Retinoblastoma Cells Research Articles

Study on the role of MYCN in retinoblastoma by inhibiting p53 and activating wnt/βcatenin/Fra-1 signaling pathway by reducing DKK3.

Retinoblastoma (RB) is a pediatric malignancy, typically diagnosed at birth or during early childhood. The pathogenesis of RB is marked by the amplification of the Basic Helix-Loop-Helix (BHLH) Transcription Factor MYCN, which serves as a transcriptional regulator capable of binding to Dickkopf 3 (DKK3). However, the precise role of DKK3 in the malignant progression of RB cells caused by MYCN remains elusive. In the present study, the expression of MYCN was either overexpressed or interfered in RB cells. Subsequently, the expression level of DKK3 was assessed throughquantitative real-time polymerase chain reaction and western blot analysis. Cell proliferation was evaluated using theCell Counting Kit-8 assay and 5-ethynyl-2'-deoxyuridine staining, while cell cycle progression and apoptosis were analyzed by flow cytometry and western blot analysis, respectively. Additionally, the expression of proteins involved in the Wnt/β-catenin/Fra-1/p53 signaling pathway was evaluated via western blot analysis. To gain further insights, Wnt agonists and the P53 inhibitor PFT-α were introduced into exploration. The current investigation revealed a negative correlation between the expression levels of MYCN and DKK3 in RB cells. Additionally, DKK3 overexpression inhibited cell proliferation, promoted cell apoptosis, and arrested cell cycle in RB cells with high expression of MYCN. Moreover, enhanced DKK3 expression inhibited proliferation, promoted cell cycle arrest and apoptosis of RB cells by modulating the wnt/βcatenin/Fra-1/p53 signaling pathway. Furthermore, in vivo experiments revealed that overexpression of DKK3 inhibits the growth of RB tumors. Collectively, our findings elucidate that MYCN stimulates the Wnt/β-catenin/Fra-1 pathway by suppressing DKK3 expression, ultimately suppressing p53 activity and contributing to malignant progression of RB.

Long non-coding RNA MIMT1 promotes retinoblastoma proliferation via sponging miR-153-5p to upregulate FGF2

With the rapid development of biotechnology, long non-coding RNAs (lncRNAs) have shown promising potential for cancer treatment and may become novel therapeutic targets. This study aimed to explore the roles of lncRNAs in retinoblastoma (RB). It involves analysing differentially expressed lncRNAs in RB and normal tissues from the GSE111168 and GSE125903 datasets, further validating them in RB cells. Our findings determined that lncRNA MIMT1 was upregulated in RB cell lines and tissues. In WERI-Rb1 and Y79 cells, silencing MIMT1 significantly inhibited cell proliferation, whereas MIMT1 overexpression enhanced cell proliferation. Furthermore, in vivo xenograft experiments demonstrated that MIMT1 knockdown suppressed tumour volume and weight. Subsequent mechanistic investigations showed that MIMT1 upregulates fibroblast expression of FGF2 by binding to miR-153-5p, ultimately promoting RB cell proliferation. This suggest that MIMT1 functions as an oncogene in RB and potentially serves as a molecular marker for diagnostic and prognostic assessments. Thus, the MIMT1/miR-153-5p/FGF2 pathway is a promising therapeutic target for RB treatment.

Liposome co-delivery of quercetin and doxorubicin in inhibiting retinoblastoma by regulating epithelial-mesenchymal transition process

