scholarly journals microRNA-136-5p from bone marrow mesenchymal stem cell-derived exosomes facilitates fracture healing by targeting LRP4 to activate the Wnt/β-catenin pathway

2021 ◽  
Vol 10 (12) ◽  
pp. 744-758
Author(s):  
Haichi Yu ◽  
Jun Zhang ◽  
Xiaoning Liu ◽  
Yingzhi Li
Keyword(s):  
Bone Marrow ◽  
Fracture Healing ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Luciferase Reporter ◽  
Tartrate Resistant Acid Phosphatase ◽  
Osteoblast Proliferation ◽  
Luciferase Reporter Gene ◽  
Gene Assay ◽  
Proliferation And Differentiation

Aims Exosomes derived from bone marrow mesenchymal stem cells (BMSCs) have been reported to be a promising cellular therapeutic approach for various human diseases. The current study aimed to investigate the mechanism of BMSC-derived exosomes carrying microRNA (miR)-136-5p in fracture healing. Methods A mouse fracture model was initially established by surgical means. Exosomes were isolated from BMSCs from mice. The endocytosis of the mouse osteoblast MC3T3-E1 cell line was analyzed. CCK-8 and disodium phenyl phosphate microplate methods were employed to detect cell proliferation and alkaline phosphatase (ALP) activity, respectively. The binding of miR-136-5p to low-density lipoprotein receptor related protein 4 (LRP4) was analyzed by dual luciferase reporter gene assay. HE staining, tartrate-resistant acid phosphatase (TRAP) staining, and immunohistochemistry were performed to evaluate the healing of the bone tissue ends, the positive number of osteoclasts, and the positive expression of β-catenin protein, respectively. Results miR-136-5p promoted fracture healing and osteoblast proliferation and differentiation. BMSC-derived exosomes exhibited an enriched miR-136-5p level, and were internalized by MC3T3-E1 cells. LRP4 was identified as a downstream target gene of miR-136-5p. Moreover, miR-136-5p or exosomes isolated from BMSCs (BMSC-Exos) containing miR-136-5p activated the Wnt/β-catenin pathway through the inhibition of LRP4 expression. Furthermore, BMSC-derived exosomes carrying miR-136-5p promoted osteoblast proliferation and differentiation, thereby promoting fracture healing. Conclusion BMSC-derived exosomes carrying miR-136-5p inhibited LRP4 and activated the Wnt/β-catenin pathway, thus facilitating fracture healing. Cite this article: Bone Joint Res 2021;10(12):744–758.

scholarly journals LncRNA NORAD promotes bone marrow stem cell differentiation and proliferation by targeting miR-26a-5p in steroid-induced osteonecrosis of the femoral head

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Dapeng Fu ◽  
Sheng Yang ◽  
Jianmin Lu ◽  
Haoyi Lian ◽  
Kairong Qin
Keyword(s):  
Bone Marrow ◽  
Femoral Head ◽  
Stem Cell Differentiation ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Inhibition Of Proliferation ◽  
Gene Assay ◽  
Proliferation And Apoptosis ◽  
Proliferation And Differentiation ◽  
Differentiation And Proliferation

Abstract Background Steroid-induced osteonecrosis of the femoral head (SONFH) is a devastating orthopedic disease, which seriously affects the quality of life of patients. The study aims to investigate the effects of LncRNA NORAD on SONFH. Methods Human bone marrow-derived mesenchymal stem cells (hBMSCs) were isolated from the proximal femur of patients during routine orthopedic surgery and then cultured with dexamethasone (Dex) and transfected with NORAD overexpression vector, siRNA-NORAD and miR-26a-5p mimics. The mRNA expression of NORAD, miR-26a-5p, OPG, RANK, and RANKL was detected by RT-qPCR. Cell proliferation and apoptosis was measured by CCK-8 assay and flow cytometry, respectively. The protein expression of RUNX2, OPG, RANK, and RANKL was detected by western blot. The dual-luciferase reporter gene assay was performed to confirm the binding between NORAD and miR-26a-5p. Results NORAD expression was downregulated in SONFH tissues, while miR-26a-5p expression was upregulated. Overexpression of NORAD improved DEX-induced inhibition of proliferation and differentiation, and promotion of apoptosis in hBMSCs, while knockdown of NORAD led to the opposite results. Moreover, NORAD improved DEX-induced inhibition of proliferation and differentiation, and promotion of apoptosis by regulation of miR-26a-5p in hBMSCs. Conclusions NORAD expression was downregulated in SONFH tissues, while miR-26a-5p expression was upregulated. NORAD improved DEX-induced inhibition of proliferation and differentiation, and promotion of apoptosis by regulation of miR-26a-5p in hBMSCs.

