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Acrylamide (AA) in heat-processed foods has emerged as a global health problem, mainly carcinogenic, neurotoxic, and reproductive toxicity, and an increasing number of researchers have delved into elucidating its toxicological mechanisms. Studies have demonstrated that exposure of HepG2 by AA in a range of concentrations can induce the upregulation of miR-21 and miR-221. Monitoring the response of intracellular miRNAs can play an important role in unraveling the mechanisms of AA toxicity. Here, multicolor aggregation induced emission nano particle (AIENP) probes were constructed from three AIE dyes for simultaneous imaging of intracellular AA and AA-induced miR-21/miR-221 by combining the recognition function of AA aptamers and the signal amplification of a DNAzyme walker. The surface of these nanoparticles contains carboxyl groups, facilitating their linkage to a substrate chain modified with a fluorescent quencher group via an amide reaction. Optimization experiments were conducted to determine the optimal substrate-to-DNAzyme ratio, confirming its efficacy as a walker for signal amplification. Sensitive detection of AA, miR-21 and miR-221 was achieved in extracellular medium, with detection limits of 0.112 nM for AA, 0.007 pM and 0.003 pM for miR-21 and miR-221, respectively, demonstrating excellent selectivity. Intracellularly, ZIF-8 structure collapsed, releasing Zn2+, activating DNAzyme cleavage activity, and the fluorescence of multicolor AIENPs within HepG2 cells gradually recovered with increasing stimulation time (0-12 h) and concentrations of AA (0-500 µM). This dynamic response unveiled the relationship between AA exposure and miR-21/miR-221 expression, further validating the carcinogenicity of AA.
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Mesenchymal stem cells (MSCs) have shown great potential for the treatment of liver injuries, and the therapeutic efficacy greatly depends on their homing to the site of injury. In the present study, we detected significant upregulation of hepatocyte growth factor (HGF) in the serum and liver in mice with acute or chronic liver injury. In vitro study revealed that upregulation of miR-9-5p or miR-221-3p promoted the migration of human MSCs (hMSCs) toward HGF. Moreover, overexpression of miR-9-5p or miR-221-3p promoted hMSC homing to the injured liver and resulted in significantly higher engraftment upon peripheral infusion. hMSCs reduced hepatic necrosis and inflammatory infiltration but showed little effect on extracellular matrix (ECM) deposition. By contrast, hMSCs overexpressing miR-9-5p or miR-221-3p resulted in not only less centrilobular necrosis and venous congestion but also a significant reduction of ECM deposition, leading to obvious improvement of hepatocyte morphology and alleviation of fibrosis around central vein and portal triads. Further studies showed that hMSCs inhibited the activation of hepatic stellate cells (HSCs) but could not decrease the expression of TIMP-1 upon acute injury and the expression of MCP-1 and TIMP-1 upon chronic injury, while hMSCs overexpressing miR-9-5p or miR-221-3p led to further inactivation of HSCs and downregulation of all three fibrogenic and proinflammatory factors TGF-ß, MCP-1, and TIMP-1 upon both acute and chronic injuries. Overexpression of miR-9-5p or miR-221-3p significantly downregulated the expression of α-SMA and Col-1α1 in activated human hepatic stellate cell line LX-2, suggesting that miR-9-5p and miR-221-3p may partially contribute to the alleviation of liver injury by preventing HSC activation and collagen expression, shedding light on improving the therapeutic efficacy of hMSCs via microRNA modification.
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Células Estreladas do Fígado , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais , MicroRNAs , MicroRNAs/genética , MicroRNAs/metabolismo , Humanos , Células-Tronco Mesenquimais/metabolismo , Células Estreladas do Fígado/metabolismo , Animais , Camundongos , Transplante de Células-Tronco Mesenquimais/métodos , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/terapia , Doença Hepática Induzida por Substâncias e Drogas/genética , Masculino , Tetracloreto de Carbono/efeitos adversos , Fator de Crescimento de Hepatócito/metabolismo , Fator de Crescimento de Hepatócito/genética , Camundongos Endogâmicos C57BL , Movimento CelularRESUMO
BACKGROUND/AIM: Caudal-type homeobox transcription factor 2 (CDX2) is a master regulator of intestinal development and maintenance of the intestinal epithelium. We previously revealed that CDX2Low colorectal cancers (CRCs) were associated with poor survival and differential response to adjuvant chemotherapy. MicroRNAs (miRNAs), a class of non-coding RNAs typically composed of fewer than 25 nucleotides, are known to regulate gene expression and signaling pathways. This study aimed to identify oncogenic miRNAs induced by CDX2 in CRC. MATERIALS AND METHODS: HCT116 cells were cultured and transfected with CDX2 siRNA. The expression levels of four oncogenic miRNAs (miR-9, miR-25, miR-106b and miR-221) were quantified by RT-qPCR. To understand whether CDX2 represented a key regulator of miR-221 expression in vivo, we analyzed the relationship between CDX2 and miR-221expression levels in the TCGA COAD database (n=454). RESULTS: The expression level of miR-221 was significantly up-regulated in CDX2 knockdown cells (n=2, p<0.05). In the TCGA database, we observed an inverse correlation between CDX2 and miR-221 expression levels, consistent with our in vitro data (r=-0.114, p=0.0149). Furthermore, the expression level of miR-221 was significantly elevated in patients with CDX2Low CRC (p<0.05). CONCLUSION: Knockdown of CDX2 induces microRNA-221 up-regulation in human CRC. Further research is warranted to elucidate the molecular mechanisms underlying miR-221 up-regulation in CDX2Low CRCs.
