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AIM: We have previously characterized oncogenic properties of IGF2BP1 in HCC, and its regulation by short noncoding RNAs (ncRNAs). Recent evidence suggests that IGF2BP1 itself may regulate long ncRNAs (lncRNAs). Therefore, this study aimed at exploring the interplay between IGF2BP1 and various upstream and downstream ncRNAs and its link to HCC pathogenesis. MATERIALS AND METHODS: Bioinformatic analysis was used to identify up- and downstream ncRNAs interacting with IGF2BP1. Huh-7 cells were transfected with siRNAs against IGF2BP1 and microRNA mimics. Relative gene expression was determined using RTqPCR and IGF2BP1 protein was quantified by western blot. Luciferase binding assay was used to explore the targeting of IGF2BP1 3'UTR. HCC tumorigenesis was measured by MTT assay, BrdU-incorporation assay, colony-forming assay, and scratch assay. KEY FINDINGS: Bioinformatic analysis identified three oncogenic lncRNAs - namely H19, FOXD2-AS1, and SNHG3 - potentially regulated by IGF2BP1. Knockdown of IGF2BP1 decreased the expression of all three oncogenic lncRNAs and inhibited malignant cell behaviors. miR-186 was revealed as a possible upstream regulator of IGF2BP1. miR-186 mimics decreased IGF2BP1 mRNA and protein levels. miR-186 was significantly lower while IGF2BP1 was elevated in cancerous tissues from ten HCC patients compared to five healthy controls. In addition, miR-186 mimics caused a downregulation of the oncogenic lncRNAs H19, SNHG3, and FOXD2-AS1 and a concomitant decrease in cell viability, proliferation, migration, and clonogenicity. SIGNIFICANCE: miR-186 may exert tumor suppressor effects in HCC by repressing oncogenic lncRNAs H19, SNHG3, and FOXD2-AS1 through its effect on IGF2BP1.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , MicroRNAs , RNA Longo não Codificante , Proteínas de Ligação a RNA , Humanos , Carcinogênese/genética , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas/patologia , MicroRNAs/genética , MicroRNAs/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
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
The role of noncoding transcripts in gene expression is nowadays acknowledged to keep various diseases at bay-despite being referred to as "junk" DNA several years ago. Believed to be at the heart of multiple regulatory pathways, microRNAs (miRNAs) are small noncoding RNAs (ncRNAs) involved in posttranscriptional gene regulation. Recently, the discovery of ncRNAs that compete for shared miRNA pools has dimmed the light on the solo performance of miRNAs in genomic regulation. Indeed, several studies describe RNAs such as long noncoding RNAs, mRNAs, circular RNAs, pseudogenes, and viral RNAs as competing endogenous RNAs (ceRNAs) that sequester miRNAs, allowing for de-repression of downstream miRNA targets. Such integration between coding and noncoding transcripts forms complex ceRNA networks that when dysregulated lead to several diseases such as hepatocellular carcinoma. Here, the authors review perturbed ceRNA networks in hepatocellular carcinoma, describe the role of each in tumorigenesis, and discuss their various clinical implications.
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Carcinoma Hepatocelular/genética , Regulação da Expressão Gênica , Neoplasias Hepáticas/genética , RNA/metabolismo , Carcinogênese/genética , Carcinoma Hepatocelular/metabolismo , Humanos , Neoplasias Hepáticas/metabolismo , RNA/genética , Processamento Pós-Transcricional do RNARESUMO
Epigallocatechin gallate (EGCG) is the most abundant component in green tea extract, that has powerful antioxidant and antiviral effects. It has been previously reported to inhibit HCV entry via several mechanisms. Hence, this study aimed at further investigating the potential impact of EGCG on HCV entry through regulation of the expression of tetraspanin receptor CD81 by the novel predicted miR-548m. Liver biopsies were obtained from 29 HCV patients and 10 healthy controls for expression profiling. Huh7 cells were stimulated with EGCG and subsequently miR-548m expression was assessed. Naïve, HCV- ED43/JFH-1 and HCV-JFH-1 infected Huh7 cells were transfected by miR-548m mimics and inhibitors. Consequently, CD81 protein and mRNA levels were assessed using flow cytometry and qRT-PCR, respectively. Additionally, these cells were used to investigate HCV permissiveness into Huh7 cells using qRT-PCR for viral quantification. Direct binding confirmation of miR-548m to CD81 was done using luciferase reporter assay. In-silico analysis revealed miR-548m to have two potential binding sites in the 3'UTR of CD81 mRNA. EGCG boosted miR-548m expression in Huh7 cells. Additionally, miR-548m caused a downregulation of CD81 protein and mRNA levels as well as reduction in HCV infectivity of Huh7 cells. Luciferase binding assay confirmed the binding of miR-548m to CD81 mRNA at the two predicted binding sites. Intriguingly, miR-548m expression was not detected in healthy liver biopsies but was found in liver biopsies of HCV patients. This study shows that EGCG might act as an anti-HCV agent that reduces cellular infectivity via enhancing miR-548m expression and repressing CD81 receptor.
