scholarly journals The Herbal DrugMelampyrum pratenseL. (Koch): Isolation and Identification of Its Bioactive Compounds Targeting Mediators of Inflammation

2013 ◽  
Vol 2013 ◽  
pp. 1-10 ◽  
Author(s):  
S. Vogl ◽  
A. G. Atanasov ◽  
M. Binder ◽  
M. Bulusu ◽  
M. Zehl ◽  
...  
Keyword(s):  
Target Genes ◽  
Luciferase Reporter ◽  
Peroxisome Proliferator ◽  
Luciferase Reporter Gene ◽  
Isolation And Identification ◽  
Protein Levels ◽  
Proinflammatory Mediators ◽  
Mediators Of Inflammation ◽  
Anti Inflammatory

Melampyrum pratenseL. (Koch) is used in traditional Austrian medicine for the treatment of different inflammation-related conditions. In this work, we show that the extracts ofM. pratensestimulated peroxisome proliferator-activated receptors- (PPARs-)αand -γthat are well recognized for their anti-inflammatory activities. Furthermore, the extract inhibited the activation of the proinflammatory transcription factor NF-κB and induction of its target genes interleukin-8 (IL-8) and E-selectinin vitro. Bioassay-guided fractionation identified several active flavonoids and iridoids including melampyroside and mussaenoside and the phenolic compound lunularin that were identified in this species for the first time. The flavonoids apigenin and luteolin were distinguished as the main components accountable for the anti-inflammatory properties. Apigenin and luteolin effectively inhibited tumor necrosis factorα(TNF-α)-induced NF-κB-mediated transactivation of a luciferase reporter gene. Furthermore, the two compounds dose-dependently reduced IL-8 and E-selectin protein expression after stimulation with lipopolysaccharide (LPS) or TNF-αin endothelial cells (ECs). The iridoids melampyroside and mussaenoside prevented the elevation of E-selectin in LPS-stimulated ECs. Lunularin was found to reduce the protein levels of the proinflammatory mediators E-selectin and IL-8 in ECs in response to LPS. These data validate the ethnomedical use ofM. pratensefor the treatment of inflammatory conditions and point to the constituents accountable for its anti-inflammatory activity.

scholarly journals LncRNA HCG11 inhibits adipocyte differentiation in human adipose-derived mesenchymal stem cells by sponging miR-204-5p to upregulate SIRT1

2020 ◽  
Author(s):  
Dandan Li ◽  
Yang Liu ◽  
Wei Gao ◽  
Jiakai Han ◽  
Rongrong Yuan ◽  
...  
Keyword(s):  
Target Genes ◽  
Adipocyte Differentiation ◽  
Inflammatory Responses ◽  
Inflammatory Factors ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Protein Levels ◽  
Suppressor Effect ◽  
Adipogenic Marker

Abstract Background: LncRNAs have been discovered to play a key role in adipogenesis, vital in regulating adipose developmen t. Numerous evidences show that adipogenesis is the leading cause of obesity, while the role of lncRNA HLA complex group 11 ( HCG11 ) in adipocyte differentiation has not been elucidated. Methods: hAdMSCs were used to establish a model of cell differentiation in vitro . Expression of lncRNA HCG11 was detected by RT-qPCR analysis. Transfection of the shRNA targeting HCG11 or pcDNA-HCG11 into hAdMSCs was also assessed. The adipogenic marker proteins C/EBPα, FABP4 and PPARγ2 and important inflammatory factors IL-6 and TNF-α were detected by Western blot. Bioinformatics analysis predicted the target genes of HCG11 and mir-204-5p, which was confirmed by luciferase reporter gene analysis and RNA pull-down analysis. Results: Here we show that lncRNA HCG11 was decreased as the degree of adipogenesis. The expression of C/EBPα, FABP4 and PPARγ2 were significantly downregulated transfected with pcDNA-HCG11 in hAdMSCs at different stages, while knockdown lncRNA HCG11 can promote adipocyte differentiation. In addition, miR-204-5p was a potential target gene of HCG11 and SIRT1 could directly target miR-204-5p. Overexpression of SIRT1 or transfected with agonists of SIRT1 (Res) significantly inhibited adipogenic marker protein levels and inflammatory responses, and the proliferation of hAdMSCs were also inhibited. pcDNA-HCG11 and miR-204-5p mimic were co-transfected into hAdMSCs, we found that miR-204-5p mimic reversed the suppressor effect of pcDNA-HCG11. Conclusion: Our findings showed that HCG11 negatively regulated cell proliferation, inflammatory responses and adipogenesis by miR-204-5p/SIRT1 axis. And our findings may provide a new target for the study of adipogenesis in hAdMSCs and obesity.