scholarly journals Bone Marrow Mesenchymal Stem Cell-Derived Exosomal miR-25 Regulates the Ubiquitination and Degradation of Runx2 by SMURF1 to Promote Fracture Healing in Mice

2020 ◽  
Vol 7 ◽  
Author(s):  
Yikun Jiang ◽  
Jun Zhang ◽  
Zhengwei Li ◽  
Guoliang Jia
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Mesenchymal Stem Cell ◽  
Fracture Healing ◽  
Osteogenic Differentiation ◽  
Bioinformatics Analysis ◽  
Luciferase Reporter ◽  
Gene Assay ◽  
And Migration ◽  
Proliferation And Migration

Recent evidence has demonstrated that mesenchymal stem cells (MSCs) can release a large number of functionally specific microRNA (miRNA) microvesicles that play a role in promoting osteogenic differentiation, but the specific mechanism is not yet clear. Under such context, this study aims to elucidate the mechanism of bone marrow mesenchymal stem cell-derived exosomes (BMSC-Exo) promoting fracture healing in mice. We isolated and identified the BMSC-Exo. Bioinformatics analysis predicted high expression of miRNA in exosomes and verified the transfer of miR-25 in exosomes by immunofluorescence. Targeting relationship between miR-25 and Smad ubiquitination regulatory factor-1 (SMURF1) was predicted and verified by dual-luciferase reporter gene assay. Immunoprecipitation and protein stability assays were used to detect Runt-related transcription factor 2 (Runx2) ubiquitination and the effect of SMURF1 on Runx2 ubiquitination, respectively. The effect of miR-25 in BMSC-Exo on fracture healing in mice was assessed using X-ray imaging. alkaline phosphatase, alizarin red staining, EdU, CCK-8, and Transwell were used to evaluate the effects of exosomes transferred miR-25 on osteogenic differentiation, proliferation, and migration of osteoblasts. Bioinformatics analysis predicted that miR-25 expression in exosomes increased significantly. Moreover, the targeted regulation of SMURF1 by miR-25 was verified. SMURF1 inhibited Runx2 protein expression by promoting ubiquitination degradation of Runx2. Notably, miR-25 secreted by BMSC-Exo can accelerate osteogenic differentiation, proliferation, and migration of osteoblasts through SMURF1/Runx2 axis. Our results demonstrate that miR-25 in BMSC-Exo regulates the ubiquitination degradation of Runx2 by SMURF1 to promote fracture healing in mice.

scholarly journals MicroRNA-409-3p promotes osteoblastic differentiation via activation of Wnt/β-catenin signaling pathway by targeting SCAI

2020 ◽  
Author(s):  
Nan Chen ◽  
Hao Yang ◽  
Lijun Song ◽  
Hua Li ◽  
Yi Liu ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Osteogenic Differentiation ◽  
Signaling Pathway ◽  
Quantitative Polymerase Chain Reaction ◽  
Osteoblastic Differentiation ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Complementary Sequence ◽  
Alizarin Red ◽  
Gene Assay

