em cadeia da polimerase com transcrição reversa também foi usada para detectar a expressão -miR-221 e seus genes-alvo. Usando ANOVA, a análise estatística dos resultados mostrou inibição significativa da viabilidade celular e indução da apoptose celular da linhagem celular SCC-25 após a transfecção. Além disso, a expressão de miR-221, receptor do fator de crescimento epidérmico (EGFR) e CDKNIB/p27 foram regulados para baixo sem diferença significativa. A transfecção de SCC-25 por inibidor de miR-221 resultou no bloqueio de sua expressão, levando à interrupção do crescimento do tumor. Esses resultados comprovaram o papel eficaz dos inibidores de micro-RNA como novo agente terapêutico para carcinomas orais.
Subject(s)
Humans , Mouth Neoplasms/genetics , Carcinoma, Squamous Cell/genetics , Carcinoma, Squamous Cell/therapy , MicroRNAs/antagonists & inhibitors , MicroRNAs/genetics , MicroRNAs/therapeutic use , Mouth Neoplasms/therapy , Genetic Therapy , Apoptosis , Cell Line, Tumor , Cell ProliferationABSTRACT
BACKGROUND: Cisplatin resistance (DDP-resistance) remains one of the major causes of poor prognosis in females with ovarian cancer. Long non-coding RNAs (lncRNAs) have been shown to participate in the regulation of cellular processes, including chemoresistance. The aim of this study was to explore the role of HOX transcript antisense RNA (HOTAIR) in DDP-resistant ovarian cancer cells. METHODS: DDP-resistant ovarian cancer cell lines (SKOV3/DDP and A2780/DDP) were established. Real-time PCR, western blot, dual-luciferase reporter assay, and flow cytometry were then used to evaluate the effect of HOTAIR/miR-138-5p axis on chemoresistance of DDP-resistant ovarian cancer cells to DDP. RESULTS: We found that HOTAIR was upregulated in DDP-resistant cells, while miR-138-5p was downregulated. Knockdown of HOTAIR increased the expression of miR-138-5p in DDP-resistant cells and miR-138-5p is directly bound to HOTAIR. Upregulation of miR-138-5p induced by HOTAIR siRNA or by its mimics enhanced the chemosensitivity of DDP-resistant cells and decreased the expression of EZH2 (enhancer of zeste 2 polycomb repressive complex 2 subunit) and SIRT1 (sirtuin 1). Furthermore, the HOTAIR silencing-induced chemosensitivity of DDP-resistant cells was weakened by miR-138-5p inhibitor. CONCLUSIONS: These data demonstrate that HOTAIR acts as a sponge of miR-138-5p to prevent its binding to EZH2 and SIRT1, thereby promoting DDP-resistance of ovarian cancer cells. Our work will shed light on the development of therapeutic strategies for ovarian cancer treatment.
Subject(s)
Humans , Female , Ovarian Neoplasms/genetics , Cisplatin/pharmacology , Drug Resistance, Neoplasm/genetics , RNA, Long Noncoding/genetics , Gene Expression Regulation, Neoplastic/drug effects , Up-Regulation , Apoptosis/drug effects , MicroRNAs/antagonists & inhibitors , Cell Line, Tumor , Gene Knockout Techniques/methods , Sirtuin 1/antagonists & inhibitors , Real-Time Polymerase Chain Reaction , Enhancer of Zeste Homolog 2 Protein/antagonists & inhibitorsABSTRACT
Previous research has shown that suppression of miR-383 can prevent inflammation of the endothelium, as well as postpone the development of atherosclerosis. However, the role of miR-383 in endothelial cell apoptosis in diabetes remains unclear. The aim of this study was to investigate the function of miR-383 in high glucose-induced apoptosis and oxidative stress in endothelial cells. A series of experiments involving qualitative polymerase chain reaction, cell transfection, luciferase assay, assessment of cell death, detection of catalase and superoxide dismutase concentrations, detection of intracellular reactive oxygen species (ROS), and western blot analysis were performed in this study. We found that miR-383 expression was promoted, while NAD+-dependent deacetylase and sirtuin 1 (SIRT1) expressions were suppressed in the endothelium of the aorta in db/db mice as well as in human umbilical vein endothelial cells, which were treated with high glucose (HG). Increased expression of miR-383 decreased expression of SIRT1, while suppression of miR-383 promoted expression of SIRT1 in human umbilical vein endothelial cells (HUVECs). Furthermore, suppression of miR-383 following transfection with miR-383 suppressor repressed cell death and generation of ROS in HUVECs. SIRT1 knockdown by siRNA-SIRT1 reversed the suppressive effect of miR-383 inhibition on ROS production and cell apoptosis induced by HG treatment. Overall, the findings of our research suggested that suppression of miR-383 repressed oxidative stress and reinforced the activity of endothelial cells by upregulation of SIRT1 in db/db mice, and targeting miR-383 might be promising for effective treatment of diabetes.
