Retinoblastoma Cell Proliferation Research Articles

Cone-rod homeobox transcriptionally activates TCF7 to promote the proliferation of retinal pigment epithelial and retinoblastoma cells in vitro

AIM: To investigate the proliferation regulatory effect of cone-rod homeobox (CRX) in retinal pigment epithelium (RPE) and retinoblastoma (RB) cells to explore the potential application and side effect (oncogenic potential) of CRX-based gene therapy in RPE-based retinopathies. METHODS: Adult human retinal pigment epithelial (ARPE)-19 and human retinal pigment epithelial (RPE)-1 cells and Y79 RB cell were used in the study. Genetic manipulation was performed by lentivirus-based technology. The cell proliferation was determined by a CellTiter-Glo Reagent. The mRNA and protein levels were determined by quantitative real-time polymerase chain reaction (qPCR) and Western blot assay. The transcriptional activity of the promoter was determined by luciferase reporter gene assay. The bindings between CRX and transcription factor 7 (TCF7) promoter as well as TCF7 and the promoters of TCF7 target genes were examined by chromatin immunoprecipitation (ChIP) assay. The transcription of the TCF7 was determined by a modified nuclear run-on assay. RESULTS: CRX overexpression and knockdown significantly increased (n=3, P<0.05 in all the cells) and decreased (n=3, P<0.01 in all the cells) the proliferation of RPE and RB cells. CRX overexpression and knockdown significantly increased and deceased the mRNA levels of Wnt signaling target genes [including MYC proto-oncogene (MYC), JUN, FOS like 1 (FOSL1), CCND1, cyclin D2 (CCND2), cyclin D3 (CCND3), cellular communication network factor 4 (CCN4), peroxisome proliferator activated receptor delta (PPARD), and matrix metallopeptidase 7 (MMP7)] and the luciferase activity driven by the Wnt signaling transcription factor (TCF7). TCF7 overexpression and knockdown significantly increased and decreased the proliferation of RPE and RB cells and depletion of TCF7 significantly abolished the stimulatory effect of CRX on the proliferation of RPE and RB cells. CRX overexpression and knockdown significantly increased and decreased the mRNA level of TCF7 and the promoter of TCF7 was significantly immunoprecipitated by CRX antibody. CONCLUSION: CRX transcriptionally activates TCF7 to promote the proliferation of RPE and RB cells in vitro. CRX is a potential target for RPE-based regenerative medicine. The potential risk of this strategy, tumorigenic potential, should be considered.

Auxiliary effect of trolox on coenzyme Q10 restricts angiogenesis and proliferation of retinoblastoma cells via the ERK/Akt pathway

Reactive oxygen species (ROS) are essential for cancer signalling pathways and tumour maintenance, making ROS targeting a promising anti-cancer strategy. Coenzyme Q10 (CoQ10) has been shown to be effective against various cancers, but its impact on retinoblastoma, alone or with trolox, remains unreported. Cytotoxicity of CoQ10 alone and with trolox was evaluated in normal human retinal pigment epithelium cells (ARPE-19) and Y79 retinoblastoma cells using CCK-8. Flow cytometry was used to assess apoptosis, cell cycle, ROS, and mitochondrial membrane potential (MMP). Anti-angiogenic potential was tested using human umbilical vein endothelial cells (HUVECs) and chick chorioallantoic membrane (CAM) assays. Mechanistic studies were conducted via RT-PCR and western blotting. CoQ10, alone and with trolox, reduced Y79 cell viability, induced apoptosis through excess ROS generation, and decreased MMP significantly. Both treatments caused G2/M phase cell arrest. The CAM assay showed a significant reduction in endothelial cell proliferation, evidenced by fewer number of co-cultured HUVECs when exposed to CoQ10 or CoQ10 with trolox. The combination of CoQ10 and trolox significantly reduced VEGF-A, ERK, and Akt receptor levels, while CoQ10 alone significantly inhibited ERK and Akt phosphorylation. Together, CoQ10 and trolox reduced protein expression of VEGFA. CoQ10 alone and with trolox, induces apoptosis in Y79 retinoblastoma cells by inhibiting the ERK/Akt pathway and downregulating VEGFA. This study is the first to report the in vitro and in-ovo anti-cancer potential of CoQ10 alone or when combined with trolox, on human retinoblastoma Y79 cells.