Osteogenic differentiation is an important process of new bone formation, miR-409-3p has been reported to be upregulated in osteogenic differentiation of human bone marrow mesenchymal stem cells (MSCs). To investigate the regulatory effect of miR-409-3p on osteogenic differentiation of MSCs and its molecular mechanism, the expression of miR-409-3p in osteoblast (HCO) and bone marrow-derived MSCs (MSC-A, MSC-B, MSC-U) were detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The binding of miR-409-3p to SCAI in MSC-B was investigated by dual-luciferase reporter gene assay. MSC-B were selected to transfect with miR-409-3p analog/complementary sequence (cs), miR-409-3p analog + SCAI and miR-409-3p cs + small interfering (si)-SCAI, as well as control, respectively. The alkaline phosphatase activity, alizarin red staining, and the expression of osteogenic markers in MSC-B during osteoblastic differentiation were tested by RT-qPCR and Western blotting, respectively. The Wnt/β-catenin pathway was inhibited by dickkopf-related protein 1 to get the roles of miR-409-3p during the osteoblastic differentiation of MSC-B when transfected with miR-409-3p analog. The expression of miR-409-3p in HCO was higher than that in these three MSCs, showing an increasing time-dependent trend on the 0 and 21th day of osteoblastic differentiation. MiR-409-3p directly regulated SCAI by targeting SCAI 3′UTR. Further, miR-409-3p suppressed SCAI expression, but SCAI upregulation suppressed the osteoblastic differentiation, as well as reduced the relative mRNA/protein expression of Wnt/β-catenin signaling pathway-related genes. Importantly, disruption of Wnt signaling also blocked miR-409-3p induced osteoblastic differentiation of MSCs. Therefore, miR-409-3p promotes osteoblastic differentiation through the activation of the Wnt/β-catenin pathway by downregulating SCAI expression.

Bone Marrow Mesenchymal Stem Cells (BMSC)-Originated miR-133 Impedes Migration and Invasiveness of Glioma Cells While Enhancing Apoptosis via Targeting the Receptor for Activated C Kinase 1 (RACK1)

2021 ◽  
Vol 11 (9) ◽  
pp. 1818-1824
Author(s):  
Jiangbo Xiong ◽  
Sheng Liu ◽  
Bin Xiang ◽  
Weibo Zhang ◽  
Jun Du ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Glioma Cells ◽  
Luciferase Reporter ◽  
Mechanistic Study ◽  
Migration And Invasion ◽  
Luciferase Reporter Gene ◽  
Gene Assay ◽  
Marrow Mesenchymal Stem Cells

This study aims to dissect the effects of bone marrow mesenchymal stem cells (BMSC) on the in vitro activity of glioma cells and the underlying mechanisms. The glioma cells were transfected with miR-133 mimics, RACK1-Vector, negative control (NC) and miR-133 mimic+RACK1-Vector, respectively, and then co-cultured with BMSC followed by analysis of miR-133 expression via PCR, apoptosis via flow cytometry, proliferation via CCK-8, invasion and migration via Transwell assay, the expression of proteins involved in apoptosis, anti-apoptosis, invasiveness and RACK1 by western blot, and the targeting relationship between miR-133 and RACK1 by dual-luciferase reporter gene assay. In comparison with normal glial cells, glioma cells exhibited a significantly diminished miR-133 level. miR-133 was upregulated in glioma cells after co-culture with BMSC, along with significantly restrained proliferation rate, migration and invasion activities as well as reduced protein levels (MMP-2, Vimentin, N-cadherin and MMP-9). Mechanistic study showed that miR-133 can retard the expression of RACK1, thereby impeding the invasion, migration and proliferation activities of cells while triggering cell apoptosis. In conclusion, BMSC-originated miR-133 can impede the migration and invasion while enhancing the apoptosis of glioma cells via targeting RACK1.

Overexpression Of Mir-125a In Bone Marrow CD34+ cells Of Patients With Myelodysplastic Syndrome Is Correlated To a Poor Prognosis and May Contribute To The Pathogenesis Of The Disease Through The Modulation Of NF-Kb Activation and Enhancement Of Differentiation Arrest

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5206-5206
Author(s):  
Irene Ganan-Gomez ◽  
Yue Wei ◽  
Hui Yang ◽  
Maria Carmen Boyano-Adánez ◽  
Guillermo Garcia-Manero
Keyword(s):  
Bone Marrow ◽  
Overall Survival ◽  
Conflicts Of Interest ◽  
Fine Tuning ◽  
Luciferase Reporter ◽  
Mirna Cluster ◽  
Luciferase Reporter Gene ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Gene Assay