Subject(s)
Animals , Male , Rabbits , Endothelium, Vascular/drug effects , Apoptosis/drug effects , Oxidative Stress/drug effects , MicroRNAs/antagonists & inhibitors , Sirtuin 1/metabolism , Glucose/pharmacology , Endothelium, Vascular/cytology , Endothelium, Vascular/physiology , Signal Transduction , Cells, Cultured , Mice, Inbred C57BLABSTRACT
ABSTRACT Objective: miR-483-5p has been identified as a miRNA oncogene in certain cancers. However, its role in prostate cancer has not been sufficiently investigated. In this study, we investigated the role of miR-483-5p in prostate cancer and examined RBM5 regulation by miR-483-5p. Material and methods: Expression levels of miR-483-5p were determined by quantitative real-time PCR. The effect of miR-483-5p on proliferation was evaluated by MTT assay, cell invasion was evaluated by trans-well invasion assays, and target protein expression was determined by western blotting in LNCaP, DU-145, and PC-3 cells. Luciferase reporter plasmids were constructed to confirm the action of miR-483-5p on downstream target gene RBM5 in HEK-293T cells. Results: we observed that miR-483-5p was upregulated in prostate cancer cell lines and tissues. A miR-483-5p inhibitor inhibited prostate cancer cell growth and invasion in DU-145 and PC-3 cells. miR-483-5p directly bound to the 3' untranslated region (3'UTR) of RBM5 in HEK-293T cells. RBM5 overexpression inhibited prostate cancer cell growth and invasion in LNCaP cells. Enforced RBM5 expression alleviated miR-483-5p promotion of prostate cancer cell growth and invasion in LNCaP cells. Conclusion: The present study describes a potential mechanism underlying a miR-483-5p/RBM5 link that contributes to prostate cancer development.
Subject(s)
Humans , Male , Prostatic Neoplasms/genetics , Prostatic Neoplasms/pathology , Gene Expression Regulation, Neoplastic/genetics , Cell Cycle Proteins/metabolism , Untranslated Regions/genetics , Tumor Suppressor Proteins/metabolism , MicroRNAs/physiology , Cell Proliferation/genetics , DNA-Binding Proteins/metabolism , Real-Time Polymerase Chain Reaction , Prostatic Neoplasms/mortality , Down-Regulation , Up-Regulation , RNA-Binding Proteins/metabolism , MicroRNAs/antagonists & inhibitors , Cell Line, Tumor , Neoplasm InvasivenessABSTRACT
To investigate the function and regulation mechanism of ATP-binding cassette, subfamily G, member 2 (ABCG2) in retinoblastoma cancer stem cells (RCSCs), a long-term culture of RCSCs from WERI-Rb1 cell line was successfully established based on the high expression level of ABCG2 on the surface of RCSCs. To further explore the molecular mechanism of ABCG2 on RCSCs, a microRNA that specifically targets ABCG2 was predicted. Subsequently, miR-3163 was selected and confirmed as the ABCG2-regulating microRNA. Overexpression of miR-3163 led to a significant decrease in ABCG2 expression. Additionally, ABCG2 loss-of-function induced anti-proliferation and apoptosis-promoting functions in RCSCs, and multidrug resistance to cisplatin, carboplatin, vincristine, doxorubicin, and etoposide was greatly improved in these cells. Our data suggest that miR-3163 has a significant impact on ABCG2 expression and can influence proliferation, apoptosis, and drug resistance in RCSCs. This work may provide new therapeutic targets for retinoblastoma.