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Fator de Transcrição CDX2 , Neoplasias do Colo , Regulação Neoplásica da Expressão Gênica , MicroRNAs , Regulação para Cima , Humanos , MicroRNAs/genética , Fator de Transcrição CDX2/genética , Fator de Transcrição CDX2/metabolismo , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Neoplasias do Colo/metabolismo , Células HCT116 , Técnicas de Silenciamento de GenesRESUMO
Colorectal cancer (CRC) is one of the most common malignancies and a relevant cause of cancer-related deaths worldwide. Dysregulation of microRNA (miRNA) expression has been associated with the development and progression of various cancers, including CRC. Among them, miR-221 emerged as an oncogenic driver, whose high expression is associated with poor patient prognosis. The present study was conceived to investigate the anti-CRC activity of miR-221 silencing based on early clinical data achieved from a first-in-human study by our group. Going back from bedside to bench, we demonstrated that LNA-i-miR-221 reduces cell viability, induces apoptosis in vitro, and impairs tumor growth in preclinical in vivo models of CRC. Importantly, we disclosed that miR-221 directly targets TP53BP2, which, together with TP53INP1, is known as a positive regulator of the TP53 apoptotic pathway. We found that (1) both these genes are overexpressed following miR-221 inhibition, (2) the strong anti-tumor activity of LNA-i-miR-221 was selectively observed on TP53 wild-type cells, and (3) this activity was reduced in the presence of the TP53-inhibitor Pifitrin-α. Our data pave the way to further investigations on TP53 functionality as a marker predictive of response to miR-221 silencing, which might be relevant for clinical applications.
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AIM: Impaired wound healing in patients with diabetes can develop into nonhealing ulcerations. Because bone marrow mesenchymal stem cells (BMSCs) exosomes can promote wound healing, this study aims to investigate the mechanism of BMSCs-isolated exosomal miR-221-3p in angiogenesis and diabetic wound healing. METHODS: To mimic diabetes in vitro, human umbilical vein endothelial cells (HUVECs) were subjected to high glucose (HG). Exosomes were derived from BMSCs and identified by transmission electron microscopy (TEM), western blot analysis and dynamic light scattering (DLS). The ability to differentiate BMSCs was assessed via Oil red O staining, alkaline phosphatase (ALP) staining and alizarin red staining. The ability to internalise PKH26-labelled exosomes was assessed using confocal microscopy. Migration, cell viability and angiogenesis were tested by scratch, MTT and tube formation assays separately. The miRNA and protein levels were analysed by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) or western blotting. The relationship among miR-221-3p, FOXP1 and SPRY1 was determined using the dual-luciferase reporter, ChIP and RIP assays. RESULTS: Exosomal miR-221-3p was successfully isolated from BMSCs and delivered into HUVECs. HG was found to suppress the angiogenesis, cell viability and migration of HUVECs and exosomal miR-221-3p separated from BMSCs inhibited the above phenomenon. FOXP1 could transcriptionally upregulate SPRY1, and the silencing of FOXP1 reversed the HG-stimulated angiogenesis inhibition, cell viability and migration in HUVECs via the downregulation of SPRY1. Meanwhile, miR-221-3p directly targeted FOXP1 and the overexpression of FOXP1 reversed the positive effect of exosomal miR-221-3p on HUVEC angiogenesis. CONCLUSION: Exosomal miR-221-3p isolated from BMSCs promoted angiogenesis in diabetic wounds through the mediation of the FOXP1/SPRY1 axis. Furthermore, the findings of this study can provide new insights into probing strategies against diabetes.
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Angiogênese , Fatores de Transcrição Forkhead , Células-Tronco Mesenquimais , MicroRNAs , Neovascularização Fisiológica , Proteínas Repressoras , Cicatrização , Humanos , Movimento Celular/genética , Regulação para Baixo , Exossomos/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Fatores de Transcrição Forkhead/genética , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células-Tronco Mesenquimais/metabolismo , MicroRNAs/metabolismo , MicroRNAs/genética , Neovascularização Fisiológica/genética , Proteínas Repressoras/metabolismo , Proteínas Repressoras/genética , Cicatrização/genéticaRESUMO
The most significant and sensitive antigen protein that causes diarrhea in weaned pigs is soybean 7S globulin. Therefore, identifying the primary target for minimizing intestinal damage brought on by soybean 7S globulin is crucial. MicroRNA (miRNA) is closely related to intestinal epithelium's homeostasis and integrity. However, the change of miRNAs' expression and the function of miRNAs in Soybean 7S globulin injured-IPEC-J2 cells are still unclear. In this study, the miRNAs' expression profile in soybean 7S globulin-treated IPEC-J2 cells was investigated. Fifteen miRNAs were expressed differently. The differentially expressed miRNA target genes are mainly concentrated in signal release, cell connectivity, transcriptional inhibition, and Hedgehog signaling pathway. Notably, we noticed that the most significantly decreased miRNA was ssc-miR-221-5p after soybean 7S globulin treatment. Therefore, we conducted a preliminary study on the mechanisms of ssc-miR-221-5p in soybean 7S globulin-injured IPEC-J2 cells. Our research indicated that ssc-miR-221-5p may inhibit ROS production to alleviate soybean 7S globulin-induced apoptosis and inflammation in IPEC-J2 cells, thus protecting the cellular mechanical barrier, increasing cell proliferation, and improving cell viability. This study provides a theoretical basis for the prevention and control of diarrhea of weaned piglets.
