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Introduction: Increased de novo lipogenesis (DNL) is one of the key factors contributing to fat accumulation and non-alcoholic fatty liver disease (NAFLD). Among the critical transcription factors (TFs) regulating DNL is mTOR and its downstream lipogenic TF, SREBP1c. In recent years, it has been established that non-coding RNAs (ncRNAs) play role in both biological processes and disease pathogenesis. Our group has previously characterized microRNAs that can target and regulate the expression of both mTOR and SREBP1c. Accordingly, this study aimed to broaden our understanding of the role of ncRNAs in regulating the mTOR/SREBP1c axis to elucidate the role of the non-coding transcriptome in DNL and lipid droplet (LD) formation. Hence, short ncRNA, miR-615-5p, and long non-coding RNA (lncRNA), H19, were chosen as they were previously proven to target mTOR by our group and in the published literature, respectively. Methodology: Huh-7 cells were treated with 800 µM oleic acid (OA) to promote LD formation. Transfection of miR-615-5p mimics or H19 over-expression vectors was performed, followed by the measurement of their downstream targets, mTOR and SREBP, on the mRNA level by quantitative real-time PCR (qRT-PCR), and on the protein level by Western blot. To determine the functional impact of miR-615-5p and H19 on LD formation and triglyceride (TG) accumulation, post-transfection LDs were stained, imaged, and characterized, and TGs were extracted and quantified. Results: miR-615-5p was able to reduce mTOR and SREBP1c significantly on both the mRNA and protein levels compared to control cells, while H19 caused a reduction of both targets on the protein level only. Both miR-615-5p and H19 were able to significantly reduce the LD count and total area, as well as TG levels compared to control cells. Conclusion: To conclude, this study shows, for the first time, the impact of miR-615-5p and H19 on the mTOR/SREBP1c axis, and thus, their functional impact on LDs and TG accumulation. These findings might pave the way for using ncRNAs as potential therapeutic targets in the management of fatty liver.
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Mesenchymal stromal cells (MSCs) have shown promise in liver cancer treatment. However, when MSCs are recruited to hepatic site of injury, they acquire cancerous promoting phenotype. AIMS: To assess the influence of Hepatocellular carcinoma (HCC) microenvironment on human adipose MSCs (hA-MSCs) and predict hA-MSCs intracellular miRNAs role. MATERIALS AND METHODS: After indirect co-culturing with Huh-7 cells, hA-MSCs were characterized via cell cycle profile, proliferation and migration potentials by MTT and scratch assays respectively. Functional enrichment analysis of deregulated proteins and miRNA targets was also analyzed. KEY FINDINGS: Co-cultured hA-MSCs could acquire a cancer-associated phenotype as shown by upregulation of CAF, cancer markers, and downregulation of differentiation markers. Migration of these cancer-associated cells was increased concomitantly with upregulation of adhesion molecules, but not epithelial to mesenchymal transition markers. Co-cultured cells showed increased proliferation confirmed by downregulation in cell percentage in G0/G1, G2/M and upregulation in S phases of cell cycle. Upregulation of miR-17-5p and 615-5p in co-cultured hA-MSCs was also observed. Functional enrichment analysis of dysregulated proteins in co-cultured hA-MSCs, including our selected miRNAs targets, showed their involvement in development of cancer-associated characteristics. SIGNIFICANCE: This study suggests an interaction between tumor cells and surrounding stromal components to generate cancer associated phenotype of some CAF-like characteristics, known to favor cancer progression. This sheds the light on the use of hA-MSCs in HCC therapy. hA-MSCs modulation may be partially achieved via dysregulation of intracellular miR17-5P and 615-5p expression, suggesting an important role for miRNAs in HCC pathogenesis, and as a possible therapeutic candidate.