Regulating the process of epithelial-mesenchymal transition(EMT) is an essential strategy to inhibit tumor growth and metastasis. This study is based on the EMT process of retinoblastoma and constructs quercetin(QUE) and doxorubicin(DOX) co-loaded liposome(QD Lipo) to investigate the therapeutic effect and mechanisms of combined QUE and DOX treatment on retinoblastoma. Single-factor experiments were conducted to optimize the prescription process of QD Lipo. Eventually, spherical particles with a diameter of(108.87±1.93) nm, a PDI of 0.13±0.02, and a Zeta potential of(-34.83±1.92) mV were obtained. The encapsulation rates of QUE and DOX were 96.20%±4.40% and 91.17%±4.41%, respectively. Y79 human retinoblastoma cells were used as an in vitro cellular model, and confocal microscopy demonstrated that QD Lipo could enhance Y79 uptake efficiency. The CCK-8 assay confirmed that the optimal combination therapy effect of QUE and DOX occurred at a mass ratio of 1∶1 to 1∶2. Flow cytometry showed that QD Lipo enhanced the induction of apoptosis in Y79 cells. Western blot analysis revealed that QD Lipo significantly reduced the expression of EMT pathway-related proteins vimentin and α-SMA. Fluorescence assays detected a significant decrease in ROS levels in Y79 cells after treatment with QD. These results indicated that liposomal co-delivery of QUE and DOX can enhance drug delivery efficiency to retinoblastoma cells, inhibit the EMT process in retinoblastoma by downregulating ROS levels, and enhance the cytotoxicity of DOX against retinoblastoma.

Bmi-1 promotes the proliferation, migration and invasion, and inhibits cell apoptosis of human retinoblastoma cells via RKIP

Retinoblastoma is one of the most common ocular malignancies in children. Bmi-1, a member of the Polycomb group family of transcriptional repressors, is expressed in a variety of tumors. The purpose of our study was to explore the role of Bmi-1 in retinoblastoma. RT-qPCR and western blot were used for calculating the mRNA and protein levels of Bmi-1 and RKIP. MTT, Wound healing and Transwell assays were performed to measure the proliferation, migration and invasion in retinoblastoma cells. Cell apoptosis was detected by flow cytometry. The volume and mass of transplanted tumors were detected in nude mice. Bmi-1 was over expressed, and RKIP was low expressed in retinoblastoma cells. Bmi-1 promoted cell proliferation, migration and invasion and suppressed cell apoptosis of Y79 and SO-RB50 cells. Downregulation of Bmi-1 and overexpression of RKIP inhibited cell proliferation, migration and invasion, and increased cell apoptosis. The functions of Bmi-1 knockdown on retinoblastoma cells were blocked by RKIP knockdown, but promoted by RKIP. Down-regulated Bmi-1 inhibited xenograft tumor growth, and RKIP exacerbated this inhibitory effect. Bmi-1 served as a potential therapeutic target for improving the efficacy of clinical treatment in retinoblastoma. All the findings revealed the functions of Bmi-1/RKIP axis in retinoblastoma tumorigenesis.

Biofabrication of Centella asiatica mediated metal (Silver and Iron) nanoparticles and their enhanced antimicrobial, anticancer activity in retinoblastoma Y79 cancer cells

In recent years, metallic nanoparticles have played a significant role in nanotechnology and bio fabrication, being considered an alternative to antibiotics due to their feasibility for use in both bacterial detection and infection prevention. Moreover, recent studies have demonstrated the green process of developing nanoparticles with anticancer activity against many human cancer cell lines. In the current investigation, the biogenic synthesis of silver and iron nanoparticles using the aqueous leaf extract of Centella asiatica was performed. The leaf extracts underwent characterization for antioxidant activity, TLC analysis, phytochemical analysis, and GC-MS analysis of Centella asiatica. The leaf extracts revealed the antioxidant potential of the phytoconstituents, namely phenolic and flavonoid contents, responsible for the anticancer activity. The synthesized silver and iron nanoparticles were analysed using various techniques, including UV-Vis absorption spectroscopy, X-ray diffraction, Fourier transform infrared spectroscopy, scanning electron microscopy with energy-dispersive spectroscopy, and transmission electron microscopy. Confirmation of silver and iron nanoparticle formation was achieved through UV-Vis absorption spectra, showing absorption peaks at 362 nm and 342 nm, respectively. Fourier transform infrared spectroscopy analysis confirmed the biological moieties involved in synthesis and stability. X-ray diffraction (XRD) analysis verified the crystallinity of the nanomaterials, while scanning electron microscopy depicted spherical shapes for silver nanoparticles and similarly round shapes for iron nanoparticles. EDS analysis revealed 87.12 % silver and 40.12 % iron content in the respective nanoparticles. TEM imaging displayed predominantly spherical silver nanoparticles, sized at 50 nm. The antimicrobial efficacy of the biosynthesized silver and iron nanoparticles was evaluated using the well diffusion method, along with the determination of the minimum inhibitory concentration. Results indicated moderate effectiveness of silver nanoparticles against Escherichia coli (7±0.036) and Aspergillus niger (5±0.034) compared to iron nanoparticles. The minimum inhibitory concentrations against Staphylococcus aureus were 46.51–86.74 % for silver nanoparticles and 42.79–77.44 % for iron nanoparticles. The anticancer effect was evaluated using human retinoblastoma (Y79) cell lines. The cytotoxicity study using the MTT assay revealed that Centella asiatica-mediated silver nanoparticles exhibited significant cytotoxicity to human retinoblastoma cells, with a low IC50 value of 49.56 μg/ml. Based on the results, it is feasible to infer that biofabricated nanoparticles could potentially serve as antibacterial and antifungal compounds. Furthermore, the synthesized silver nanoparticles, followed by iron nanoparticles, showed high effectiveness against human retinoblastoma cancer cells, indicating potential for future drug discovery to inhibit retinoblastoma cancer for biomedical applications.