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Catequina/análogos & derivados , Hepacivirus/fisiologia , Hepatite C/patologia , MicroRNAs/genética , Tetraspanina 28/genética , Regiões 3' não Traduzidas , Estudos de Casos e Controles , Catequina/farmacologia , Linhagem Celular , Simulação por Computador , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Hepacivirus/efeitos dos fármacos , Hepatite C/genética , Hepatite C/metabolismo , Hepatite C/virologia , Humanos , Tetraspanina 28/metabolismo , Internalização do Vírus/efeitos dos fármacosRESUMO
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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The author would like to correct the errors in the online published article.
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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: Because there is a global shortage of intravenous immunoglobulin, there is a need for new products to fill the gap. STUDY DESIGN AND METHODS: This was a multicenter, open-label study investigating the safety and efficacy of a newly developed mini-pool intravenous immunoglobulin G for children with immune thrombocytopenia. Seventy-two patients ages 1 to 18 years with newly diagnosed (<1 month) immune thrombocytopenia who had platelet counts from 5 to 20 × 109 /L with no serious bleeding were recruited from four centers in Egypt. Eligible patients were randomized into three groups 1:1:1. Group A (n = 24) received blood group-specific mini-pool intravenous immunoglobulin in a dose equivalent to immunoglobulin 1 g/kg over 6 to 8 hours, Group B (n = 24) received standard intravenous immunoglobulin (approximately 1g/kg) as a single dose, and Group C (n = 24) did not receive any platelet-enhancing therapy. Parents signed informed consent. RESULTS: Of the patients who received mini-pool intravenous immunoglobulin, 14 achieved a complete response (CR) (58.8%), and four had a response (16.6%). Of the patients who received intravenous immunoglobulin G, 16 achieved a complete response (66.6%), and four had a response (16.6%). In Group C, eight patients achieved a complete response (33.3%), and four had a response (16.6%). The median time to response was 8, 9, and 21 days in Group A, B, and C, respectively, which was significantly higher in Group C than Groups A and B (p < 0.001). Patients in Groups A and B reported 16 adverse drug reactions. CONCLUSION: Mini-pool intravenous immunoglobulin G was well tolerated, presented no safety issues, and was effective in the treatment of immune thrombocytopenia, with efficacy comparable to that of the standard intravenous immunoglobulin G group, and it was significantly more effective than no treatment.
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Imunoglobulinas Intravenosas/administração & dosagem , Púrpura Trombocitopênica Idiopática/tratamento farmacológico , Adolescente , Criança , Pré-Escolar , Egito , Feminino , Humanos , Imunoglobulinas Intravenosas/uso terapêutico , Lactente , Masculino , Resultado do TratamentoRESUMO
Occludin (OCLN) is an essential factor for HCV entry through interacting with other surface receptors. The aim of this study was to investigate the epigenetic regulation of Occludin expression and to study its impact on viral infectivity. microRNAs expression was assessed using qRT-PCR, while OCLN protein expression was investigated by indirect immunofluorescence and Western blotting. Viral infectivity was assessed by measuring viral-load using qRT-PCR. In silico analysis predicted that miR-200c targeted the OCLN 3'UTR, which was further experimentally confirmed. miR-122 was previously validated to target the 3'UTR of OCLN and was used as a control. We report a significant down-regulation of miR-200c in liver tissues of HCV-infected patients. Ectopic expression of both miR-122 and miR-200c in Huh7 cells reduced OCLN mRNA and protein levels. Viral infectivity was significantly reduced by miR-200c but enhanced by miR-122. This work sheds light on miR-200c as a novel regulator of HCV infectivity through the regulation of OCLN.