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Biomarcadores Tumorais/metabolismo , Carcinoma Hepatocelular/patologia , Regulação Neoplásica da Expressão Gênica , Células-Tronco Mesenquimais/patologia , MicroRNAs/genética , Fenótipo , Microambiente Tumoral , Apoptose , Biomarcadores Tumorais/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/metabolismo , Ciclo Celular , Movimento Celular , Proliferação de Células , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Células-Tronco Mesenquimais/imunologia , Células-Tronco Mesenquimais/metabolismo , Células Tumorais CultivadasRESUMO
BACKGROUND: Doxorubicin (DOX) is one of the most common drugs used in cancer therapy, including Hepatocellular Carcinoma (HCC). Drug resistance is one of chemotherapy's significant problems. Emerging studies have shown that microRNAs (miRNAs) could participate in regulating this mechanism. Nevertheless, the impact of miRNAs on HCC chemoresistance is still enigmatic. OBJECTIVE: Investigating the role of microRNA-520c-3p (miR-520c-3p) in the enhancement of the anti-tumor effect of DOX against HepG2 cells. METHODS: Expression profile for liver-related miRNAs (384 miRNAs) has been analyzed on HepG2 cells treated with DOX using qRT-PCR. miR-520c-3p, the most deregulated miRNA, was selected for combination treatment with DOX. The expression level for LEF1, CDK2, CDH1, VIM, Mcl-1 and p53 was evaluated in miR-520c-3p transfected cells. Cell viability, colony formation, wound healing as well as apoptosis assays have been demonstrated. Furthermore, Mcl-1 protein level was measured using the western blot technique. RESULTS: The present data indicated that miR-520c-3p overexpression could render HepG2 cells chemo-sensitive to DOX through enhancing its suppressive effects on proliferation, migration, and induction of apoptosis. The suppressive effect of miR-520c-3p involved altering the expression levels of some key regulators of cell cycle, proliferation, migration and apoptosis, including LEF1, CDK2, CDH1, VIM, Mcl-1 and p53. Interestingly, Mcl-1 was found to be one of the potential targets of miR-520c-3p, and its protein expression level was down-regulated upon miR-520c-3p overexpression. CONCLUSION: Our data referred to the tumor suppressor function of miR-520c-3p that could modulate the chemosensitivity of HepG2 cells towards DOX treatment, providing a promising therapeutic strategy in HCC.
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Antibióticos Antineoplásicos/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/genética , Doxorrubicina/farmacologia , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , MicroRNAs/genética , Apoptose/efeitos dos fármacos , Carcinoma Hepatocelular/patologia , Ciclo Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genes Supressores de Tumor/efeitos dos fármacos , Células Hep G2 , Humanos , Neoplasias Hepáticas/patologiaRESUMO
BACKGROUND: Identification of factors to detect and improve chemotherapy.response in cancer is the main concern. microRNA-372-3p (miR-372-3p) has been demonstrated to play a crucial role in cellular proliferation, apoptosis and metastasis of various cancers including Hepatocellular Carcinoma (HCC). However, its contribution towards Doxorubicin (Dox) chemosensitivity in HCC has never been studied. OBJECTIVE: This study aims to investigate the potential role of miR-372-3p in enhancing Dox effects on HCC cell line (HepG2). Additionally, the correlation between miR-372-3p and HCC patients who received Transarterial Chemoembolization (TACE) with Dox treatment has been analyzed. METHODS: Different cell processes were elucidated by cell viability, colony formation, apoptosis and wound healing assays after miR-372-3p transfection in HepG2 cells Furthermore, the miR-372-3p level has been estimated in the blood of primary HCC patients treated with TACE/Dox by quantitative real-time PCR assay. Receiver Operating Curve (ROC) analysis for serum miR-372-3p was constructed for its prognostic significance. Finally, the protein level of Mcl-1, the anti-apoptotic player, has been evaluated using western blot. RESULTS: We found a significantly higher level of miR-372-3p in the blood of the responder group of HCC patients who received TACE with Dox than of non-responders. Ectopic expression of miR-372-3p reduced cell proliferation, migration and significantly induced apoptosis in HepG2 cells which was coupled with a decrease of anti-apoptotic protein Mcl-1. CONCLUSION: Our study demonstrated that miR-372-3p acts as a tumor suppressor in HCC and can act as a predictor biomarker for drug response. Furthermore, the data referred for the first time its potential role in drug sensitivity that might be a therapeutic target for HCC.