ALYREF/THOC4 expression and cell growth modulation in retinoblastoma

In this study, we tested the hypothesis that ALYREF/THOC4, a poor prognostic factor in different cancer types, has potential as a drug target and prognostic biomarker for retinoblastoma (RB). Immunostaining (IHC), Western blot, and RT-qPCR analyses detected overexpression of ALYREF in the RB cell lines Y79, RB143, WERI-RB1, and RB116. IHC analysis on RB tumor array showed that 11/14 of RB tumors were ALYREF+ to varying degrees, with eight tumors at maximum 3+ intensity. The IHC analysis also detected ALYREF+ cells in normal retina, mainly in the inner nuclear and ganglion cell layer, while some tumor-bearing human eyes were ALYREF+ in the optic nerve suggesting a role in optic invasion/tumor invasion. The expression of ALYREF within the tumor itself, in the optic nerve, as well as in adjacent “normal” retina, suggest that this pattern of expression may lead to ALYREF being a potentially useful prognostic indicator for RB, as it is for other tumors. siRNA knockdown of ALYREF resulted in a 40 % decrease in cell growth in both WERI-RB1 and Y79 cells (p<0.05) and this was associated with decreased expression of mRNAs for the cell proliferation markers Ki67 and PCNA (p<0.005). These results suggest a role for ALYREF in RB cell growth regulation and its potential as both a target and a biomarker for tumor growth inhibition by anti-cancer therapies.

FABP4 Is an Indispensable Factor for Regulating Cellular Metabolic Functions of the Human Retinal Choroid.

The purpose of the current study was to elucidate the physiological roles of intraocularly present fatty acid-binding protein 4 (FABP4). Using four representative intraocular tissue-derived cell types, including human non-pigmented ciliary epithelium (HNPCE) cells, retinoblastoma (RB) cells, adult retinal pigment epithelial19 (ARPE19) cells and human ocular choroidal fibroblast (HOCF) cells, the intraocular origins of FABP4 were determined by qPCR analysis, and the intracellular functions of FABP4 were investigated by seahorse cellular metabolic measurements and RNA sequencing analysis using a specific inhibitor for FABP4, BMS309403. Among these four different cell types, FABP4 was exclusively expressed in HOCF cells. In HOCF cells, both mitochondrial and glycolytic functions were significantly decreased to trace levels by BMS309403 in a dose-dependent manner. In the RNA sequencing analysis, 67 substantially up-regulated and 94 significantly down-regulated differentially expressed genes (DEGs) were identified in HOCF cells treated with BMS309403 and those not treated with BMS309403. The results of Gene Ontology enrichment analysis and ingenuity pathway analysis (IPA) revealed that the DEGs were most likely involved in G-alpha (i) signaling, cAMP-response element-binding protein (CREB) signaling in neurons, the S100 family signaling pathway, visual phototransduction and adrenergic receptor signaling. Furthermore, upstream analysis using IPA suggested that NKX2-1 (thyroid transcription factor1), HOXA10 (homeobox A10), GATA2 (gata2 protein), and CCAAT enhancer-binding protein A (CEBPA) were upstream regulators and that NKX homeobox-1 (NKX2-1), SFRP1 (Secreted frizzled-related protein 1) and TREM2 (triggering receptor expressed on myeloid cells 2) were causal network master regulators. The findings in this study suggest that intraocularly present FABP4 originates from the ocular choroid and may be a critical regulator for the cellular homeostasis of non-adipocyte HOCF cells.