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Apoptose , Globulinas , Glycine max , Mucosa Intestinal , MicroRNAs , Proteínas de Soja , Animais , MicroRNAs/genética , MicroRNAs/metabolismo , Suínos , Linhagem Celular , Glycine max/genética , Glycine max/química , Glycine max/metabolismo , Mucosa Intestinal/metabolismo , Proteínas de Soja/genética , Proteínas de Soja/metabolismo , Globulinas/genética , Globulinas/metabolismo , Proteínas de Armazenamento de Sementes/genética , Células Epiteliais/metabolismo , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Antígenos de PlantasRESUMO
Background: Cancer-derived exosomes facilitate chemoresistance by transferring RNAs, yet their role in exosomal microRNA-221-3p (miR-221-3p) regulation of Adriamycin resistance in breast cancer (BC) remains unclear. Methods: Adriamycin-resistant BC cells were developed from MCF-7 and MDA-MB-231 cells by incremental Adriamycin exposure. The miR-221-3p levels were quantified by quantitative reverse transcription-polymerase chain reaction. Subsequently, exosomes were isolated and incubated with BC cells, and exosome-mediated Adriamycin sensitivity was evaluated using Cell Counting Kit-8, colony formation, and flow cytometry assays. Sensitive cells were cocultured with miR-221-3p inhibitor-treated cells to assess Adriamycin resistance. Moreover, the interaction between miR-221-3p and phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1) was validated using a dual luciferase reporter gene assay. Mimics and inhibitors were used to determine the effects of miR-221-3p on Adriamycin resistance. Results: Elevated levels of miR-221-3p expression were observed in Adriamycin-resistant BC cells and exosomes. Sensitive cells were cocultured with exosomes from resistant cells, resulting in increased half-maximal inhibitory concentration value and proliferation, and reduced Adriamycin-induced apoptosis. However, the effects of coculturing sensitive cells with Adriamycin-resistant cells were significantly weakened by miR-221-3p inhibitor transfection in Adriamycin-resistant cells. PIK3R1 was found to be a target of miR-221-3p, and miR-221-3p mimics enhanced Adriamycin resistance in sensitive cells. miR-221-3p inhibitors increased the expression of PIK3R1, p-AKT, c-Myc, HK2, and PKM2, decreased FOXO3 expression, and weakened the Adriamycin resistance in resistant cells. Conclusions: miR-221-3p can be transferred between BC cells through exosomes. High levels of miR-221-3p were found to target PIK3R1 and promoted Adriamycin resistance in BC cells. [Figure: see text].
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Manganese (Mn)-induced pulmonary toxicity and the underlying molecular mechanisms remain largely enigmatic. Further, in recent years, microRNAs (miRNAs) have emerged as regulators of several pollutants-mediated toxicity. In this context, our study aimed at elucidating whether miRNAs are involved in manganese (II) chloride (MnCl2) (Mn2+)-induced cytotoxicity in lung epithelial cells. Growth inhibition of Mn2+ towards normal human bronchial epithelial (BEAS-2B) and adenocarcinomic human alveolar basal epithelial (A549) cells was analyzed by MTT assay following 24 or 48 h treatment. Reactive oxygen species (ROS) generation, mitochondrial membrane potential (ΔΨm), cell cycle arrest, and apoptosis were evaluated by flow cytometry. RT-qPCR and Western blot were performed to analyze the expression of cyclins, anti-oxidant genes, and miRNAs. We used small RNA sequencing to investigate Mn2+-induced changes in miRNA expression patterns. In both cell lines, Mn2+ treatment inhibited growth in a dose-dependent manner. Further, compared with vehicle-treated cells, Mn2+ (250 µM) treatment induced ROS generation, cell cycle arrest, apoptosis, and decreased ΔΨm as well as altered the expression of cyclins and anti-oxidant genes. Sequencing data revealed that totally 296 miRNAs were differentially expressed in Mn2+-treated cells. Among them, miR-221-3p was one of the topmost down-regulated miRNAs in Mn2+-treated cells. We further confirmed this association in A549 cells. In addition, transient transfection was performed to study gain-of-function experiments. Forced expression of miR-221-3p significantly improved cell viability and reduced Mn2+-induced cell cycle arrest and apoptosis in BEAS-2B cells. In conclusion, miR-221-3p may be the most likely target that accounts for the cytotoxicity of Mn2+-exposed lung epithelial cells.