GCN2-SLC7A11 axis coordinates autophagy, cell cycle and apoptosis and regulates cell growth in retinoblastoma upon arginine deprivation

BackgroundArginine deprivation was previously shown to inhibit retinoblastoma cell proliferation and induce cell death in vitro. However, the mechanisms by which retinoblastoma cells respond to arginine deprivation remain to be elucidated.MethodsThe human-derived retinoblastoma cell lines Y79 and WERI-Rb-1 were subjected to arginine depletion, and the effects on inhibiting cell growth and survival were evaluated. This study investigated potential mechanisms, including autophagy, cell cycle arrest and apoptosis. Moreover, the roles of the general control nonderepressible 2 (GCN2) and mechanistic target of rapamycin complex 1 (mTORC1) signaling pathways in these processes were examined.ResultsWe demonstrated that arginine deprivation effectively inhibited the growth of retinoblastoma cells in vitro. This treatment caused an increase in the autophagic response. Additionally, prolonged arginine deprivation induced G2 cell cycle arrest and was accompanied by an increase in early apoptotic cells. Importantly, arginine depletion also induced the activation of GCN2 and the inhibition of mTOR signaling. We also discovered that the activation of SLC7A11 was regulated by GCN2 upon arginine deprivation. Knockdown of SLC7A11 rendered retinoblastoma cells partially resistant to arginine deprivation. Furthermore, we found that knockdown of GCN2 led to a decrease in the autophagic response in WERI-Rb-1 cells and arrested more cells in S phase, which was accompanied by fewer apoptotic cells. Moreover, knockdown of GCN2 induced the constant expression of ATF4 and the phosphorylation of 70S6K and 4E-BP1 regardless of arginine deprivation.ConclusionsCollectively, our findings suggest that the GCN2‒SLC7A11 axis regulates cell growth and survival upon arginine deprivation through coordinating autophagy, cell cycle arrest, and apoptosis in retinoblastoma cells. This work paves the way for the development of a novel treatment for retinoblastoma.

PRMT1 mediates the proliferation of Y79 retinoblastoma cells by regulating the p53/p21/CDC2/cyclin B pathway

Retinoblastoma (RB) is the most common intraocular malignancy among children and presents a certain mortality risk, especially in low- and middle-income countries. Clarifying the molecular mechanisms underlying the onset and progression of retinoblastoma is vital for devising effective cancer treatment approaches. PRMT1, a major type I PRMT, plays significant roles in cancer development. However, its expression and role in retinoblastoma are still unclear. Our research revealed a marked increase in PRMT1 levels in both retinoblastoma tissues and Y79 cells. The overexpression of PRMT1 in Y79 cells promoted their growth and cell cycle progression. Conversely, the suppression of PRMT1 hindered the growth of Y79 cells and impeded cell cycle progression. Mechanistically, PRMT1 mediated the growth of Y79 retinoblastoma cells by targeting the p53/p21/CDC2/Cyclin B pathway. Additionally, the ability of PRMT1 knockdown to suppress cell proliferation was also observed in vivo. Overall, PRMT1 could function as a potential target for therapeutic treatment in individuals with retinoblastoma.

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.

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].

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.

Antitumoral Potential of the Histone Demethylase Inhibitor GSK-J4 in Retinoblastoma.