Aurora Kinase A Is Overexpressed in Human Retinoblastoma and Correlates with Histopathologic High-Risk Factors: Implications for Targeted Therapy

Retinoblastoma (RB) is an intraocular malignancy initiated by loss of RB1 function and/or dysregulation of MYCN oncogene. RB is primarily treated with chemotherapy; however, systemic toxicity and long-term side effects remain a significant challenge necessitating the identification of specific molecular targets. Aurora kinase A (AURKA), a critical cell cycle regulator, contributes to cancer pathogenesis especially in RB1-deficient and MYCN dysregulated tumors. Our immunohistochemistry study in patient specimens (n=67) discovered that AURKA is overexpressed in RB and elevated expression correlates with one or more histopathological high-risk factors such as tumor involvement of the optic nerve, choroid, sclera and/or anterior segment. More specifically, AURKA is ubiquitously expressed in majority of the advanced-stage RB tumors that show a sub-optimal response to chemotherapy. shRNA-mediated depletion/pharmacological inhibition studies in cell lines, patient-derived cells, in-vivo xenografts, and enucleated patient specimens confirm that RB cells are highly sensitive to a lack of functional AURKA. In addition, we deciphered that AURKA and MYCN associate with each other to regulate their levels in RB cells. Overall, our results demonstrate a previously unknown upregulation of AURKA in RB, facilitated by its crosstalk with MYCN, and elevated levels of this kinase may indicate unfavorable prognosis in tumors refractory to chemotherapy. This study provides a rationale and confirms that therapeutic targeting of elevated AURKA in RB could be a potential treatment approach.

Retracted] Downregulation of CKS1B restrains the proliferation, migration, invasion and angiogenesis of retinoblastoma cells through the MEK/ERK signaling pathway.

Following the publication of the above paper, it has been drawn to the Editors' attention by a concerned reader that certain of the lumen formation assay data shown in Fig. 5A on p. 112 were strikingly similar to data appearing in different form in another article written by different authors at different research institute, which had already been published in the journal Biomedicine & Pharmacotherapy prior to the submission of this paper to International Journal of Molecular Medicine, and which has also subsequently been retracted. In view of the fact that the contentious data had already apparently been published previously, the Editor of International Journal of Molecular Medicine has decided that this paper should be retracted from the Journal. After having been in contact with the authors, they agreed with the decision to retract the paper. The Editor apologizes to the readership for any inconvenience caused. [International Journal of Molecular Medicine 44: 103‑114, 2019; DOI: 10.3892/ijmm.2019.4183].

Deciphering metabolic heterogeneity in retinoblastoma unravels the role of monocarboxylate transporter 1 in tumor progression