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Apoptose , Células Epiteliais , Pulmão , MicroRNAs , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Células A549 , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Apoptose/efeitos dos fármacos , Pulmão/efeitos dos fármacos , Pulmão/patologia , Pulmão/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Compostos de Manganês , Manganês/toxicidade , Linhagem Celular , Cloretos/toxicidade , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Relação Dose-Resposta a DrogaRESUMO
INTRODUCTION: The repertoire of microRNAs (miRNAs) in thyroid carcinomas starts to be elucidated. Among differentiated thyroid carcinomas (DTCs), papillary thyroid carcinoma (PTC) is the most frequent. The assessment of miRNAs expression may contribute to refine the pre-surgical diagnosis in order to obtain a personalized and more effective treatment for patients. AIMS: This study aims to evaluate (1) the miRNAs in a series of DTCs, and their association with the presence of selected genetic mutations in order to improve diagnosis and predict the biologic behavior of DTC/PTC. (2) The reliability of molecular tests in Ultrasound-guided Fine Needle Aspiration Cytology (US-FNAC) for a more precise preoperative diagnosis. MATERIAL AND METHODS: This series includes 176 samples (98 cytology and 78 histology samples) obtained from 106 patients submitted to surgery, including 13 benign lesions (controls) and 93 DTCs (cases). The microRNA expression was assessed for miR-146b, miR-221, miR-222, and miR-15a through quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). The results were analyzed by the 2-ΔΔCT method, using miR16 as an endogenous control. Regarding PTC diagnosis, the discriminative ability of miRNAs expression was assessed by the area under the Receiver Operating Characteristic Curve (AUC). In PTCs, the association of miRNAs expression, clinicopathological features, and genetic mutations (BRAF, RAS, and TERTp) was evaluated. RESULTS/DISCUSSION: All the analyzed miRNAs presented a tendency to be overexpressed in DTCs/PTCs when compared with benign lesions, both in cytology and histology samples. In cytology, miRNAs expression levels were higher in malignant tumors than in benign tumors. In histology, the discriminative abilities regarding PTC diagnosis were as follows: miR-146b (AUC 0.94, 95% CI 0.87-1), miR-221 (AUC 0.79, 95% CI 0.68-0.9), miR-222 (AUC 0.76, 95% CI 0.63-0.89), and miR-15a (AUC 0.85, 95% CI 0.74-0.97). miR-146b showed 89% sensitivity (se) and 87% specificity (sp); miR-221 se = 68.4, sp = 90; miR-222 se = 73, sp = 70; and mi-R15a se = 72, sp = 80. MicroRNAs were associated with worst-prognosis clinicopathological characteristics in PTCs (p < 0.05), particularly for miR-222. Our data reveal a significant association between higher expression levels of miR-146b, miR-221, and miR-222 in the presence of the BRAF mutation (p < 0.001) and miR-146b (p = 0.016) and miR-221 (p = 0.010) with the RAS mutation, suggesting an interplay of these mutations with miRNAs expression. Despite this study having a relatively small sample size, overexpression of miRNAs in cytology may contribute to a more precise preoperative diagnosis. The miRNAs presented a good discriminative ability in PTC diagnosis. The association between the miRNAs expression profile and genetic alterations can be advantageous for an accurate diagnosis of DTCs/PTCs in FNAC.
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Carcinoma Papilar , MicroRNAs , Neoplasias da Glândula Tireoide , Humanos , MicroRNAs/metabolismo , Proteínas Proto-Oncogênicas B-raf/genética , Reprodutibilidade dos Testes , Carcinoma Papilar/diagnóstico , Carcinoma Papilar/genética , Carcinoma Papilar/patologia , Neoplasias da Glândula Tireoide/diagnóstico , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/patologia , Câncer Papilífero da Tireoide/diagnóstico , Câncer Papilífero da Tireoide/genética , BiomarcadoresRESUMO
AIM: To explore the potential role of microRNA miR-221-5p on the angiopoietin-1 (Ang-1)/Ang-2/Tie-2 signaling axis after subarachnoid hemorrhage (SAH) in a rat model. METHODS: Aspects of the rat's behavior were measured using the Kaoutzanis scoring system to test neurological responses. This included feeding behavior, body contraction, motor, and eye-opening responses. Brain sections were studied using transmission electron microscopy and Evans blue extravasation. Levels of Ang-1, Ang-2, and Tie-2 were determined by Western blot, while miR-221-5p was quantified using stem-loop real-time quantitative PCR (RT-qPCR). RESULTS: The SAH group responded worse to the neurological response test than the sham-operated group. The intercellular space was widened in the SAH group, but not in the sham-operated group. Evans blue dye leaked significantly more into brain tissue cells of the SAH group. Stem-loop qRT-PCR showed elevated miR-221-5p levels. Additionally, Ang-1 and Tie-2 were reduced but Ang-2 expression was increased after SAH. This led to a significant reduction of the Ang-1/Ang-2 ratio in the brain tissue, which was associated with the destruction of the blood-brain barrier. CONCLUSION: The data indicate that miR-221-5p might regulate blood-brain barrier dysfunction through the Ang-1/Ang-2/Tie-2 signaling axis, suggesting that it should be further investigated as a potential novel biomarker.