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Antibióticos Antineoplásicos/administração & dosagem , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/genética , Doxorrubicina/administração & dosagem , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , MicroRNAs/genética , Antibióticos Antineoplásicos/farmacologia , Antibióticos Antineoplásicos/uso terapêutico , Carcinoma Hepatocelular/diagnóstico , Quimioembolização Terapêutica , Doxorrubicina/farmacologia , Doxorrubicina/uso terapêutico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genes Supressores de Tumor/efeitos dos fármacos , Células Hep G2 , Humanos , Neoplasias Hepáticas/diagnóstico , PrognósticoRESUMO
HCV entry involves a complex interplay between viral and host molecules. During post-binding interactions, the viral E2 complexes with CD81 receptor for delivery to the tight junction proteins CLDN1 and OCLN, which aid in viral internalization. Targeting HCV entry receptors represents an appealing approach to inhibit viral infectivity. This study aimed at investigating the impact of targeting CLDN1 by microRNAs on HCV infectivity. miR-155 was previously shown to target the 3'UTR of CLDN1 mRNA. Therefore, miR-155 was used as a control in this study. In-silico analysis and luciferase reporter assay were utilized to identify potential targeting miRNAs. The impact of the identified miRNAs on CLDN1 mRNA and protein expression was examined by qRT-PCR, indirect immunofluorescence and western blotting, respectively. The role of the selected miRNAs on HCV infectivity was assessed by measuring the viral load following the ectopic expression of the selected miRNAs. miR-182 was identified in-silico and by experimental validation to target CLDN1. Both miR-155 and miR-182 inhibited CLDN1 mRNA and protein expression in infected Huh7 cells. Ectopic expression of miR-155 increased, while miR-182 reduced the viral load. In conclusion, despite repressing CLDN1, the impact of miR-155 and miR-182 on HCV infectivity is contradictory. Ectopic miR-182 expression is suggested as an upstream regulator of the entry factor CLDN1, harnessing HCV infection.
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miR-let-7a is a tumor suppressor miRNA with reduced expression in most cancers. Methylation of MIRLET7A3 gene was reported to be the cause of this suppression in several cancers; however, it was not explicitly investigated in hepatocellular carcinoma (HCC). We aimed at investigating miR-let-7a expression and molecular mode in HCC, identifying drug-targetable networks, which might be affected by its abundance. Our results illustrated a significant repression of miR-let-7a, which correlated with hypermethylation of its gene of origin MIRLRT7A3. This was further supported by the induction of miR-let-7a expression upon treatment of HCC cells with a DNA-methyltransferase inhibitor. Using a computational approach, insulin-like growth factor (IGF)-II and IGF-2 mRNA binding proteins (IGF2BP)-2/-3 were identified as potential targets for miR-let-7a that was further confirmed experimentally. Indeed, miR-let-7a mimics diminished IGF-II as well as IGF2BP-2/-3 expression. Direct binding of miR-let-7a to each respective transcript was confirmed using a luciferase reporter assay. In conclusion, this study suggests that DNA hypermethylation leads to epigenetic repression of miR-let-7a in HCC cells, which induces the oncogenic IGF-signaling pathway.
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BACKGROUND AND AIM: The activity of natural killer (NK) cells is known to be decreased in systemic lupus erythematosus (SLE) patients. Nevertheless, the exact contribution of NK cells in the pathogenesis of SLE is still inconclusive. MicroRNAs (miRNAs), are small noncoding RNA molecules that play a fundamental role in regulating NK cell function. The objective of this study was to investigate the expression of miRNAs that might potentially target an essential activating receptor, NKG2D in peripheral blood mononuclear cells (PBMCs) and NK cells of SLE patients. METHODS: In silico analysis revealed miR-27a* to potentially target NKG2D messenger RNA (mRNA), hence PBMCs and NK cells were isolated from blood samples of SLE patients and healthy controls. Next, the cells were transfected using mimics and antagomirs, after which miRNA/mRNA were quantified using real time quantitative reverse transcription polymerase chain reaction. RESULTS: The results of this study showed that miR-27a* is overexpressed in the PBMCs and NK cells of SLE patients. In contrast, NKG2D was found to be downregulated in PBMCs and NK cells of SLE patients. Forcing the expression of miR-27a* in PBMCs and NK cells enhances the expression of NKG2D in SLE patients. Furthermore, the ligand of NKG2D, ULBP2, was found to be downregulated in the PBMCs of SLE patients. CONCLUSION: The altered expression of the triad, miR-27a* as well as NKG2D and ULBP2, is thought to be characteristic for NK cells in SLE patients. Hence, the ability of miR-27a* to alter the expression of NKG2D may provide a new groundwork for understanding the role of miRNAs in NK cells of SLE patients.