BackgroundTumors exhibit metabolic heterogeneity, influencing cancer progression. However, understanding metabolic diversity in retinoblastoma (RB), the primary intraocular malignancy in children, remains limited.MethodsThe metabolic landscape of RB was constructed based on single-cell transcriptomic sequencing from 11 RB and 5 retina samples. Various analyses were conducted, including assessing overall metabolic activity, metabolic heterogeneity, and the correlation between hypoxia and metabolic pathways. Additionally, the expression pattern of the monocarboxylate transporter (MCT) family in different cell clusters was examined. Validation assays of MCT1 expression and function in RB cell lines were performed. The therapeutic potential of targeting MCT1 was evaluated using an orthotopic xenograft model. A cohort of 47 RB patients was analyzed to evaluate the relationship between MCT1 expression and tumor invasion.ResultsDistinct metabolic patterns in RB cells, notably increased glycolysis, were identified. This metabolic heterogeneity correlated closely with hypoxia. MCT1 emerged as the primary monocarboxylate transporter in RB cells. Disrupting MCT1 altered cell viability and energy metabolism. In vivo studies using the MCT1 inhibitor AZD3965 effectively suppressed RB tumor growth. Additionally, a correlation between MCT1 expression and optic nerve invasion in RB samples suggested prognostic implications.ConclusionsThis study enhances our understanding of RB metabolic characteristics at the single-cell level, highlighting the significance of MCT1 in RB pathogenesis. Targeting MCT1 holds promise as a therapeutic strategy for combating RB, with potential prognostic implications.

Identification of miR-20a as A Potential Discerning Biomarker for Non-Invasive versus Invasive Retinoblastoma.

Intraocular retinoblastoma (RB) is common in kids. Although the cause of this disease is a mutation in the RB1 gene, the formed cancerous mass in different patients is seen in non-invasive states, limited to the ocular cavity or in invasive states distributed to other parts of the body. Because this tumor's aggressiveness cannot be predicted early, these patients receive systemic chemotherapy with multiple drugs. Treating non-invasive and invasive tumors separately reduces chemical drug side effects. The aim of this study was to identify diagnostic biomarkers by separating miRNAs in blood serum from invasive and non-invasive RB patients. In this experimental study, selected three gene expression omnibus (GEO) datasets. Two were related to serum and tumor tissue miRNAs, and one was related to non-invasive and invasive RB gene expression. Examined RB gene-miRNA relationships. Then, we performed real-time polymerase chain reaction (PCR) on candidate miRNAs in the Y79 cell line and patient blood samples in non-invasive and invasive retinoblastoma. Fourteen high-expression and 7 low-expression miRNAs resulted. MiR-181, miR-135a, miR-20a, miR-373, and miR-191 were common genes with differential genes between invasive and non-invasive retinoblastoma. Only MiR-181 was upregulated in the Y79 RB cell line. Other candidate miRNAs expressed less. Invasive retinoblastomas increased serum miR-20a and miR-191. Integrated and regular bioinformatics analyses found important miRNAs in patients' and miR-20a, miR- 191, and miR-135a can distinguish non-invasive and invasive retinoblastoma, suggesting further research.

RETINOBLASTOMA CELLS OVEREXPRESSING THE MRPS18-2 PROTEIN COULD BE DIFFERENTIATED in vitro

The mitochondrial ribosomal protein MRPS18-2 is involved in cell cycle regulation through its interaction with the retinoblastoma-associated protein, RB. Earlier we have shown that this protein plays an important role in homeostasis of normal and tumor cells, embryogenesis, and in the maintenance of cell stemness. We also found that the MRPS18-2 protein is transactivated by a transcription factor KLF4, one of the Yamanaka factors, inducing cell pluripotency. The aim of the present work was to study the functional consequences of overexpression of the MRPS18-2 and RB proteins in the retinoblastoma cell line (WERI RB 27), concerning putative directed differentiation in vitro. Methods. Direct multipotent differentiation was conducted with cocktails of chemicals for osteogenic, chondrogenic, and adipogenic lineages. Ca+2 ions were stained with Alizarin Red, triglycerides – with Oil Red O, and glycosaminoglycans - with Alcian blue. Results. Parental cells WERI RB-27 did not differentiate upon treatment with any of chemical cocktails, cells remained in suspension and did not change morphology. At the same time, cells that overexpressed MRPS18-2 have demonstrated their differentiation ability into osteo-, chondro-, and adipo-lineages. Conclusions. Thus, we have shown that overexpression of the MRPS18-2 protein in WERI RB 27 cells leads to changes in cell morphology and to ability of the directed multipotent differentiation in vitro. Hence, the MRPS18-2 protein plays a significant role in cancerogenesis and cell stemness.