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MicroRNAs , Hemorragia Subaracnóidea , Ratos , Animais , Barreira Hematoencefálica , Angiopoietina-1/genética , Angiopoietina-1/metabolismo , Azul Evans/metabolismo , MicroRNAs/metabolismoRESUMO
Alterations in microRNA (miRNA) expression have been reported in different cancers. We assessed the expression of 754 oncology-related miRNAs in esophageal adenocarcinoma (EAC) samples and evaluated their correlations with clinical parameters. We found that miR-221 and 483-3p were consistently upregulated in EAC patients vs. controls (Wilcoxon signed-rank test: miR-221 p < 0.0001; miR-483-3p p < 0.0001). Kaplan-Meier analysis showed worse cancer-related survival among all EAC patients expressing high miR-221 or miR-483-3p levels (log-rank p = 0.0025 and p = 0.0235, respectively). Higher miR-221 or miR-483-3p levels also correlated with advanced tumor stages (Mann-Whitney p = 0.0195 and p = 0.0085, respectively), and overexpression of miR-221 was associated with worse survival in low-risk EAC patients. Moreover, a significantly worse outcome was associated with the combined overexpression of miR-221 and miR-483-3p (log-rank p = 0.0410). To identify target genes affected by miRNA overexpression, we transfected the corresponding mimic RNA (miRVANA) for either miR-221 or miR-483-3p in a well-characterized esophageal adenocarcinoma cell line (OE19) and performed RNA-seq analysis. In the miRNA-overexpressing cells, we discovered a convergent dysregulation of genes linked to apoptosis, ATP synthesis, angiogenesis, and cancer progression, including a long non-coding RNA associated with oncogenesis, i.e., MALAT1. In conclusion, dysregulated miRNA expression, especially overexpression of miR-221 and 483-3p, was found in EAC samples. These alterations were connected with a lower cancer-specific patient survival, suggesting that these miRNAs could be useful for patient stratification and prognosis.
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The circular RNA/microRNA/mRNA axis is a new layer of non-coding RNA(ncRNA)-based regulatory gene expression networks upstream of numerous cell signaling pathways. Circular RNAPAN3 (circPAN3) is involved in autophagy, fibrosis and apoptosis which are responsible for the reduction incardiac functional capacityfollowingmyocardial infarction(MI). However, the molecular mechanism of circPAN3 association with apoptosis is unknown. In addition, the relationship between quercetin as a cardioprotective factor in MI and circular RNA-dependent regulatory pathways has not yet been elucidated. MI was induced in Wistar rats using the left anterior descending artery (LAD) ligation method. One day after surgery, quercetin (30 mg/kg) was injected intraperitoneal (IP) every other day for two weeks. The expression of circPAN3 was increased in the MI group (P < 0.05). The increase in circPAN3 was accompanied by a decrease in miR-221 (P < 0.0001), an increase in PTEN (P < 0.0001), and cleaved caspase 3 (P < 0.001). Quercetin effectively reduced the expression of circPAN3 (P < 0.05), PTEN (P < 0.0001), and cleaved caspase 3 (P < 0.001), and increased the expression of miR-221 (P < 0.0001) and the ratio of p-AKT to p-PI3K (P < 0.001). The circPAN3/miR-221/PTEN pathway is an ncRNA-dependent apoptotic pathway in MI cardiac tissue. Quercetin effectively modulated this pathway, resulting in a reduction of cardiac tissue death and improvement in cardiac function after MI. This suggests that the circPAN3/miR-221 axis plays a role in apoptosis in MI, and quercetin can act as a protective candidate by modulating this pathway.