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Regulação da Expressão Gênica/efeitos dos fármacos , Hepacivirus/fisiologia , Hepatócitos/efeitos dos fármacos , MicroRNAs/farmacologia , Ocludina/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Linhagem Celular Tumoral , Hepatócitos/metabolismo , Hepatócitos/virologia , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Ocludina/genética , RNA Viral , Replicação ViralRESUMO
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
Aims: To examine the regulation of SREBP-1c and CAV1 by microRNA-29a (miR-29a) in cells infected with hepatitis C virus (HCV) in an attempt to control HCV-induced non-alcoholic fatty liver disease. Methods: In order to examine the manipulation of SREBP-1c and CAV1 by miR-29a, oleic acid (OA)-treated JFH-I-infected Huh-7 cells were used. OA was added 24 h post-transfection and gene expression was investigated by qRT-PCR at 48 h post treatment. The functional impact of the observed alteration in SREBP-1c and CAV1 expression was analyzed by examining lipid droplet (LD) and triglyceride (TG) content at 72 h post-OA treatment using light microscopy and spectrophotometry, respectively. Viral load was quantified by qRT-PCR at 72 h post-transfection. Results: OA treatment induced the expression of miR-29a and SREBP-1c, as compared to untreated cells. Forced miR-29a expression led to a significant up-regulation of SREBP-1c as well as CAV1 compared to mock untransfected cells. Ectopic expression of miR-29a resulted in a marked increase in LDs and their respective TGs, while miR-29a antagomirs decreased both the LD and TG content compared to mock untransfected cells. Moreover, forcing the expression of miR-29a in JFH-1 HCV-infected Huh-7 cells resulted in 53% reduction in viral titers compared to mock untransfected Huh-7 cells. Conclusion: Inducing miR-29a expression significantly induces SREBP-1c and CAV1 expression, thereby increasing LDs as well as their respective TGs. Nonetheless, forcing the expression of miR-29a resulted in reduction of HCV RNA levels in Huh-7 cells.
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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
OBJECTIVES: Natural killer cells are immune safeguards against HCV infection. PU.1 is a pivotal transcription factor in the development of NK cells. This study aimed at studying the regulatory effect of miRNAs on both development and function of NK cells isolated from HCV patients. METHODS: NK cells were isolated from 17 chronic HCV patients and 12 healthy controls; after which miRNA and mRNA were quantified using qRT-PCR. Manipulating miRNA expression using mimics and antagomirs, was performed followed by investigating downstream targets as well as viral abundance. RESULTS: PU.1 expression levels were upregulated in NK cells of HCV patients. In silico analysis revealed PU.1 to be a potential downstream target of miR-29a(∗), where miR-29a(∗) overexpression in NK cells caused a significant downregulation in PU.1 mRNA. Forcing miR-29a(∗) caused a downregulation of the cytotoxicity determinant NK activating receptor (NKG2D) via upregulation of miR-155. Moreover, perforin-1 mRNA was found to be downregulated upon forcing the expression of miR-29a(∗) in NK cells of HCV patients. This decrease in NK cytolytic function was accompanied by an 80% viral load increase in cocultured HCVcc cell models. CONCLUSIONS: This study showed that HCV infection might abrogate NK cytotoxic potential through altering PU.1, NKG2D receptor and perforin molecules.