Gastric Inhibitory Polypeptide Receptor (GIPR) Overexpression Reduces the Tumorigenic Potential of Retinoblastoma Cells.

Retinoblastoma (RB) is the most common malignant intraocular tumor in early childhood. Gene expression profiling revealed that the gastric inhibitory polypeptide receptor (GIPR) is upregulated following trefoil factor family peptide 1 (TFF1) overexpression in RB cells. In the study presented, we found this G protein-coupled transmembrane receptor to be co-expressed with TFF1, a new diagnostic and prognostic RB biomarker for advanced subtype 2 RBs. Functional analyses in two RB cell lines revealed a significant reduction in cell viability and growth and a concomitant increase in apoptosis following stable, lentiviral GIPR overexpression, matching the effects seen after TFF1 overexpression. In chicken chorioallantoic membrane (CAM) assays, GIPR-overexpressing RB cells developed significantly smaller CAM tumors. The effect of GIPR overexpression in RB cells was reversed by the GIPR inhibitor MK0893. The administration of recombinant TFF1 did not augment GIPR overexpression effects, suggesting that GIPR does not serve as a TFF1 receptor. Investigations of potential GIPR up- and downstream mediators suggest the involvement of miR-542-5p and p53 in GIPR signaling. Our results indicate a tumor suppressor role of GIPR in RB, suggesting its pathway as a new potential target for future retinoblastoma therapy.

Role of Protein Tyrosine Phosphatase Receptor Type E (PTPRE) in Chemoresistant Retinoblastoma.

Protein tyrosine phosphatase receptor type E (PTPRE) is a member of the "classical" protein tyrosine phosphatase subfamily and regulates a variety of cellular processes in a tissue-specific manner by antagonizing the function of protein tyrosine kinases. PTPRE plays a tumorigenic role in different human cancer cells, but its role in retinoblastoma (RB), the most common malignant eye cancer in children, remains to be elucidated. Etoposide-resistant RB cell lines and RB patients display significant higher PTPRE expression levels compared to chemosensitive counterparts and the healthy human retina, respectively. PTPRE promotor methylation analyses revealed that PTPRE expression in RB is not regulated via this mechanism. Lentiviral PTPRE knockdown (KD) induced a significant decrease in growth kinetics, cell viability, and anchorage-independent growth of etoposide-resistant Y79 and WERI RB cells. Caspase-dependent apoptosis rates were significantly increased and a re-sensitization for etoposide could be observed after PTPRE depletion. In vivo chicken chorioallantoic membrane (CAM) assays revealed decreased tumor formation capacity as well as reduced tumor size and weight following PTPRE KD. Expression levels of miR631 were significantly downregulated in etoposide-resistant RB cells and patients. Transient miR631 overexpression resulted in significantly decreased PTPRE levels and concomitantly decreased proliferation and increased apoptosis levels in etoposide-resistant RB cells. These impacts mirror PTPRE KD effects, indicating a regulation of PTPRE via this miR. Additionally, PTPRE KD led to altered phosphorylation of protein kinase SGK3 and-dependent on the cell line-AKT and ERK1/2, suggesting potential PTPRE downstream signaling pathways. In summary, these results indicate an oncogenic role of PTPRE in chemoresistant retinoblastoma.

Retraction Note: Curcumin inhibits proliferation, migration, invasion and promotes apoptosis of retinoblastoma cell lines through modulation of miR-99a and JAK/STAT pathway

Retraction Note: Curcumin inhibits proliferation, migration, invasion and promotes apoptosis of retinoblastoma cell lines through modulation of miR-99a and JAK/STAT pathway

An anti-GD2 aptamer-based bifunctional spherical nucleic acid nanoplatform for synergistic therapy targeting MDM2 for retinoblastoma