Subject(s)
Humans , 3' Untranslated Regions , ATP Binding Cassette Transporter, Subfamily G, Member 2/antagonists & inhibitors , Antagomirs/metabolism , Antineoplastic Agents/toxicity , Apoptosis/drug effects , Base Sequence , Cell Line, Tumor , Cell Proliferation/drug effects , Drug Resistance, Neoplasm/drug effects , Gene Silencing , MicroRNAs/antagonists & inhibitors , Neoplasm Proteins/antagonists & inhibitors , Neoplastic Stem Cells/metabolism , Retinoblastoma/metabolism , Sequence Alignment , TransfectionABSTRACT
Background: Ruthenium (Ru) tetraamines are being increasingly used as nitric oxide (NO) carriers. In this context, pharmacological studies have become highly relevant to better understand the mechanism of action involved. Objective: To evaluate the vascular response of the tetraamines trans-[RuII(NH3)4(Py)(NO)]3+, trans-[RuII(Cl)(NO) (cyclan)](PF6)2, and trans-[RuII(NH3)4(4-acPy)(NO)]3+. Methods: Aortic rings were contracted with noradrenaline (10−6 M). After voltage stabilization, a single concentration (10−6 M) of the compounds was added to the assay medium. The responses were recorded during 120 min. Vascular integrity was assessed functionally using acetylcholine at 10−6 M and sodium nitroprusside at 10−6 M as well as by histological examination. Results: Histological analysis confirmed the presence or absence of endothelial cells in those tissues. All tetraamine complexes altered the contractile response induced by norepinephrine, resulting in increased tone followed by relaxation. In rings with endothelium, the inhibition of endothelial NO caused a reduction of the contractile effect caused by pyridine NO. No significant responses were observed in rings with endothelium after treatment with cyclan NO. In contrast, in rings without endothelium, the inhibition of guanylate cyclase significantly reduced the contractile response caused by the pyridine NO and cyclan NO complexes, and both complexes caused a relaxing effect. Conclusion: The results indicate that the vascular effect of the evaluated complexes involved a decrease in the vascular tone induced by norepinephrine (10−6 M) at the end of the incubation period in aortic rings with and without endothelium, indicating the slow release of NO from these complexes and suggesting that the ligands promoted chemical stability to the molecule. Moreover, we demonstrated that the association of Ru with NO is more stable when the ligands pyridine and cyclan ...
Fundamento: As tetra-aminas de rutênio cada vez mais se destacam como carreadoras da molécula de óxido nítrico. Desse modo, estudos farmacológicos tornam-se altamente relevantes, afim de melhor compreender o mecanismo de ação envolvido. Objetivo: Avaliar a resposta vascular das tetra-aminas trans-[RuII(NH3)4(Py)(NO)]3+, trans-[RuII(Cl)(NO)(Cyclan)](PF6)2 e trans-[RuII(NH3)4(4-acPy)(NO)]3+. Métodos: Anéis de aorta foram pré-contraídos com noradrenalina (10-6M). Após estabilização da tensão, concentração única (10-6M) dos compostos foi adicionada ao banho de incubação. As respostas foram registradas ao longo de 120 minutos. A integridade vascular foi avaliada funcionalmente (acetilcolina 10-6M; nitroprussiato de sódio 10-6M) e histologicamente Resultados: A análise histológica confirmou a presença ou não de células endoteliais nos tecidos analisados. Todos os complexos alteraram a resposta contrátil induzida pela noradrenalina, resultando em aumento de tônus seguido de efeito relaxante. Em anéis com endotélio, a inibição do óxido nítrico endotelial causou redução do efeito contrátil da piridina óxido nítrico. Não foram observadas respostas significativas em anéis com endotélio referente ao composto cyclan óxido nítrico. Por outro lado, em anéis sem endotélio, a inibição da guanilato ciclase reduziu significativamente a resposta contrátil dos complexos piridina óxido nítrico e cyclan óxido nítrico, levando ambos os compostos a um efeito relaxante. Conclusão: Os resultados obtidos demonstram que o efeito vascular dos complexos avaliados apresentaram diminuição no tônus vascular induzido pela noradrenalina (10-6M) ao final do tempo de incubação, em anéis com e sem endotélio, indicando liberação lenta da molécula de óxido nítrico do composto estudado e sugerindo que os ligantes causaram estabilidade química à molécula. Demonstramos que a ligação rutênio óxido nítrico é mais estável quando utilizamos os ligantes piridina e cyclan para a formulação ...
Subject(s)
Animals , Humans , Mice , Apoptosis/physiology , MicroRNAs/physiology , Endothelial Cells/physiology , MicroRNAs/antagonists & inhibitors , MicroRNAs/metabolism , Molecular Targeted Therapy/methods , Neoplasms/physiopathology , Ribonuclease III/deficiency , Ribonuclease III/physiology , Up-Regulation , Vascular Endothelial Growth Factor A/physiologyABSTRACT
BACKGROUND: Low survival rate of transplanted cells compromises the efficacy of cell therapy. Hexokinase II (HKII) is known to have anti-apoptotic activity through its interaction with mitochondria. The objective was to identify miRNAs targeting HKII and investigate whether miRNA-mediated modulation of HKII could improve the survival of mesenchymal stem cells (MSCs) exposed to H2O2. The expression of HKII in MSCs exposed to H2O2 was evaluated, and HKII-targeting miRNA was screened based on miRNA-target prediction databases. The effect of H2O2 on the expression of the selected HKII-targeting miRNA was examined and the effect of modulation of the selected HKII-targeting miRNA using anti-miRNA on H2O2-induced apoptosis of MSC was evaluated. RESULTS: H2O2 (600 µM) induced cell death of MSCs and decreased mitochondrial HKII expression. We have identified miR-181a as a HKII-targeting miRNA and H2O2 increased the expression of miR-181a in MSCs. Delivery of anti-miR-181a, which neutralizes endogenous miR-181a, significantly attenuated H2O2-induced decrease of HKII expression and disruption of mitochondrial membrane potential, improving the survival of MSCs exposed to H2O2. CONCLUSIONS: These findings suggest that H2O2-induced up-regulation of miR-181a contributes to the cell death of MSCs by down-regulating HKII. Neutralizing miR-181a can be an effective way to prime MSCs for transplantation into ischemic tissues.