The purpose of this study was to investigate the antitumor effects of GSK-J4 on retinoblastoma, as well as its related biological functions and molecular mechanisms. The antitumor effect of GSK-J4 on retinoblastoma was evaluated by in vitro and in vivo assays. CCK-8, EdU incorporation, and soft agar colony formation assays were performed to examine the effect of GSK-J4 on cell proliferation. Flow cytometry was used to evaluate the effect of GSK-J4 on the cell cycle and apoptosis. RNA-seq and Western blotting were conducted to explore the molecular mechanisms of GSK-J4. An orthotopic xenograft model was established to determine the effect of GSK-J4 on tumor growth. GSK-J4 significantly inhibited retinoblastoma cell proliferation both in vitro and in vivo, arrested the cell cycle at G2/M phase, and induced apoptosis. Mechanistically, GSK-J4 may suppress retinoblastoma cell growth by regulating the PI3K/AKT/NF-κB signaling pathway. The antitumor effects of GSK-J4 were noticeable in retinoblastoma and were at least partially mediated by PI3K/AKT/NF-κB pathway suppression. Our study provides a novel strategy for the treatment of retinoblastoma.

Prox1 Suppresses Proliferation and Drug Resistance of Retinoblastoma Cells via Targeting Notch1.

Retinoblastoma (RB) is a prevalent type of eye cancer in youngsters. Prospero homeobox 1 (Prox1) is a homeobox transcriptional repressor and downstream target of the proneural gene that is relevant in lymphatic, hepatocyte, pancreatic, heart, lens, retinal, and cancer cells. The goal of this study was to investigate the role of Prox1 in RB cell proliferation and drug resistance, as well as to explore the underlying Notch1 mechanism. Human RB cell lines (SO-RB50 and Y79) and a primary human retinal microvascular endothelial cell line (ACBRI-181) were used in this study. The expression of Prox1 and Notch1 mRNA and protein in RB cells was detected using quantitative real time-polymerase chain reaction (RT-qPCR) and Western blotting. Cell proliferation was assessed after Prox1 overexpression using the Cell Counting Kit-8 and the MTS assay. Drug-resistant cell lines (SO-RB50/vincristine) were generated and treated with Prox1 to investigate the role of Prox1 in drug resistance. We employed pcDNA-Notch1 to overexpress Notch1 to confirm the role of Notch1 in the protective function of Prox1. Finally, a xenograft model was constructed to assess the effect of Prox1 on RB in vivo. Prox1 was significantly downregulated in RB cells. Overexpression of Prox1 effectively decreased RB cell growth while increasing the sensitivity of drug-resistant cells to vincristine. Notch1 was involved in Prox1's regulatory effects. Notch1 was identified as a target gene of Prox1, which was found to be upregulated in RB cells and repressed by increased Prox1 expression. When pcDNA-Notch1 was transfected, the effect of Prox1 overexpression on RB was removed. Furthermore, by downregulating Notch1, Prox1 overexpression slowed tumor development and increased vincristine sensitivity in vivo. These data show that Prox1 decreased RB cell proliferation and drug resistance by targeting Notch1, implying that Prox1 could be a potential therapeutic target for RB.

METTL14-Mediated m6a Modification of CDKN2A Promotes the Development of Retinoblastoma by Inhibiting the p53 Pathway.

The methyltransferase 14, N6-adenosine-methyltransferase subunit (METTL14) and Cyclin-dependent kinase inhibitor 2A (CDKN2A) have been identified as involved in the regulation of various cancer progression, while their mechanism and regulatory effect in retinoblastoma (RB) is still unclear. Cell colony formation, CCK-8 as well as Western blotting were used to evaluate the proliferation, apoptosis as well as p53 protein level of RB cell line. The METTL14 and CDKN2A levels were detected by qRT-PCR or Western blotting when METTL14 was up-regulated or CDKN2A was down-regulated. MeRIP and Pearson analysis were performed to confirm the regulatory relationship between METTL14 among CDKN2A. We found that the levels of CDKN2A and METTL14 were abundant in RB samples, as well as RB cells. METTL14 enhances N6-methyladenosine (m6A) modification of CDKN2A to upregulate its mRNA and protein levels. The proliferation of RB cells can be inhibited by silencing CDKN2A, which promotes apoptosis and p53 protein level. Furthermore, high-expression of METTL14 eliminated the anti-tumor effect of CDKN2A silencing in RB progression in vitro. CDKN2A is mediated by METTL14-m6A modified and restrains p53 pathway activation to accelerate the malignancy of RB. This points to the METTL14-m6A-CDKN2A-p53 pathway axis as a possible prospective target for the future RB treatment.