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Células Matadoras Naturais/metabolismo , Leucócitos Mononucleares/metabolismo , Lúpus Eritematoso Sistêmico/genética , MicroRNAs/genética , Subfamília K de Receptores Semelhantes a Lectina de Células NK/genética , Regiões 3' não Traduzidas , Regiões 5' não Traduzidas , Adolescente , Idade de Início , Antagomirs/genética , Antagomirs/metabolismo , Sítios de Ligação , Estudos de Casos e Controles , Células Cultivadas , Feminino , Proteínas Ligadas por GPI/sangue , Proteínas Ligadas por GPI/genética , Regulação da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/sangue , Peptídeos e Proteínas de Sinalização Intercelular/genética , Células Matadoras Naturais/imunologia , Leucócitos Mononucleares/imunologia , Lúpus Eritematoso Sistêmico/sangue , Lúpus Eritematoso Sistêmico/diagnóstico , Lúpus Eritematoso Sistêmico/imunologia , Masculino , MicroRNAs/metabolismo , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , TransfecçãoRESUMO
Hepatitis C Virus (HCV) promotes lipid droplet (LD) formation and perturbs the expression of the LD associated PAT proteins ADRP and TIP47, to promote its own lifecycle. HCV enhances TIP47 and suppresses ADRP by displacing it from LD surface in infected cell models. We have previously shown that suppression of TIP47 by miR-148a and miR-30a decreased intracellular LDs and HCV RNA. Thus, this study aimed at examining whether this microRNA-mediated suppression of HCV would limit HCV-dependent displacement of ADRP from LDs. ADRP expression was examined in 21 HCV-infected liver biopsies and 9 healthy donor liver tissues as well as in HCV-infected Huh7 cells using qRT-PCR. miR-148a and miR-30a expression was manipulated using specific oligos in JFH-1 infected, oleic acid treated cells, to study their impact on ADRP expression using qRT-PCR, and immunofluorescence microscopy. Intracellular HCV RNA was assessed using qRT-PCR. ADRP is down regulated in patients as well as HCVcc-JFH-I infected cell models. Forcing the expression of both miRNAs induced ADRP on the mRNA and protein levels. This study shows that HCV suppresses hepatic ADRP expression in infected patients and cell lines. Forcing the expression of miR-148a and miR-30a limits the suppressive effect of HCV on ADRP. J. Med. Virol. 89:653-659, 2017. © 2016 Wiley Periodicals, Inc.
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Hepacivirus/patogenicidade , Interações Hospedeiro-Patógeno , MicroRNAs/metabolismo , Perilipina-2/antagonistas & inibidores , Adulto , Biópsia , Linhagem Celular , Feminino , Perfilação da Expressão Gênica , Hepatite C/patologia , Hepatócitos/virologia , Humanos , Fígado/patologia , Masculino , Microscopia de Fluorescência , Pessoa de Meia-Idade , Perilipina-3/metabolismo , RNA Viral/análise , Reação em Cadeia da Polimerase em Tempo RealRESUMO
BACKGROUND AND AIM: NK-cells are the principle defense line against different malignancies. Their activation status is determined by the balance between activating and inhibitory receptors such as NKG2D and NKG2A, respectively. MicroRNAs are crucial post-transcriptional regulators of gene expression, playing key roles in modulating NK-cell development and function. The aim of this study is to investigate the role of miRNAs in regulating the activation and cytotoxic function of NK-cells in HCC. METHODS: In silico analysis was performed to predict a potential miRNA that might target NKG2D and NKG2A mRNAs. NK-cells were isolated from HCC patients and healthy controls, after which miRNA and mRNA were quantified. Manipulating miRNA expression was performed followed by investigating downstream targets and the cytotoxic activity of NK-cells against Huh-7 cell lines. RESULTS: NK-cells of HCC patients showed miR-182 overexpression compared to controls. NKG2D and NKG2A were upregulated and downregulated, respectively, in HCC NK-cells. Upon forcing miR-182 expression in the HCC NK-cells, upregulation of both receptors was observed. Finally, miR-182 was reported to induce NK-cell cytotoxicity represented in Perforin-1 upregulation and increase in cytolytic killing of co-cultured Huh-7 cells. CONCLUSION: Our findings suggest that miR-182 may augment NK-cell cytotoxicity against liver cancer via modulating NKG2D and NKG2A expressions.