scholarly journals Peroxisome-Proliferator Receptor γ Represses Hepatic Sex Hormone-Binding Globulin Expression

Endocrinology ◽  
2009 ◽  
Vol 150 (5) ◽  
pp. 2183-2189 ◽  
Author(s):  
David M. Selva ◽  
Geoffrey L. Hammond
Keyword(s):  
Hepg2 Cells ◽  
Promoter Activity ◽  
Binding Activity ◽  
Sex Hormone Binding Globulin ◽  
Luciferase Reporter ◽  
Peroxisome Proliferator ◽  
Luciferase Reporter Gene ◽  
Pparγ Agonist ◽  
Gene Assay

Plasma SHBG production by the liver is influenced by its metabolic state, and hepatocyte nuclear factor-4α regulates SHBG expression in response to changes in lipogenesis. Peroxisome-proliferator receptors (PPARs) also regulate glucose homeostasis and fatty acid metabolism. The human SHBG promoter contains a PPAR-response element (PPAR-RE), and plasma SHBG levels increase in polycystic ovarian syndrome patients treated with the PPARγ agonist, rosiglitazone. In addition, plasma SHBG levels are associated with a genetic polymorphism in the PPARγ-2 coding sequence that alters its transcriptional activity. Therefore, we set out to determine whether PPARγ influences hepatic production of SHBG by using human HepG2 hepatoblastoma cells as an in vitro model. Surprisingly, treatment of HepG2 cells with rosiglitazone reduced SHBG production and SHBG promoter activity (as assessed in a luciferase reporter gene assay) by 20–25%, whereas the PPARγ antagonist, GW9662, increased both by 2- to 3-fold. The effects of PPARγ agonists and antagonists on SHBG promoter activity were substantially diminished when the PPAR-RE in the SHBG promoter was mutated. A PPARγ small interfering RNA also increased SHBG production by HepG2 cells as well as SHBG promoter activity, and the latter was accentuated by cotreatment with GW9662. Importantly, overexpression of a PPARγ-2 Pro12 variant in HepG2 cells was more effective at reducing SHBG promoter activity, when compared with PPARγ-2 Ala12, consistent with its superior PPAR-RE binding activity. We conclude that PPARγ represses human SHBG expression in liver cells, and that differences in PPARγ levels and activity contribute directly to variations in plasma SHBG levels.

scholarly journals Curcumin induces paraoxonase 1 in cultured hepatocytes in vitro but not in mouse liver in vivo

2010 ◽  
Vol 105 (2) ◽  
pp. 167-170 ◽  
Author(s):  
Charlotte Schrader ◽  
Christina Schiborr ◽  
Jan Frank ◽  
Gerald Rimbach
Keyword(s):  
Cultured Cells ◽  
Paraoxonase 1 ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Protein Levels ◽  
Huh7 Cells ◽  
Gene Assay ◽  
B6c3f1 Mice

Paraoxonase 1 (PON1) is an enzyme that is mainly synthesised in the liver and protects LDL from oxidation, thereby exhibiting antiatherogenic properties. Using a luciferase reporter gene assay, we tested curcumin for its ability to induce PON1 in Huh7 hepatocytes in culture. Curcumin ( ≥ 10 μmol/l) dose-dependently induced PON1 transactivation in Huh7 cells. However, dietary supplementation of female B6C3F1 mice with curcumin (500 mg/kg diet) for 2 weeks did not increase the hepatic PON1 mRNA and protein levels. No curcumin was detectable in the plasma of the 12 h fasted mice. In conclusion, curcumin may be a potent PON1 inducer in cultured cells in vitro, but not in the liver of curcumin-fed mice because of its low concentrations in vivo.