MiR-452-5p facilitates retinoblastoma cell growth and invasion via the SOCS3/JAK2/STAT3 pathway.

Retinoblastoma (RB) is an intraocular tumor in children. Accumulated evidence confirmsthat microRNAs (miRNAs) exert critical functions in RB. This research aimed to investigate the miR-452-5p function in RB. MiR-452-5p expressions in RB were tested with quantitative real-time polymerase chain reaction (PCR). MiR-452-5p functions in RB were evaluated via Cell Counting Kit-8, 5-Ethynyl-2'-deoxyuridine assay, flow cytometry, Western blot, and Transwell. MiR-452-5p mechanism in RB was assessed using bioinformatics software Starbase and dual-luciferase reporter gene assay. Meanwhile, miR-452-5p function in RB in vivo was examined by constructing tumor xenografts in nude mice, immunohistochemistry, and Western blot assays. MiR-452-5p was overexpressed in RB tissues and cells, and miR-452-5p expression was positively correlated with RB clinicopathology including the Largest tumor base (mm) and Differentiation. Functionally, miR-452-5p knockdown restrained RB cell proliferation, invasion, epithelial-mesenchymal transition (EMT), and facilitated cell apoptosis. Mechanistically, suppressors of cytokine signaling (SOCS3) knockdown restored the inhibitory effects of miR-452-5p knockdown on RB cells. Meanwhile, in vivo studies further corroborated that miR-452-5p knockdown reduced RB tumor growth, EMT, and accelerated apoptosis in vivo. Also, miR-452-5p knockdown increased SOCS3 protein levels, and decreased phosphorylated Janus kinase 2/Janus kinase 2 (JAK2), phosphorylated signal transducer and activator of transcription 3/signal transducer and activator of transcription 3 (STAT3) in vivo. MiR-452-5p accelerated RB cell growth and invasion by SOCS3/JAK2/STAT3.

Quercetin suppresses retinoblastoma cell proliferation and invasion and facilitates oxidative stress-induced apoptosis through the miR-137/FNDC5 axis

Retinoblastoma (RB) constitutes a prevalent malignancy in clinic and usually occurs in children under the age of 5 years old. The increased frequency of malignant tumor metastases and the delayed diagnosis and treatment caused unsatisfactory therapeutic efficiency. Quercetin was formerly identified to impede tumor growth in certain malignancies. Our study attempted to investigate the effects and mechanisms of quercetin in Rb development, in order to provide an effective clinical therapeutic approach. Rb cell lines (WER1-RB1 and Y79) were incubated with different concentrations of quercetin, and then cell proliferation, invasion, apoptosis, and oxidative stress were determined. It was showed that quercetin restrained Rb cell proliferation and invasion, and induced cell apoptosis and oxidative stress in a dose dependent manner. Moreover, we found that quercetin incubation upregulated miR-137 expression in Rb cells. MiR-137 inhibition abrogated quercetin-mediated inhibition of Rb cell progression. Furthermore, dual-luciferase reporter gene assay validated that fibronectin type III domain-containing protein 5 (FNDC5) was a target for miR-137. MiR-137 overexpression restrained proliferation and invasion, and enhanced apoptosis and oxidative stress in Rb cells, whereas FNDC5 overexpression abrogated these effects. Additionally, nude mice were injected with WER1-RB1 cells to establish a xenograft tumor model, and then treated with 50 or 100 mg/kg quercetin. Quercetin treatment mitigated xenograft tumor growth in nude mice. In conclusion, quercetin restrained proliferation and invasion, and induced apoptosis and oxidative stress in Rb cells through regulating the miR-137/FNDC5 pathway. We expected that our study could provide an effective approach for Rb treatment. However, quercetin and miR-137 may have off-target effects in Rb cells, and our study still has certain limitations. Therefore, we will investigate the effects of quercetin on other signaling pathways in Rb cells and explore the application of combination therapy in follow-up experiments, in order to provide a rigorous research basis for the treatment of Rb with quercetin.