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MicroRNAs , Infarto do Miocárdio , Ratos , Animais , Caspase 3/metabolismo , Quercetina/farmacologia , RNA Circular/metabolismo , Ratos Wistar , Infarto do Miocárdio/tratamento farmacológico , Infarto do Miocárdio/genética , Infarto do Miocárdio/metabolismo , Apoptose/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Miócitos Cardíacos/metabolismoRESUMO
BACKGROUND: Breast cancer is one of the most common diseases worldwide that affects women of reproductive age. miR-221 and miR-222 are two highly homogeneous microRNAs that play pivotal roles in many cellular processes and regulate the Wnt/ß-catenin signaling pathway. Curcumin (CUR), a yellow polyphenolic compound, targets numerous signaling pathways relevant to cancer therapy. The main aim of this study was to compare the ability of chitosan curcumin nanoparticle (CC-CUR) formulation with the curcumin in modulating miR-221 and miR-222 expression through Wnt/ß-catenin signaling pathway in MCF-7, MDA-MB-231 and SK-BR-3 breast cancer cell lines. METHOD: Chitosan-cyclodextrin-tripolyphosphate containing curcumin nanoparticles (CC-CUR) were prepared. Cytotoxicity of the CUR and CC-CUR was evaluated. Experimental groups including CC-CUR, CUR and negative control were designed. The expression of miR-221 and miR-222 and Wnt/ß-catenin pathway genes was measured. RESULTS: The level of miR-221 and miR-222 and ß-catenin genes decreased in MCF-7 and MDA-MB-231 cells and WIF1 gene increased in all cells in CC-CUR group. However, the results in SK-BR-3 cell line were unexpected; since miRs and WIF1 gene expressions were increased following CC-CUR administration and ß-catenin decreased by administration of CUR. CONCLUSION: Although the composite form of curcumin decreased the expression of miR-221 and miR-222 in MCF-7 and MDA cells, with significant decreasing of ß-catenin and increasing of WIF1 gene in almost all three cell lines, we can conclude than this formulation exerts its effect mainly through the Wnt/ß-catenin pathway. These preliminary findings may pave the way for the use of curcumin nanoparticles in the treatment of some known cancers.
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Neoplasias da Mama , Quitosana , Curcumina , MicroRNAs , Feminino , Humanos , beta Catenina/genética , beta Catenina/metabolismo , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Quitosana/farmacologia , Curcumina/farmacologia , Células MCF-7 , MicroRNAs/metabolismo , Via de Sinalização Wnt/genética , NanopartículasRESUMO
miR-221-3p has been reported to attenuate the osteogenic differentiation of annulus fibrosus cells (AFs), which has been implicated in intervertebral disk degeneration (IVDD) development. This study aimed to elucidate miR-221-3p's role in osteogenic differentiation and apoptosis of AFs in an IVDD model. After successfully establishing an IVDD rat model by annulus fibrosus needle puncture, AFs were isolated. Bioinformatics, dual-luciferase reporter, and AGO2-RNA immunoprecipitation (RIP) assays predicted and confirmed the potential miR-221-3p lncRNA and gene target. Functional analyses were performed after AF transfection to explore the roles of the identified lncRNA and gene. Western blotting, Alkaline phosphatase (ALP), and Alizarin red and TUNEL staining were performed to investigate AF apoptosis and osteogenic differentiation with different transfections. Compared with AFs isolated from sham rats, IVDD-isolated Afs exhibited stronger osteogenic potential and higher apoptosis rates accompanied by miR-221-3p downregulation. The growth arrest-specific transcript 5 (GAS5) was identified as miR-221-3p's target lncRNA, which was highly expressed in IVDD. GAS5 overexpression facilitated AF apoptosis and osteogenic differentiation, whereas silencing GAS5 had the opposite effect. SRY box-related11 (SOX11) was identified as a downstream miR-221-3p target gene in IVDD. GASS silencing-induced suppression of AF apoptosis and osteogenic differentiation could be reversed by SOX11 overexpression. Our findings uncovered a lncRNA GAS5/miR-221-3p/SOX11 axis in Afs under IVDD, which may help implement novel IVDD therapeutic strategies.
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Degeneração do Disco Intervertebral , MicroRNAs , RNA Longo não Codificante , Animais , Ratos , Apoptose/genética , Diferenciação Celular/genética , Fibroblastos , Degeneração do Disco Intervertebral/genética , MicroRNAs/genética , Osteogênese/genética , RNA Longo não Codificante/genéticaRESUMO
BACKGROUND: Tumor cell spheroids are organized multicellular structures that form during the expansive growth of carcinoma cells. Spheroids formation is thought to contribute to metastasis by supporting growth and survival of mobile tumor cell populations. METHODS AND RESULTS: We investigated how spheroid architecture affects OXPHOS activity, microRNA expression, and intraperitoneal survival of an ovarian carcinoma cell line using high resolution respirometry, quantitative RT-PCR, and a rodent intraperitoneal growth model. Rates of oxidative phosphorylation/respiration per cell of cells growing as spheroids were nearly double those of a variant of the same cell type growing in suspension as loosely aggregated cells. Further, inhibition of spheroid formation by treatment with CDH2 (N-cadherin) siRNA reduced the rate of OXPHOS to that of the non-spheroid forming variant. Cells growing as spheroids showed greatly enhanced expression of miR-221/222, an oncomiR that targets multiple tumor suppressor genes and promotes invasion, and reduced expression of miR-9, which targets mitochondrial tRNA-modification enzymes and inhibits OXPHOS. Consistent with greater efficiency of ATP generation, tumor cells growing as spheroids injected into the nutrient-poor murine peritoneum survived longer than cells growing in suspension as loosely associated aggregates. CONCLUSIONS: The data indicate that growth in spheroid form enhances the OXPHOS activity of constituent tumor cells. In addition, spheroid architecture affects expression of microRNA genes involved in growth control and mitochondrial function. During the mobile phase of metastasis, when ovarian tumor cells disperse through nutrient-poor environments such as the peritoneum, enhanced OXPHOS activity afforded by spheroid architecture would enhance survival and metastatic potential.