scholarly journals Plasma Exosomal Mir-423-5p Is Involved in the Occurrence and Development of Bicuspid Aortopathy via TGF-β/SMAD2 Pathway

2021 ◽  
Vol 12 ◽  
Author(s):  
Hongqiang Zhang ◽  
Dingqian Liu ◽  
Shichao Zhu ◽  
Fanshun Wang ◽  
Xiaoning Sun ◽  
...  
Keyword(s):  
Aortic Valve ◽  
Target Genes ◽  
Gene Prediction ◽  
Luciferase Reporter ◽  
Small Rna Sequencing ◽  
Protein Levels ◽  
Increased Risk ◽  
Exosomal Mirnas ◽  
Low Cardiovascular Risk

Objectives: Patients with bicuspid aortic valve (BAV) are at increased risk for ascending aortic dilation (AAD). Our study was aimed at systemically analyzing the expression profile and mechanism of circulating plasma exosomal microRNAs (miRNAs) related to BAV and AAD.Methods: We isolated plasma exosomes from BAV patients (n=19), BAV patients with AAD (BAVAD, n=26), and healthy tricuspid aortic valve individuals with low cardiovascular risk (TAVnon, n=16). We applied a small RNA sequencing approach to identify the specific plasma exosomal miRNAs associated with BAV (n=8) and BAVAD (n=10) patients compared with healthy TAVnon (n=6) individuals. The candidate differentially expressed (DE) miRNAs were selected and validated by RT-qPCR in the remaining samples. GO and KEGG pathway enrichment analyses were performed to illustrate the functions of target genes. Western blot analysis and luciferase reporter assay were conducted in human aortic vascular smooth muscle cells (VSMCs) to verify the results of target gene prediction in vitro.Results: The expression levels of three up-regulated (miR-151a-3p, miR-423-5p, and miR-361-3p) and two down-regulated (miR-16-5p and miR-15a-5p) exosomal miRNAs were significantly altered in BAV disease. Additionally, miR-423-5p could be functionally involved in the occurrence and development of BAV and its complication BAVAD by regulating TGF-β signaling. miR-423-5p could target to SMAD2 and decreased the protein levels of SMAD2 and P-SMAD2.Conclusion: Plasma exosomal miR-423-5p regulated TGF-β signaling by targeting SMAD2, thus exerting functions in the occurrence and development of BAV disease and its complication bicuspid aortopathy.

scholarly journals LncRNA Malat1 regulates iPSC-derived β-cell differentiation by targeting the miR-15b-5p/Ihh axis

2021 ◽  
Author(s):  
Yao Wang ◽  
Xinxing Lin ◽  
Jin Lv ◽  
Jiachen Zhu ◽  
Haowen Fan ◽  
...  
Keyword(s):  
Stem Cell ◽  
Target Genes ◽  
Induced Pluripotent Stem Cell ◽  
Luciferase Reporter ◽  
Β Cells ◽  
Pancreatic Β Cells ◽  
Protein Levels ◽  
Lncrna Malat1

Abstract Background: Differentiation of induced pluripotent stem cell (iPSC)-derived β-like cells is a novel strategy for treatment of type 1 diabetes. Elucidation of the regulatory mechanisms of long noncoding RNAs (lncRNAs) in β-like cells derived from iPSCs is important for understanding the development of the pancreas and pancreatic β-cells and may improve the quality of β-like cells for stem cell therapy.Methods: β-like cells were derived from iPSCs in a three-step protocol. RNA sequencing and bioinformatics analysis were carried out to screen the differentially expressed lncRNAs and identify the putative target genes separately. LncRNA Malat1 was chosen for further research. Series of loss and gain of functions experiments were performed to study the biological function of this lncRNA. Quantitative real-time PCR (qRT-PCR), Western blot analysis and immunofluorescence (IF) staining were carried out to separately detect the functions of pancreatic β-cells at the mRNA and protein levels. Cytoplasmic and nuclear RNA fractionation and fluorescence in situ hybridization (FISH) were used to determine the subcellar location of lncRNA Malat1 in β-like cells. Flow cytometry and enzyme-linked immunosorbent assays (ELISAs) were performed to examine the differentiation and insulin secretion of β-like cells after stimulation with different glucose concentrations. Structural interactions between lncRNA Malat1 and miR-15b-5p and between miR-15b-5p and Ihh were detected by dual luciferase reporter assays (LRAs).Results: We found that the expression of lncRNA Malat1 declined during differentiation, and overexpression of this lncRNA notably impaired the differentiation and maturation of β-like cells derived from iPSCs in vitro and in vivo. Localized to the cytoplasm, lncRNA Malat1 could function as a competing endogenous RNA (ceRNA) of miR-15b-5p to regulate the expression of Ihh according to bioinformatics prediction, mechanistic analysis and downstream experiments.Conclusion: This study established an unreported regulatory network of lncRNA Malat1 and the miR-15b-5p/Ihh axis during the differentiation of iPSCs into β-like cells. In addition to acting as an oncogene promoting tumorigenesis, lncRNA Malat1 may be an effective and novel target for treatment of diabetes in the future.