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Carcinoma Hepatocelular/imunologia , Células Matadoras Naturais/fisiologia , Neoplasias Hepáticas/imunologia , MicroRNAs/genética , Subfamília C de Receptores Semelhantes a Lectina de Células NK/metabolismo , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Carcinoma Hepatocelular/genética , Linhagem Celular Tumoral , Biologia Computacional , Citotoxicidade Imunológica/genética , Regulação da Expressão Gênica , Humanos , Neoplasias Hepáticas/genética , Subfamília C de Receptores Semelhantes a Lectina de Células NK/genética , Subfamília K de Receptores Semelhantes a Lectina de Células NK/genética , Transgenes/genéticaRESUMO
BACKGROUND AND AIM: Natural killer cells are part of the innate immunity involved in viral eradication and were shown to be greatly affected by HCV infection. Epigenetic regulation of NK cell function by microRNAs was not efficiently studied before and was never studied in HCV infection; therefore the aim of this study was to assess for the first time the role of microRNAs in regulating the function of NK cells of HCV-infected patients and hence viral replication in the target HCV-infected Huh7 cells. METHODOLOGY: NK cells were isolated from PBMCs of HCV-infected patients as well as controls, and HCV-infected liver biopsies as well as Huh7 cells infected with the virus were used. For the infection of Huh7 cells, first viral vector was in-vitro transcribed into viral RNA that was then used to infect naÑve Huh7 cells. Supernatant from the infected cells was then collected and used for further infection. For manipulation of NK cells or Huh7 cells, miR-182 mimics and inhibitors were transfected via lipofection method. RNA was extracted from each cell population, reverse transcribed. Gene expression as well as viral load was quantified using qPCR. RESULTS: Screening of NKG2A and NKG2D between patients and controls showed no difference in expression of NKG2A, while NKG2D was found to be downregulated. In view of that, bioinformatics analysis was performed and showed that miR-182 has potential binding sites on both the inhibitory receptor NKG2A and the activating receptor NKG2D, and on its ligand ULBP2, as well as on the viral genome itself. In NK cells of HCV-infected patients, miR-182 was found to be over-expressed compared to controls; its ectopic expression was found to decrease NKG2D mRNA level, while miR-182 inhibitors were able to decrease NKG2A mRNA compared to untransfected cells. In addition, co-culturing genotype 4 or 2 HCV-infected Huh7 cells with miR-182 mimicked NK cells of HCV-infected patients showed decreased viral replication, suggesting an enhanced NK cell function. On the other hand, miR-182 and ULBP2 were both found to be downregulated in HCV liver tissues and HCV-infected Huh7 cells compared to their controls. miR-182 mimics were found to decrease ULBP2 mRNA and increase viral replication in genotypes 4 and 2 HCV-infected target (Huh7) cells compared to controls, while miR-182 inhibitor decreased viral replication in the cell models. CONCLUSION: miR-182 was never investigated before, neither in HCV infection nor in NK cells, and we found it to have dysregulated expression in both liver tissues and NK cells of HCV-infected patients compared to control. In addition to that, miR-182 was found to have a contradicting effect in both effector cell and its HCV-infected target cell regarding HCV replication.
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Hepacivirus/fisiologia , Hepatite C/imunologia , Hepatócitos/fisiologia , Células Matadoras Naturais/fisiologia , MicroRNAs/metabolismo , Linhagem Celular , Epigênese Genética , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Regulação da Expressão Gênica , Hepatite C/genética , Hepatócitos/virologia , Especificidade de Hospedeiro , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Células Matadoras Naturais/virologia , Ativação Linfocitária , MicroRNAs/genética , Subfamília C de Receptores Semelhantes a Lectina de Células NK/genética , Subfamília C de Receptores Semelhantes a Lectina de Células NK/metabolismo , Subfamília K de Receptores Semelhantes a Lectina de Células NK/genética , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Especificidade de Órgãos , Carga Viral , Replicação ViralRESUMO
This study aimed at identifying potential microRNAs that modulate hepatic lipid droplets (LD) through targeting the Tail interacting protein of 47 kDa (TIP47) in HCV infection. Bioinformatics analysis revealed that miR-148a and miR-30a potentially target TIP47. Expression profiling showed that both microRNAs were downregulated, while TIP47 was upregulated in liver biopsies of HCV-infected patients. Forcing the expression of both microRNAs in JFH-I infected, oleic acid-treated Huh7 cells, significantly suppressed TIP47 expression and reduced cellular LDs with marked decrease in viral RNA. This study shows that miR-148a and miR-30a, regulate TIP47 expression and LDs in HCV infected cells.