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MicroRNAs , Neoplasias Ovarianas , Animais , Feminino , Humanos , Camundongos , Caderinas/genética , Linhagem Celular Tumoral , MicroRNAs/genética , MicroRNAs/metabolismo , Neoplasias Ovarianas/patologia , Fosforilação Oxidativa , Esferoides Celulares/metabolismoRESUMO
Aims: Heme oxygenase (HO-1) affords protection against ischaemia/reperfusion (I/R) injury; however, its effects on testicular I/R injury remain poorly explored. Herein, we aimed to examine the effects of HO-1 on testicular I/R injury and elucidate the underlying mechanism. Methods: Using the TALEN technique, we knocked out the HO-1 gene from rats. In vivo: Thirty hmox+/+ and 30 hmox-/- rats were randomly assigned to six groups: sham-operated (sham), I/R (the left testicle torsion/detorsion) 0 d,I/R 1d, I/R 3d, I/R 7d and I/R 28d. In vitro: GC-1 were suffered from: control,H/R (oxygen-deprivation/reoxygenation),H/R + HO-1 siRNA,H/R + c-Jun siRNA or H/R + HO-1 siRNA + c-jun.We performed immunofluorescence and immunohistochemistry experiments to detect HO-1 nuclear translocation. Flow cytometry was used to detect cell apoptosis and analyse the cell cycle. High-resolution miRNA, mRNA sequencing, reverse transcription-quantitative PCR, and western blotting were performed to identify testicular I/R injury-related genes strongly conserved in HO-1 knockout rats. A double luciferase reporter assay was performed to verify the relationship between C-jun and miR-221/222. Main findings: In vivo, HO-1 improved the pathological damage induced by testicular I/R. In GC-1 cells, we confirmed the nuclear translocation of HO-1 and its protective effect against hypoxia/reoxygenation (H/R) damage. Accordingly, HO-1 protein itself, rather than heme metabolites, might play a key role in testicular I/R. Gene sequencing was performed to screen for miR221/222 and its downstream gene, thymocyte selection-associated high mobility group box (TOX). HO-1 increased c-Jun phosphorylation in the H/R group, knocked down c-Jun in GC-1 cells, and decreased miR-221/222 expression. Inhibition of HO-1 expression decreased the expression of c-Jun and miR-221/222, which was rescued by adding c-Jun. Dual-luciferase reporter assay confirmed the interaction between c-Jun and miR-221/222. Conclusions: HO-1 could exert a protective effect against testicular I/R via the phosphorylated c-Jun-miR-221/222-TOX pathway.
RESUMO
BACKGROUND: MicroRNA-221-3p (miR-221-3p) facilitates the advancement of breast cancer (BC) through the induction of epithelial-mesenchymal transition (EMT). Our research aimed to utilize bioinformatics to discover possible EMT-related target genes (ETGs) of miR-221-3p and examine their roles in breast cancer. METHODS: We employed bioinformatics techniques to identify ten key ETGs of miR-221-3p. Subsequently, we conducted an extensive analysis of both miR-221-3p and the ten ETGs, including clinical significance and immune characteristics. RESULTS: The expression of miR-221-3p was notably higher in Basal-like BC compared to other subtypes and adjacent normal tissue. Our pathway analysis suggested that miR-221-3p might regulate EMT through the MAPK signaling pathway by targeting its ETGs. Among the ETGs, seven core genes (EGFR, IGF1, KDR, FGF2, KIT, FGFR1, and FGF1) exhibited downregulation in BC. Conversely, ERBB2, SDC1, and MMP14 showed upregulation in BC and displayed potential diagnostic value. The analysis of prognostication indicated that increased levels of SDC1 and MMP14 were correlated with an unfavorable prognosis, whereas elevated expression of KIT was associated with a more favorable prognosis. The infiltration of various immune cells and the expression of immune checkpoint genes (ICGs) exhibited positive correlations with most ETGs and miR-221-3p. SDC1 exhibited a greater tumor mutational burden (TMB) score, while ERBB2, KDR, FGF2, KIT, FGFR1, and FGF1 showed lower TMB scores. Furthermore, decreased ERBB2 and KDR expression levels were correlated with elevated microsatellite instability (MSI) scores. Elevated expression of ETGs was linked to decreased mRNA stemness indices (mRNAsi), whereas miR-221-3p displayed the opposite pattern. Most ETGs and miR-221-3p expression exhibited a negative correlation with IC50 values for drugs. Among the ETGs, amplification was the most significant genetic alteration, except for IGF1. CONCLUSION: In conclusion, miR-221-3p acts as a unique indicator for Basal-like BC. The examination revealed ten essential ETGs of miR-221-3p, some of which show potential as diagnostic and prognostic markers. The in-depth examination of these ten ETGs and miR-221-3p indicates their participation in the development of BC, emphasizing their promise as innovative targets for therapy in BC patients.