Exercise-associated generation of PPARγ ligands activates PPARγ signaling events and upregulates genes related to lipid metabolism

2012 ◽  
Vol 112 (5) ◽  
pp. 806-815 ◽  
Author(s):  
A. W. Thomas ◽  
N. A. Davies ◽  
H. Moir ◽  
L. Watkeys ◽  
J. S. Ruffino ◽  
...  
Keyword(s):  
Training Program ◽  
Target Genes ◽  
Exercise Program ◽  
Exercise Bout ◽  
Luciferase Reporter ◽  
Peroxisome Proliferator ◽  
Luciferase Reporter Gene ◽  
Peroxisome Proliferator Activated Receptor ◽  
Beneficial Effects ◽  
Exercise Induced

The aim of the present study was to test the hypotheses that exercise is associated with generation of peroxisome proliferator-activated receptor-γ (PPARγ) ligands in the plasma and that this may activate PPARγ signaling within circulating monocytes, thus providing a mechanism to underpin the exercise-induced antiatherogenic benefits observed in previous studies. A cohort of healthy individuals undertook an 8-wk exercise-training program; samples were obtained before (Pre) and after (Post) standardized submaximal exercise bouts (45 min of cycling at 70% of maximal O2 uptake, determined at baseline) at weeks 0, 4, and 8. Addition of plasma samples to PPARγ response element (PPRE)-luciferase reporter gene assays showed increased PPARγ activity following standardized exercise bouts (Post/Pre = 1.23 ± 0.10 at week 0, P < 0.05), suggesting that PPARγ ligands were generated during exercise. However, increases in PPARγ/PPRE-luciferase activity in response to the same standardized exercise bout were blunted during the training program (Post/Pre = 1.18 ± 0.14 and 1.10 ± 0.10 at weeks 4 and 8, respectively, P > 0.05 for both), suggesting that the relative intensity of the exercise may affect PPARγ ligand generation. In untrained individuals, specific transient increases in monocyte expression of PPARγ-regulated genes were observed within 1.5–3 h of exercise (1.7 ± 0.4, 2.6 ± 0.4, and 1.4 ± 0.1 fold for CD36, liver X receptor-α, and ATP-binding cassette subfamily A member 1, respectively, P < 0.05), with expression returning to basal levels within 24 h. In contrast, by the end of the exercise program, expression at the protein level of PPARγ target genes had undergone sustained increases that were not associated with an individual exercise bout (e.g., week 8 Pre/ week 0 Pre = 2.79 ± 0.61 for CD36, P < 0.05). Exercise is known to upregulate PPARγ-controlled genes to induce beneficial effects in skeletal muscle (e.g., mitochondrial biogenesis and aerobic respiration). We suggest that parallel exercise-induced benefits may occur in monocytes, as monocyte PPARγ activation has been linked to beneficial antidiabetic effects (e.g., exercise-induced upregulation of monocytic PPARγ-controlled genes is associated with reverse cholesterol transport and anti-inflammatory effects). Thus, exercise-triggered monocyte PPARγ activation may constitute an additional rationale for prescribing exercise to type 2 diabetes patients.