Gentiopicroside inhibits retinoblastoma cell proliferation, invasion, and tumorigenesis in nude mice by suppressing the PI3K/AKT pathway.

Retinoblastoma is a prevalent pediatric intraocular tumor. The suppressive effect of gentiopicroside (GPS) has been reported on various tumors. This study sought to determine the effect of GPS on retinoblastoma cell proliferation, apoptosis, invasion, and epithelial-mesenchymal transition (EMT), and tumorigenesis in nude mice. The effect and mechanism of GPS on growth, apoptosis, invasion, and EMT were determined by cell counting kit-8 (CCK-8), western blot, flow cytometry, and transwell assays in retinoblastoma cells. Y79 cells were injected into the vitreous cavity of BALB/c‑nude mice to construct a retinoblastoma mouse model. Tumor growth and mouse weight were monitored for sequential 5weeks. The effect of GPS in vivo was assessed by immunohistochemistry (IHC), terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL), and western blot assays. GPS decreased the cell viability of both Y79 and Weri-Rb1 cells with the IC50 of 18.85μM and 27.57μM, respectively. Besides, GPS reduced the relative expression of proteins involved in proliferation and EMT, and the number of invading cells, while increased the apoptosis rate and the relative expressions of apoptosis proteins in retinoblastoma cells. Mechanically, GPS decreased the relative protein level of PI3K/AKT pathway, which was then recovered after 740 Y-P was applied. Correspondingly, 740 Y-P reversed the inhibitory effect of GPS on growth, invasion, and EMT, and the increased effect of GPS on apoptosis. Additionally, GPS decreased tumor volume and weight as well as the relative level of Ki-67, VEGF, p-PI3K/PI3K, and p-AKT/AKT, while increased the apoptosis rate in vivo. GPS inhibited retinoblastoma cell proliferation and invasion via deactivating the PI3K/AKT pathway in both cell and animal models.

LncRNA LOXL1-AS1 promotes proliferation and invasion and inhibits apoptosis in retinoblastoma by regulating the MAPK signaling pathway.

Retinoblastoma (RB) is an intraocular malignancy that is most common in children and rare in adults. Addressing novel biomarkers and therapeutic targets for RB to modulate tumor progression has become a challenge. The aim of the present study was to investigate the function of long non-coding RNAs (LncRNAs) LOXL1-AS1 in RB cell proliferation and metastasis. It was found that LOXL1-AS1 was overexpressed in RB tissues and cells. In order to evaluate cell viability and colony formation potential, the knockdown of LOXL1-AS1 has been established. Knockdown of LOXL1-AS1 was also inhibited cells migration and invasion. In addition, the proportion of cells in the G2/M phase of the sh-LOXL1-AS1 group increased significantly, and the proportion of cells in the sh-NC group decreased significantly. In the xenograft model of RB, the tumors in the sh-LOXL1-AS1 group grow slowly compared to the sh-NC group. Western blot analysis revealed that LOXL1-AS1 can regulate the progression of RB cells through MAPK signaling pathway in vitro and in vivo. These results indicated that LncRNA LOXL1-AS1 promotes proliferation, invasion and inhibits apoptosis of retinoblastoma by regulating MAPK signaling pathway, and might be expected to be a novel basis for clinical diagnosis and treatment.

KCNQ1OT1 promotes retinoblastoma progression by targeting miR-339-3p that suppresses KIF23.

Long noncoding RNAs (lncRNAs) are involved in tumor formation and development. KCNQ1OT1 regulates the malignant proliferation of retinoblastoma (RB), but the specific mechanism remains to be further investigated. The KCNQ1OT1, miR-339-3p and KIF23 expression levels in RB were detected by qRT-PCR and western blotting. The cell viability, proliferation, migration ability and caspase-3 activity of RB cells were evaluated by CCK-8, BrdU, transwell and caspase-3 activity analysis. Western blot was used to detect the Bax and Bcl-2 protein expression in RB cells. The binding relationship between KCNQ1OT1, miR-339-3p and KIF23 was detected by luciferase, RIP and RNA pull-down assay. KCNQ1OT1 and KIF23 were up-regulated frequently in RB, and miR-339-3p was down-regulated. Functional studies showed that downregulation of KCNQ1OT1 or KIF23 inhibited the survival and migration of RB cells, and facilitated apoptosis. Interference with miR-339-3p showed the opposite effect. Mechanisms suggested that KCNQ1OT1 exited its oncogenic activity by positively regulating the expression of KIF23 and sponging miR-339-3p. KCNQ1OT1/miR-339-3p/KIF23 may be a new biomarker for the diagnosis and treatment of RB.