Assuntos
Neoplasias da Mama , MicroRNAs , Humanos , Feminino , MicroRNAs/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Metaloproteinase 14 da Matriz/genética , Linhagem Celular Tumoral , Relevância Clínica , Fator 1 de Crescimento de Fibroblastos/genética , Fator 2 de Crescimento de Fibroblastos/metabolismo , Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica , Proliferação de Células/genética , Movimento Celular/genéticaRESUMO
BACKGROUND: Gastric cancer (GC) is one of the most common digestive malignancies. Although miR-221-3p was defined as a novel biomarker in many types of cancer, the relationship between its expression differences and the clinicopathological characteristics and prognosis of GC patients was yet to be fully understood. METHODS AND RESULTS: TCGA database was utilized to predict the potential biological function of miR-221-3p in GC. QRT-PCR and RNA FISH were performed to detect the expression levels of miR-221-3p in GC. The miR-221-3p expression levels in GC tissues and cells were significantly higher than those in paracancerous tissues (p < 0.001) and normal gastric mucosal cells (p < 0.05). Higher expression levels of miR-221-3p were associated with tumor diameter ≥ 4 cm (χ2 = 5.519, p = 0.019), cTNM stage (III + IV) (χ2 = 28.013, p = 0.000), lymph node metastasis (χ2 = 23.272, p = 0.000) and distant metastasis (χ2 = 7.930, p = 0.005). Kaplan-Meier survival analysis showed a better prognosis for GC patients with miR-221-3p low expression(HR = 4.520, 95% CI: 1.844-11.075). CONCLUSIONS: miR-221-3p is highly expressed in GC tissues, which plays an important role in tumorigenesis, invasion and metastasis. miR-221-3p may become an important biomarker and potential molecular therapeutic target for patients with GC.
Assuntos
MicroRNAs , Neoplasias Gástricas , Humanos , Neoplasias Gástricas/genética , Carcinogênese , Transformação Celular Neoplásica , Biomarcadores , MicroRNAs/genéticaRESUMO
OBJECTIVE: To investigate the effects of miR-221 and miR-222 and high glucose on human periodontal ligament (PL) cells morphology, cytoskeleton, adhesion, and migration. BACKGROUND: Chronic hyperglycemia is common in uncontrolled diabetes mellitus (DM) and plays a central role in long-term DM complications, such as impaired periodontal healing. We have previously shown that high glucose increases apoptosis of human PL cells by inhibiting miR-221 and miR-222 and consequently augmenting their target caspase-3. However, other effects of miR-221/222 downregulation on PL cells are still unknown. METHODS: Cells from young humans' premolar teeth were cultured for 7 days under 5 or 30 mM glucose. Directional and spontaneous migration on fibronectin were studied using transwell and time-lapse assays, respectively. F-actin staining was employed to study cell morphology and the actin cytoskeleton. MiR-221 and miR-222 were inhibited using antagomiRs, and their expressions were evaluated by real-time RT-PCR. RESULTS: High glucose inhibited PL cells early adhesion, spreading, and migration on fibronectin. Cells exposed to high glucose showed reduced polarization, velocity, and directionality. They formed several simultaneous unstable and short-lived protrusions, suggesting impairment of adhesion maturation. MiR-221 and miR-222 inhibition also reduced migration, decreasing cell directionality but not significantly cell velocity. After miR-221 and miR-222 downregulation cells showed morphological resemblance with cells exposed to high glucose. CONCLUSION: High glucose impairs human PL cells migration potentially through a mechanism involving reduction of microRNA-221 and microRNA-222 expression. These effects may contribute to the impairment of periodontal healing, especially after surgery and during guided regeneration therapies.
Assuntos
MicroRNAs , Humanos , MicroRNAs/metabolismo , Fibronectinas/farmacologia , Ligamento Periodontal/metabolismo , Movimento Celular , Glucose/farmacologia , Células CultivadasRESUMO
Parkinson's disease (PD) is a neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra, leading to various motor and non-motor symptoms. Several cellular and molecular mechanisms such as alpha-synuclein (α-syn) accumulation, mitochondrial dysfunction, oxidative stress and neuroinflammation are involved in the pathogenesis of this disease. MicroRNAs (miRNAs) play important roles in post-transcriptional gene regulation. They are typically about 21-25 nucleotides in length and are involved in the regulation of gene expression by binding to the messenger RNA (mRNA) molecules. miRNAs like miR-221 play important roles in various biological processes, including development, cell proliferation, differentiation and apoptosis. miR-221 promotes neuronal survival against oxidative stress and neurite outgrowth and neuronal differentiation. Additionally, the role of miR-221 in PD has been investigated in several studies. According to the results of these studies, (1) miR-221 protects PC12 cells against oxidative stress induced by 6-hydroxydopamine; (2) miR-221 prevents Bax/caspase-3 signalling activation by stopping Bim; (3) miR-221 has moderate predictive power for PD; (4) miR-221 directly targets PTEN, and PTEN over-expression eliminates the protective action of miR-221 on p-AKT expression in PC12 cells; and (5) miRNA-221 controls cell viability and apoptosis by manipulating the Akt signalling pathway in PD. This review study suggested that miR-221 has the potential to be used as a clinical biomarker for PD diagnosis and stage assignment.