Retinoblastoma (RB) is a type of pediatric solid tumor in the fundus. The lack of precision therapies combined with the difficulty of delivering small interfering RNA (siRNA) into the eyes means that there is currently no nucleic acid-based therapy for RB in clinical practice. Here, we reported on anti-GD2 and glutathione-responsive spherical nucleic acids (SNAs), loaded with siRNA and the inhibitor NVP-CGM097, which jointly blocked the oncogenic factor n in RB cells (Y79 and WERI-RB-1). The SNAs were formed through the self-assembly of bifunctional cholesterol amphiphiles containing aptamers that specifically targeted GD2-positive RB cells, allowing for the formation of an SNA with a dense DNA shell. The aptamer/siRNA component functioned both as a carrier and a payload, enhancing the specific recognition and delivery of both components and constituting an active agent for MDM2 regulation. Following SNA endocytosis by RB cells, siRNA and NVP-CGM097 were released from the SNA particles by glutathione, which synergistically blocked the MDM2-p53 pathway, increasing p53 protein content and inducing cell apoptosis. This study showed a potent antitumor effect following intravitreal injection of SNAs in Y79 tumor-bearing mice through clinical manifestation and tumor pathological analysis. In hematological analysis and hepatotoxicity assays, SNAs were safer for mice than melphalan, the favored drug for treating RB in clinical practice. Our results illustrated the potential of intravitreally injected SNAs as a precision medicine for treating RB.

Protein expression of nucleolar protein 12 in the retina and its implication in protection of retina from UV irradiation damage

Nucleolar protein 12 (NOL12), one of the nucleolar proteins which are primarily expressed in the nucleolus and play key roles in RNA metabolism, cell proliferation, cell cycle, and cell survival, is widely expressed in various species and multiple organs. Although it has been reported that the mRNA of Drosophila NOL12 homolog viriato is expressed in the eyes of Drosophila, the protein expression of NOL12 in mammalian eyes remains to be elucidated. In this study, we showed through immunohistochemistry that NOL12 was present in the rat retina, with predominant distribution in the cytoplasm of the retinal neuronal cells. In the human retinoblastoma cell line WERI-Rb1, we found that altering NOL12 expression led to a change in WERI-Rb1 cell viability. Knocking down NOL12 expression decreased cell viability. In contrast, overexpressing NOL12 increased cell viability. Furthermore, increasing NOL12 expression inhibited ultraviolet (UV)-induced apoptosis. These findings demonstrated that NOL12 may play an important protective role in retinal cells. In the WERI-Rb1 cells exposed to UV irradiation, we detected that NOL12 was degraded, but this degradation could be attenuated by a pan-Caspase inhibitor. Notably, the inhibitory effect of NOL12 against UV-induced apoptosis could be restrained by increasing the expression of ATR serine/threonine kinase (ATR), a kinase that, when activated by severe DNA damage, can result in apoptosis. We also found that upregulating NOL12 inhibited the activation of ATR caused by UV irradiation. Additionally, inhibiting ATR activity reduced apoptosis resulting from both silencing NOL12 expression and UV exposure. Thus, NOL12 may protect against UV irradiation-induced retinal damage by inhibiting ATR activity.

LncRNA NEAT1 promotes angiogenesis of retinoblastoma cells through regulation of the miR-106a/HIF-1α axis

ObjectiveTo explore the role and mechanisms of lncRNA nuclear enriched abundant transcript 1 (NEAT1) in angiogenesis of retinoblastoma (RB) cells. MethodsThis study investigated the roles of NEAT1 in RB progression. The RNA expression levels of NEAT1, miR-106a, and hypoxia-inducible factor-1alpha (HIF-1α) examined by quantitative reverse transcription polymerase chain reaction (RT-qPCR) were compared between RB cells and normal retinal pigment epithelial (RPE) cells. The binding sites between NEAT1 and miR-106a, and between miR-106a and HIF-1α were predicted by the TargetScan database and verified using the dual-luciferase reporter assay. By transfection of overexpression plasmid or shRNA of NEAT1, and/or treatment of miR-106a inhibitor or mimics, proliferation, invasion, and angiogenesis of RB cells (measured by the MTT assay, the Transwell assay, and the tube formation assay, respectively) were compared between groups. Group comparisons were analyzed using one-way analysis of variance (ANOVA), and Tukey's post-hoc test was employed for further statistical assessment. P-value less than 0.05 was considered statistically significant. ResultsThe RNA expression levels of NEAT1 and HIF-1α were upregulated in RB cells, whereas the expression level of miR-106a was downregulated compared with RPE cells. NEAT1 overexpression or miR-106a knockdown advanced proliferation, invasion, and tube formation of RB cells. As a target of NEAT1, miR-106a could sponge HIF-1α to downregulate HIF-1α expression level. Functional analyses indicated that miR-106a knockdown reversed the inhibitory effects of NEAT1 silencing on the proliferation, invasion, and tube formation of RB cells. Furthermore, miR-106a overexpression suppressed RB cell angiogenesis by downregulating HIF-1α expression level. ConclusionNEAT1 promoted proliferation, invasion, and angiogenesis of RB cells through upregulation of HIF-1α expression level by sponging miR-106a, demonstrating that NEAT1 may be a novel target for RB treatment.