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Hepacivirus/genética , Gotículas Lipídicas/metabolismo , MicroRNAs/genética , Proteínas de Transporte Vesicular/genética , Regiões 3' não Traduzidas/genética , Adulto , Idoso , Sequência de Bases , Western Blotting , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/virologia , Linhagem Celular Tumoral , Epigênese Genética , Feminino , Regulação Neoplásica da Expressão Gênica , Hepacivirus/fisiologia , Interações Hospedeiro-Patógeno/genética , Humanos , Fígado/metabolismo , Fígado/patologia , Fígado/virologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/virologia , Masculino , Pessoa de Meia-Idade , Perilipina-3 , Interferência de RNA , RNA Viral/genética , RNA Viral/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência do Ácido Nucleico , Proteínas de Transporte Vesicular/metabolismoRESUMO
Hepatocellular carcinoma (HCC) is characterized by the aberrant expression of a number of genes that govern crucial signaling pathways. The insulin-like growth factor (IGF) axis is important in this context, and the precise regulation of expression of members of this axis is known to be lost in HCC. miR-155 is a well-established oncogene in numerous types of cancer. However, to the best of our knowledge, its effect on the regulation of the IGF axis has not been investigated to date. The present study aimed to elucidate the interactions between miR-155 and key components of the IGF axis, in addition to examining its effect on HCC development. Reverse transcription-quantitative polymerase chain reaction was used to measure the expression of miR-155 in HCC and cirrhotic tissues, in addition to HCC cell lines. Furthermore, the effect of the induction of miR-155 expression on the expression of three members of the IGF axis [IGF II, IGF type-1 receptor (IGF-1R) and IGF-binding protein 3 (IGFBP-3)], was analyzed. Finally, the effect of miR-155 on HCC cell proliferation, migration and clonogenicity was also examined. Quantification of the expression of miR-155 demonstrated that it is upregulated in HCC. Induction of the expression of miR-155 in HCC cell lines led to the upregulation of IGF-II and IGF-IR, and the downregulation of IGFBP-3. In addition, the proliferation, migration and clonogenicity of HCC was increased following induction of miR-155 expression. miR-155 is an oncomiR, which upregulates the oncogenes, IGF-II and IGF-IR, and downregulates the tumor suppressor, IGFBP-3, thereby resulting in increased HCC cell carcinogenicity. Therefore, miR-155 may be a therapeutic target in HCC.
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Gender discrepancies in immune response to HCV infections and during HCV therapy exist and previous findings including those from this research team indicate the female sex hormone, 17ß-estradiol (E2), to be one probable cause of such inconsistencies. Also, it was recently demonstrated that estrogen receptor modulator Tamoxifen (TAM) exerts an upmodulating/enhancing effect on the TLR7 and JAK-STAT pathways in PBMCs of premenopausal females infected with HCV. Pursuing this work, a discrepancy was noticed in the results from male patients, therefore this study aimed to determine whether the effects of TAM previously observed in the PBMCs of women would hold true in PBMCs from males infected with HCV. Isolated PBMCs were pooled and relative expression of the TLR7 was quantified using RTqPCR. Sets of PBMCs were treated with exogenous interferon alpha (IFNα) or the TLR7 ligand, Imiquimod; these stimulations were performed with and without E2 and TAM pretreatment and the relative gene expressions of TLR7 and MxA were measured. Pretreatment with E2 and IFNα downregulated TLR7 (**P = 0.0080) and TAM further decreased this expression significantly (*P = 0.0284). TAM pretreatment also caused a significant downregulation in MxA expression in Imiquimod-stimulated PBMCs (*P = 0.0218). In conclusion, TAM displays several paradoxical effects in PBMCs of males infected with HCV compared to those of females. Contrary to the previous study involving premenopausal females, in PBMCs of infected males TAM may decrease IFNα release as indicated by reduced MxA expression possibly via the suppression of TLR7 expression.