Subject(s)
Humans , Apoptosis , MicroRNAs/metabolism , Mesenchymal Stem Cells/pathology , Glioma/pathology , Hexokinase/metabolism , Hydrogen Peroxide/toxicity , Membrane Glycoproteins/genetics , Membrane Glycoproteins/metabolism , Cell Differentiation , Cell Movement , Cell Survival , Reactive Oxygen Species , Semaphorins/genetics , Semaphorins/metabolism , MicroRNAs/antagonists & inhibitors , Mesenchymal Stem Cells/drug effects , Mesenchymal Stem Cells/enzymology , Real-Time Polymerase Chain Reaction , Glioma/metabolism , Hydrogen Peroxide/administration & dosage , Mitochondria/enzymology , Neoplasm InvasivenessABSTRACT
BACKGROUND: Gliomas are the most common primary tumors in the central nervous system. Due to complicated signaling pathways involved in glioma progression, effective targets for treatment and biomarkers for prognosis prediction are still scant. RESULTS: In this study we revealed that a new microRNA (miR), the miR-221, was highly expressed in the glioma cells, and suppression of miR-221 resulted in decreased cellular proliferation, migration, and invasion in glioma cells. Mechanistic experiments validated that miR-221 participates in regulating glioma cells proliferation and invasion via suppression of a direct target gene, the Semaphorin 3B (SEMA3B). The rescue experiment with miR-221 and SEMA3B both knockdown results in significant reversion of miR-221 induced phenotypes. CONCLUSION: Taken together, our findings highlight an unappreciated role for miR-221 and SEMA3B in glioma.
Subject(s)
Humans , Brain Neoplasms/pathology , Membrane Glycoproteins/pharmacology , Apoptosis , Semaphorins/pharmacology , MicroRNAs/antagonists & inhibitors , Cell Proliferation , Glioma/pathology , Brain Neoplasms/metabolism , Membrane Glycoproteins/genetics , Signal Transduction , Gene Expression Regulation, Neoplastic , Cell Movement , Blotting, Western , Semaphorins/genetics , MicroRNAs/metabolism , Cell Line, Tumor , Real-Time Polymerase Chain Reaction , Glioma/metabolism , Luciferases/metabolism , Neoplasm InvasivenessABSTRACT
BACKGROUND/AIMS: Intraductal papillary mucinous neoplasms (IPMN) are precursor lesions of fatal pancreatic cancer. Physiological function of microRNA is to regulate the stability and translation of mRNA. The aberrant microRNA expression is commonly observed in many cancers. The aim of this study was to analyze the expression pattern of microRNA in IPMN and evaluate the role of the microRNA. METHODS: Using two paraffin-embedded IPMN tissues, microRNA expression of normal tissue, IPMN adenoma and carcinoma were compared by cDNA-mediated annealing, selection, extension and ligation microarray assay. Using real time PCR, expression levels of aberrantly up-regulated microRNAs were assessed in another 20 IPMNs, four pancreatic cancer cell lines (Panc1, MiaPaCa-2, XPA-3, BxPC-3) and immortalized pancreatic ductal cell line (HPNE). Effect of suppressing highly over-expressed two microRNAs in pancreatic cancer cell lines with anti-microRNA inhibitors were evaluated using CCK-8 assay. RESULTS: Among aberrantly expressed 122 microRNAs in IPMN, miR-552, miR-25*, miR-183, miR-1300, miR-196a, miR-182*, and miR-30c-1* were consistently increased more than 3-fold. On average, miR-196a and miR-183 increased 10,824 folds and 26,519 folds in four pancreatic cancer cell lines compared with HPNE. These two microRNAs were also over-expressed in 20 IPMNs compared with HPNE. After applying anti-miRNA inhibitors, cell survival of four pancreatic cancer cell lines decreased by 24.5% with anti-miR-196a and by 14.2% with anti-miR-183 on average. CONCLUSIONS: Aberrant expression of 122 microRNAs was observed in IPMN. Two microRNAs, miR-196a and miR-183-increased in IPMN and pancreatic cancer cell lines compared with immortalized dancreatic ductal cell line. The inhibitions of these microRNAs repressed cell proliferation of pancreatic cancer cell lines.