ACOT7 promotes retinoblastoma resistance to vincristine by regulating fatty acid metabolism reprogramming

The rate of vincristine (VCR) resistance in the treatment of retinoblastoma (RB) is relatively high, and the exact role and mechanism of autophagy and fatty acid (FA) metabolism in RB are still unknown. The aim of this study was to elucidate the molecular mechanism by which acyl-CoA thioesterase 7 (ACOT7) regulates FA metabolism and autophagy, which may lead to potential therapeutic strategies for RB. In the present study, the relationship between FA metabolism and cellular drug sensitivity was evaluated through ACOT7 overexpression or inhibition tests in RB-resistant cells. The lipase inhibitor orlistat and the autophagy inhibitor CQ were used to determine the effects of ACOT7 on FA metabolism, autophagy, and cellular drug sensitivity, as well as the therapeutic value of ACOT7 targeting. The results showed that ACOT7 was upregulated in VCR-resistant RB cells, significantly enhancing cell resistance and indicating that ACOT7 may serve as a biomarker for VCR resistance in RB cells. Knockdown of ACOT7 inhibited FA metabolism and reduced cell viability in VCR-resistant RB cells. The effect of ACOT7 overexpression was opposite to that of ACOT7 knockdown, and ACOT7 overexpression promoted autophagy in VCR-resistant RB cells. After treatment with orlistat or CQ, FA metabolism in VCR-resistant RB cells decreased, cell viability and autophagy were inhibited, EMT was inhibited, and the sensitivity of RB cells to VCR was increased. In conclusion, ACOT7 knockdown can mediate FA metabolism to inhibit autophagy and the migration of RB cells, thereby improving the sensitivity of RB cells to VCR.

Construction of a Functional Nucleic Acid-Based Artificial Vesicle-Encapsulated Composite Nanoparticle and Its Application in Retinoblastoma-Targeted Theranostics.

Retinoblastoma (RB) is an aggressive tumor of the infant retina. However, the ineffective targeting of its theranostic agents results in poor imaging and therapeutic efficacy, which makes it difficult to identify and treat RB at an early stage. In order to improve the imaging and therapeutic efficacy, we constructed an RB-targeted artificial vesicle composite nanoparticle. In this study, the MnO2 nanosponge (hMNs) was used as the core to absorb two fluorophore-modified DNAzymes to form the Dual/hMNs nanoparticle; after loaded with the artificial vesicle derived from human red blood cells, the RB-targeted DNA aptamers were modified on the surface, thus forming the Apt-EG@Dual/hMNs complex nanoparticle. The DNA aptamer endows this nanoparticle to target the nucleolin-overexpressed RB cell membrane specifically and enters cells via endocytosis. The nanoparticle could release fluorophore-modified DNAzymes and supplies Mn2+ as a DNAzyme cofactor and a magnetic resonance imaging (MRI) agent. Subsequently, the DNAzymes can target two different mRNAs, thereby realizing fluorescence/MR bimodal imaging and dual-gene therapy. This study is expected to provide a reliable and valuable basis for ocular tumor theranostics.