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Antagonistas de Estrogênios/farmacologia , Hepatite C Crônica/imunologia , Leucócitos Mononucleares/efeitos dos fármacos , Proteínas de Resistência a Myxovirus/biossíntese , Tamoxifeno/farmacologia , Receptor 7 Toll-Like/biossíntese , Células Cultivadas , Regulação para Baixo , Feminino , Perfilação da Expressão Gênica , Humanos , Leucócitos Mononucleares/imunologia , Masculino , Proteínas de Resistência a Myxovirus/antagonistas & inibidores , Reação em Cadeia da Polimerase em Tempo Real , Receptor 7 Toll-Like/antagonistas & inibidoresRESUMO
The biological differences between males and females advocate the ultimate need for gender-specific medicine. The variation in response to viral infection as well as therapy among different genders makes it very intriguing to reveal the responsible factors for causing this discrepancy. HCV is one of the most noxious infectious diseases, however the impact of gender on the response to HCV has received negligible attention in the literature. The controversial studies concerning the effect of gender on the outcome of interferon-based therapy urge a need to judge the gender discrepancy in host factors responsible for both interferon release and action. The main aim of this review is to disentangle the interplay between sex hormones and several viral and host factors responsible for viral clearance in an attempt to clarify the role of gender in modulating the response to HCV as well as interferon-based therapy.
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Hormônios Esteroides Gonadais/metabolismo , Disparidades nos Níveis de Saúde , Hepacivirus/metabolismo , Hepatite C/metabolismo , Animais , Antivirais/uso terapêutico , Feminino , Hepacivirus/imunologia , Hepacivirus/patogenicidade , Hepatite C/diagnóstico , Hepatite C/tratamento farmacológico , Hepatite C/imunologia , Humanos , Interferons/uso terapêutico , Masculino , Fatores Sexuais , Resultado do TratamentoRESUMO
BACKGROUND & AIMS: Hepatitis C virus (HCV) uses several host factors to infect and replicate in human hepatocytes. Cyclophilin A (CypA) is required for viral replication, and CypA inhibitors are in development. We investigated the effects of nonsynonymous single nucleotide polymorphisms (SNPs) in the region of peptidyl-prolyl isomerase A (PPIA) that encodes CypA on HCV infection and replication of human hepatocytes. METHODS: We used a combination of virologic, biochemical, and genetic approaches to investigate the effects of PPIA variants on HCV replication in cultured Huh-7.5 cells. We reduced levels of CypA in these cells using small hairpin RNAs (shRNAs). RESULTS: Using shRNAs, we showed that CypA was required for replication of HCV in Huh-7.5 cells and identified 3 SNPs in PPIA that protected cells from HCV entry or replication. Levels of HCV RNA were reduced 3-4 log in cells homozygous for the variant alleles; release of new particles was also reduced, but viral entry was not affected. The effects of the variant alleles were recessive and stronger for preventing replication of full-length HCV genomes than subgenomes. CypA inhibitors prevented replication of residual HCV in hepatocytes. The variants appeared to destabilize the CypA protein; the single amino acid changes led to rapid degradation of the protein. CONCLUSIONS: We identified variants in PPIA that destabilize its product, CypA, and prevent HCV infection and replication. These findings indicate mechanisms by which some cells might be resistant to HCV infection and that CypA is a good therapeutic target.