Abstract Myelodysplastic syndromes (MDS) are a group of clonal malignancies characterized by impaired proliferation and differentiation of hematopoietic stem cells and precursors. The involvement of toll-like receptor (TLR)-mediated signalling in the modulation of myeloid differentiation and its participation in the pathogenesis of MDS are well documented (Wei et al 2013). Increased signaling through this pathway leads to the constitutive activation of NF-kB, which regulates the production of cytokines and mediates cell proliferation and apoptosis (Starczynowski 2010). In addition to the expression of proteins involved in inflammation, the TRL pathway also induces the expression of microRNAs (miRNAs) which participate in the fine-tuning of the inflammatory response (Kawai and Akira 2010). miR-125a and miR-125b are known modulators of hematopoiesis (Gerrits et al. 2012) and have been reported to be involved in several lymphoid and myeloid diseases. Little is known about their role in the pathogenesis of MDS. Interestingly, NF-kB-activating ability has been described for both miR-125a/b (Kim et al. 2012), and miR-125b appears to be upregulated by NF-kB within a positive feedback loop (Zhou et al. 2009; Tan et al. 2012). The aim of this work was to analyze the expression of miR-125a/b in MDS CD34+ cells and to study their relationship with the TLR pathway and differentiation. For this purpose, we analyzed the expression of miR-125a/b by qPCR in bone marrow CD34+ cells of 48 MDS patients, compared it with expression in healthy donors and studied the correlation with overall survival. In our study, we included miR-99b, which is clustered with miR-125a in the genome. Levels of TLR pathway components were detected by qPCR and correlated to those of the miRNAs. Activation of NF-kB was determined in Meg-01 and KG1 cells by the luciferase reporter gene assay, using a vector containing NF-kB responsive elements. Differentiation was studied in K562 and MDS-L cells through colony formation assays combined with analyses of the expression of specific markers by qPCR. For these experiments we used miRNA analogs and a miR-125a anti-sense oligonucleotide. Our results showed that miR-125a, but not miR-125b, is strongly overexpressed in MDS patients (∼15-fold of controls; P<0.01) and that miR-125a levels are significantly and negatively correlated to overall survival of MDS patients (P<0.05). Moreover, expression of miR-99b is also directly connected to the progression of the disease (P<0.05). Both miR-125a and miR-99b cooperate in vitro in the activation of NF-kB (P<0.001); however, we observed a negative correlation between miR-99b/miR-125a expression and levels of TLR2, TLR7 and their downstream proteins MyD88 and JMJD3 (P<0.05), suggesting that NF-kB activation by the miRNA cluster occurs in the absence of TLR signaling. Furthermore, we observed a ∼4-fold increase in NF-kB activity after miR-125a inhibition in the presence of a TLR2 agonist (P<0.001), indicating that miR-125a acts as an NF-kB inhibitor upon TLR stimulation. These results show that miR-125a is involved in the fine-tuning of NF-kB activity and that its effects may depend on the status of the TLR pathway. We then investigated the role of miR-125a in hematopoiesis and found that this miRNA contributes to the blockade of differentiation in the cell lines studied. Therefore, miR-125a could be involved in the pathogenesis or progression of MDS through the modulation of NF-kB activity and differentiation arrest. Thus, this miRNA could be a good prognostic marker and is a potential therapeutic target in MDS. Disclosures: No relevant conflicts of interest to declare.

scholarly journals MiR-128-3p inhibits vascular smooth muscle cell proliferation and migration by repressing FOXO4/MMP9 signaling pathway

2020 ◽  
Vol 26 (1) ◽  
Author(s):  
Chuan Qu ◽  
Xin Liu ◽  
Yan Guo ◽  
Yuhong Fo ◽  
Xiuhuan Chen ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Inflammatory Cytokines ◽  
Density Lipoprotein ◽  
Lipoprotein Cholesterol ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Gene Assay ◽  
And Migration ◽  
Proliferation And Migration