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

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

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

AAnti-Inflammatory Effects of Plasma Circulating Exosomes Obtained from Normal-Weight and Obese Subjects on Hepatocytes

2020 ◽  
Vol 20 ◽  
Author(s):  
Reza Afrisham ◽  
Sahar Sadegh-Nejadi ◽  
Reza Meshkani ◽  
Solaleh Emamgholipour ◽  
Molood Bagherieh ◽  
...  
Keyword(s):  
Inflammatory Cytokines ◽  
Hepg2 Cells ◽  
Human Liver ◽  
Liver Cells ◽  
Normal Weight ◽  
Protein Levels ◽  
Human Liver Cells ◽  
Tnf Α ◽  
Anti Inflammatory

Introduction: Obesity is a disorder with low-grade chronic inflammation that plays a key role in the hepatic inflammation and steatosis. Moreover, there are studies to support the role of exosomes in the cellular communications, the regulation of metabolic homeostasis and immunomodulatory activity. Accordingly, we aimed to evaluate the influence of plasma circulating exosomes derived from females with normal-weight and obesity on the secretion of inflammatory cytokines in human liver cells. Methods: Plasma circulating exosomes were isolated from four normal (N-Exo) and four obese (O-Exo) women. The exosomes were characterized and approved for CD63 expression (common exosomal protein marker) and morphology/size using the western blot and TEM methods, respectively. The exosomes were used for stimulation of HepG2 cells in vitro. After 24 h incubation, the protein levels of TNF-α,IL-6, and IL-1β were measured in the culture supernatant of HepG2 cells using the ELISA kit. Results: The protein levels of IL-6 and TNF-α in the cells treated with O-Exo and N-Exo reduced significantly in comparison with control group (P=0.039 and P<0.001 respectively), while significance differences were not found between normal and obese groups (P=0.808, and P=0.978 respectively). However, no significant differences were found between three groups in term of IL-1β levels (P=0.069). Based on the correlation analysis, the protein levels of IL-6 were positively correlated with TNF-α (r 0.978, P<0.001). Conclusion: These findings suggest that plasma circulating exosomes have probably anti-inflammatory properties independently from body mass index and may decrease the secretion of inflammatory cytokines in liver. However, further investigations in vitro and in vivo are needed to address the anti-inflammatory function of N-Exo and O-Exo in human liver cells and/or other cells.

scholarly journals Long noncoding RNA SNHG14 promotes hepatocellular carcinoma progression by regulating miR-876-5p/SSR2 axis

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Zhibin Liao ◽  
Hongwei Zhang ◽  
Chen Su ◽  
Furong Liu ◽  
Yachong Liu ◽  
...  
Keyword(s):  
Noncoding Rna ◽  
Animal Experiments ◽  
Luciferase Reporter ◽  
Western Blot Assay ◽  
Downstream Target ◽  
Protein Levels ◽  
Elevated Expression ◽  
Rna Immunoprecipitation

Abstract Background Aberrant expressions of long noncoding RNAs (lncRNAs) have been demonstrated to be related to the progress of HCC. The mechanisms that SNHG14 has participated in the development of HCC are obscure. Methods Quantitative real-time PCR (qRT-PCR) was used to measure the lncRNA, microRNA and mRNA expression level. Cell migration, invasion and proliferation ability were evaluated by transwell and CCK8 assays. The ceRNA regulatory mechanism of SNHG14 was evaluated by RNA immunoprecipitation (RIP) and dual luciferase reporter assay. Tumorigenesis mouse model was used to explore the roles of miR-876-5p in vivo. The protein levels of SSR2 were measured by western blot assay. Results In this study, we demonstrated that SNHG14 was highly expressed in HCC tissues, meanwhile, the elevated expression of SNHG14 predicted poor prognosis in patients with HCC. SNHG14 promoted proliferation and metastasis of HCC cells. We further revealed that SNHG14 functioned as a competing endogenous RNA (ceRNA) for miR-876-5p and that SSR2 was a downstream target of miR-876-5p in HCC. Transwell, CCK8 and animal experiments exhibited miR-876-5p inhibited HCC progression in vitro and in vivo. By conducting rescue experiments, we found the overexpression of SSR2 or knocking down the level of miR-876-5p could reverse the suppressive roles of SNHG14 depletion in HCC. Conclusion SNHG14 promotes HCC progress by acting as a sponge of miR-876-5p to regulate the expression of SSR2 in HCC.

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

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

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

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