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Ciclofilina A/metabolismo , Hepacivirus/fisiologia , Hepatite C/virologia , Hepatócitos/virologia , Polimorfismo de Nucleotídeo Único , Internalização do Vírus , Replicação Viral , Biomarcadores/metabolismo , Linhagem Celular Tumoral , Ciclofilina A/genética , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Hepatócitos/metabolismo , Humanos , ImmunoblottingRESUMO
MicroRNAs (miRNAs) are short noncoding RNAs that act as post-transcriptional regulators. The low complementarity required between the sequences of a miRNA and its target mRNA enables a single miRNA to act on a large range of targets. Thus miRNAs have an intersecting complex effect that spans a multiplicity of pathways and processes. In this review, the different roles of a vital miRNA, miR-181a, in physiological and pathological developments are collated in an attempt to highlight the intersections of such processes and to show how the deregulation of miR-181a could in one context drive malignancy, whereas in another it can lead to autoimmunity. Such deregulation could be related to the faulty levels of one of its own targets, p53, which was recently reported to control an array of miRNAs, one of which is miR-181a. This sheds light on a hidden loop of chaos behind chronic diseases such as autoimmunity and cancer.
Assuntos
Regulação Neoplásica da Expressão Gênica , MicroRNAs/metabolismo , Neoplasias/metabolismo , Processamento Pós-Transcricional do RNA , RNA Mensageiro/metabolismo , RNA Neoplásico/metabolismo , Animais , Autoimunidade/genética , Regulação para Baixo/genética , Humanos , MicroRNAs/genética , Neoplasias/genética , RNA Mensageiro/genética , RNA Neoplásico/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Sex has been reported to influence the rates of viral clearance in hepatitis C virus (HCV)-infected patients. However, little is known regarding the influence of sex on the host genetic response to HCV, which is mediated by the expression of interferon (IFN)-stimulated genes (ISGs) after the activation of janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway by IFN. Thus, we investigated gender differences in MxA genetic profile, which is a downstream reliable marker for JAK/STAT pathway activation. In all, 40 untreated HCV-infected patients were subclassified into premenopausal, postmenopausal, and male patients. The peripheral blood mononuclear cells (PBMCs) from premenopausal women showed the highest MxA gene expression compared to both postmenopausal females and males before and after IFN stimulation. The prestimulation of PBMCs with 17beta-estradiol prior to IFN treatment resulted in a decrease of MxA expression in all groups of patients. That was confirmed by the reversal of this effect using estrogen antagonist ICI182/780. This study demonstrates for the first time the presence of gender variations in the genetic response to chronic HCV infection and to interferon treatment. It also clarifies that estrogen is not the key player in enhancing the JAK/STAT pathway.
Assuntos
Estrogênios/metabolismo , Proteínas de Ligação ao GTP/genética , Hepatite C Crônica/tratamento farmacológico , Hepatite C Crônica/genética , Interações Hospedeiro-Patógeno/genética , Interferons/metabolismo , Interferons/uso terapêutico , Estradiol/análogos & derivados , Estradiol/farmacologia , Antagonistas de Estrogênios/farmacologia , Feminino , Fulvestranto , Perfilação da Expressão Gênica , Hepacivirus/imunologia , Humanos , Janus Quinases/metabolismo , Leucócitos Mononucleares/metabolismo , Masculino , Proteínas de Resistência a Myxovirus , Pós-Menopausa/metabolismo , Pré-Menopausa/metabolismo , RNA/isolamento & purificação , Fatores Sexuais , Transdução de Sinais/fisiologia , Transcrição GênicaRESUMO
Hepatitis C virus (HCV) is characterized by a narrow host range and high interindividual variability in the clinical course of infection. Both of these traits are thought to be largely due to genetic variation between species and between individual hosts. The tight junction component occludin (OCLN) is essential for HCV entry into host cells, and the differences between human and murine OCLN are thought to account in part for the inability of HCV to infect mice and hence preclude their use as a convenient small-animal model. This study assesses the impact of genetic variation in OCLN on cell culture-grown HCV (HCVcc) using a newly generated and characterized OCLN(low) subclone of the Huh-7.5 cell line (Huh-7.5 subclone in which endogenous OCLN expression has been downregulated by a short hairpin RNA). We report the frequency of coding nonsynonymous single nucleotide polymorphisms, i.e., polymorphisms resulting in amino acid exchanges, present in the human population and determine their ability to function as HCV (co)receptors. Moreover, we show that murine OCLN can sustain HCVcc entry, albeit with about 5-fold reduced efficiency compared to that of human OCLN. This reduction in efficiency is due solely to two amino acid residues previously identified by others using an HCV pseudoparticle approach. Finally, we use the Huh-7.5/OCLN(low) cell line to show that HCV spread between neighboring cells is strictly dependent on OCLN.