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Citotoxicidade Imunológica/genética , Hepacivirus/imunologia , Hepatite C/genética , Hepatite C/imunologia , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , MicroRNAs/genética , Proteínas Proto-Oncogênicas/genética , Transativadores/genética , Regiões 3' não Traduzidas , Estudos de Casos e Controles , Linhagem Celular , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Hepatite C Crônica/genética , Hepatite C Crônica/imunologia , Humanos , Subfamília K de Receptores Semelhantes a Lectina de Células NK/genética , Interferência de RNA , RNA Mensageiro/genéticaRESUMO
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
Hepatits C virus (HCV) genotype 4 (GT4) shows low treatment response rates and discrepancies when compared to other genotypes. However, the reason underlying these discrepancies remains unclear due to the limited number of studies on GT4. microRNA-155 (miR-155) is a noteworthy example of a discrepancy in GT4, as it was found to be upregulated in genotypes 1, 2 and 3 HCV infection, but downregulated in GT4-HCV-infected peripheral blood mononuclear cells (PBMCs). The present study aimed to investigate the expression of miR-155 in PBMCs, serum and liver tissues of GT4-HCV-infected patients. miR-155 expression was assessed using reverse transcription-quantitative polymerase chain reaction in GT4-HCV-infected PBMCs, serum and liver tissues, as well as GT2- and GT4-infected Huh7 cells, and compared to the healthy controls. There was no difference in miR-155 expression observed between naïve GT4-HCV patients and healthy controls in the PBMCs and serum. In HCV-infected liver tissues, however, a significant downregulation was observed. The unique miR-155 expression pattern during GT4 infection was confirmed in the infected Huh7 cell lines when compared to GT2 infection. Clinical data showed a positive correlation between liver transaminases and serum miR-155 expression. In addition, serum miR-155 expression was significantly lower in naïve non-responders (NRs) than naïve sustained virological responders (SVRs), and in post-treatment NRs compared to post-treatment SVRs. In conclusion, miR-155 was not only proven to be a genotype-specific microRNA that is not induced during GT4-HCV infection, but also a good prognostic factor and predictor of response to treatment enabling a non-invasive differentiation between NRs and SVRs during GT4-HCV infection.
RESUMO
OBJECTIVE: The tetraspanin CD81 is one of the main receptors involved in hepatitis C virus entry. Herein, we aimed to explore the role of microRNAs in regulating CD81 receptor expression and function. PATIENTS AND METHODS: Bioinformatics analysis was carried out to select potential mircroRNAs that binds CD81 3'untranslated region. Liver biopsies taken from 28 HCV genotype- 4 patients and 10 healthy donors were screened. Naïve, JFH1 and ED43/JFH1- infected- Huh7 cells were transfected with mimics and inhibitors followed by analyzing CD81 protein and mRNA expression. This was done using flow cytometry and Q-RT PCR, respectively. HCV entry into Huh7 cells was investigated post-transfection. Binding confirmation was done using luciferase reporter vector harboring wild/mutant target sites of microRNA. The impact of Epigallocatechin-gallate on modulating microRNA/CD81 expression was assessed. RESULTS: Bioinformatics revealed that CD81 is a potential down-stream target for miR-194. A significant inverse correlation was found between miR-194 and CD81 expression in liver biopsies of HCV patients. Forcing the expression of miR-194 showed a down-regulation of CD81 protein, mRNA expression and significantly abrogated the HCV infectivity of Huh7 cells. Stimulation with EGCG enhanced mir-194 expression and down-regulated CD81 expression. CONCLUSION: This study showed that mir-194 hinders HCV entry through targeting CD81 receptors.
Assuntos
Hepacivirus/metabolismo , MicroRNAs/metabolismo , Tetraspanina 28/metabolismo , Catequina/análogos & derivados , Catequina/farmacologia , Biologia Computacional , Citometria de Fluxo , Hepacivirus/genética , Hepatite C , Humanos , Fígado/patologia , Transplante de Fígado , MicroRNAs/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para CimaRESUMO
microRNA (miRNA) expression in organs does not always represent their quantity in serum. A disparity in the expression of miR-181a has been reported in the tissues and serum of hepatocellular carcinoma (HCC) patients. Since hepatitis C virus (HCV) is a major cause of HCC and miR-181a has never been studied in HCV, the present study aimed to investigate the miR-181a expression profile in genotype 4 (GT4)-HCV patients to evaluate whether this pattern is also apparent in HCV. RNA was extracted from liver tissues, peripheral mononuclear cells (PBMCs) and serum samples from GT4-HCV-infected patients and healthy donors to evaluate the relative miR-181a expression using quantitative reverse transcription-polymerase chain reaction. miR-181a was significantly higher in the serum of naïve patients compared to controls, and an inverse correlation with the viral load and liver enzymes was apparent. By contrast, no difference in miR-181a expression was observed in the liver tissues and PBMCs of patients compared to controls. This expression observed in HCV is conflicting to that previously reported in HCC. The study also demonstrates a significant upregulation of miR-181a post-interferon/ribavirin treatment in the serum of sustained virological responders (SVRs) compared to non-responders and treatment-naïve SVRs. In conclusion, miR-181a may be considered to be a possible prognostic marker in GT4-HCV infection.