Abstract Background MicroRNAs (miRNAs) have been identified as important participants in the development of atherosclerosis (AS). The present study explored the role of miR-128-3p in the dysfunction of vascular smooth muscle cells (VSMCs) and the underlying mechanism. Methods Human VSMCs and ApoE knockout (ApoE−/−) C57BL/6J mice were used to establish AS cell and animal models, respectively. Expression levels of miR-128-3p, forkhead box O4 (FOXO4) and matrix metallopeptidase 9 (MMP9) were detected using qRT-PCR and Western blot, respectively. CCK-8, BrdU, and Transwell assays as well as flow cytometry analysis were performed to detect the proliferation, migration and apoptosis of VSMCs. Levels of inflammatory cytokines and lipids in human VSMCs, mice serum and mice VSMCs were also determined. The binding site between miR-128-3p and 3′UTR of FOXO4 was confirmed using luciferase reporter gene assay. Results MiR-128-3p was found to be decreased in AS patient serum, ox-LDL-treated VSMCs, AS mice serum and VSMCs of AS mice. Transfection of miR-128-3p mimics suppressed the proliferation and migration of VSMCs, accompanied by the promoted apoptosis and the decreased levels of inflammatory cytokines. Further experiments confirmed the interaction between miR-128-3p and FOXO4. Augmentation of FOXO4 or MMP9 reversed the effects of miR-128-3p. Besides, miR-128-3p inhibited triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) but increased high-density lipoprotein cholesterol (HDL-C) in the serum of AS mice. Conclusion MiR-128-3p repressed the proliferation and migration of VSMCs through inhibiting the expressions of FOXO4 and MMP9.

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.

scholarly journals Sevoflurane inhibits proliferation, invasion, but enhances apoptosis of lung cancer cells by Wnt/β-catenin signaling via regulating lncRNA PCAT6/ miR-326 axis

2020 ◽  
Vol 15 (1) ◽  
pp. 159-172
Author(s):  
Guoning Su ◽  
Zhibing Yan ◽  
Min Deng
Keyword(s):  
Lung Cancer ◽  
Cancer Cells ◽  
Volatile Anesthetic ◽  
Lung Cancer Cells ◽  
Luciferase Reporter ◽  
Dependent Manner ◽  
Luciferase Reporter Gene ◽  
Western Blot Assay ◽  
Gene Assay ◽  
Cancer Tissues

AbstractSevoflurane was frequently used as a volatile anesthetic in cancer surgery. However, the potential mechanism of sevoflurane on lung cancer remains largely unclear. In this study, lung cancer cell lines (H446 and H1975) were treated by various concentrations of sevoflurane. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assessment and colony formation assay were performed to detect the cell viability and proliferation, separately. Also, transwell assay or flow cytometry assay was applied as well to evaluate the invasive ability or apoptosis in lung cancer cells, respectively. Western blot assay was employed to detect the protein levels of β-catenin and Wnt5a. Moreover, quantitative real-time polymerase chain reaction (qRT-PCR) was used to examine the expression level of prostate cancer-associated transcript 6 (PCAT6) and miR-326 in lung cancer tissues and cells. The target interaction between miR-326 and PCAT6 or Wnt5a was predicted by bioinformatics analysis and verified by the dual-luciferase reporter gene assay. Sevoflurane inhibited the abilities on viability, proliferation, invasion, and activation of Wnt/β-catenin signaling, but promoted apoptosis of H446 and H1975 cells in a dose-dependent manner. The expression of PCAT6 was increased in lung cancer tissues and cells, except for that of miR-326. Besides, sevoflurane could lead to expressed limitation of PCAT6 or improvement of miR-326. This process presented a stepwise manner. Up-regulation of PCAT6 restored the suppression of sevoflurane on abilities of proliferation, invasion, rather than apoptosis, and re-activated the Wnt5a/β-catenin signaling in cells. Moreover, the putative binding sites between miR-326 and PCTA6 or Wnt5a were predicted by starBase v2.0 software online. PCAT6 suppressing effects on cells could be reversed by pre-treatment with miR-326 vector. The promotion of Wnt5a inverted effects led from miR-326 or sevoflurane. Our study indicated that sevoflurane inhibited the proliferation, and invasion, but enhanced the apoptosis in lung cancer cells by regulating the lncRNA PCAT6/miR-326/Wnt5a/β-catenin axis.

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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