Abstract LB267: Nitric oxide suppression by secreted frizzled-related protein 2 drives retinoblastoma

Abstract Retinoblastoma is a cancer of the infant retina primarily driven by the loss of the Rb tumor suppressor gene, which is undruggable. Here, we identified an autocrine signaling, mediated by secreted frizzled-related protein 2 (SFRP2), which suppresses nitric oxide and enables retinoblastoma growth. SFRP2 silencing resulted in rapid production of nitric oxide, leading to p53-dependent growth arrest of retinoblastoma cells. We show that coxsackievirus and adenovirus receptor (CXADR) is the cell surface receptor for SFRP2 in retinoblastoma cells, that CXADR functions as a "dependence receptor," transmitting a growth inhibitory signal in the absence of SFRP2, and that the balance between SFRP2 and CXADR determines nitric oxide production. Accordingly, high SFRP2 RNA expression correlates with high-risk histopathologic features in retinoblastoma. Targeting SFRP2 signaling by SFRP2-binding peptides or by a pharmacological inhibitor rapidly induced nitric oxide and profoundly inhibited retinoblastoma growth in orthotopic xenograft models. These results reveal a cytokine signaling pathway that regulates nitric oxide production and retinoblastoma cell proliferation, and is amenable to therapeutic intervention. Citation Format: Panneerselvam Jayabal, Xiuye Ma, Kathryn M. Bondra, Yidong Chen, Peter J. Houghton, Yuzuru Shiio. Nitric oxide suppression by secreted frizzled-related protein 2 drives retinoblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB267.

The effect of lncRNA ADPGK-AS1 on the proliferation and apoptosis of retinoblastoma cells by targeting miR-200b-5p

Objective: To explore the effect of lncRNA ADPGK-AS1 on the proliferation and apoptosis of retinoblastoma cells and its possible mechanism. Methods: The tumor tissues of 31 patients with retinoblastoma admitted to Henan Provincial Eye Hospital from February to June 2020 and their corresponding normal tissues adjacent to the cancer were collected. The expression levels of lncRNA ADPGK-AS1 and miR-200b-5p in retinoblastoma tissues and normal adjacent tissues were detected by real-time fluorescence quantitative polymerase chain reaction (qRT-PCR). Human retinal epithelial cell ARPE-19, human retinoblastoma cell Y-79 and WERI-Rb-1 were cultured in vitro. The expression levels of lncRNA ADPGK-AS1 and miR-200b-5p were detected by qRT-PCR. Y-79 cells were randomly divided into si-con group, si-lncRNA ADPGK-AS1 group, miR con group, miR-200b-5p group, si-lncRNA ADPGK-AS1+ anti-miR con group, and si-lncRNA ADPGK-AS1+ anti-miR-200b-5p group. The proliferation, cloning and apoptosis of cells in each group were detected by tetramethylazol blue method, plate cloning test and flow cytometry, respectively. The targeting relationship between lncRNA ADPGK-AS1 and miR-200b-5p was detected by double luciferase report test, and the expression level of cleaved-caspase-3 protein was detected by western blot. Results: Compared with the adjacent tissues, the expression of lncRNA ADPGK-AS1 in retinoblastoma tissues was increased (P<0.05), while the expression of miR-200b-5p was decreased (P<0.05). Compared with ARPE-19 cells, the expression of lncRNA ADPGK-AS1 in Y-79 and WERI-Rb-1 cells was increased (P<0.05), while the expression of miR-200b-5p was decreased (P<0.05). Compared with the si-con group, the cell viability of the si-lncRNA ADPGK-AS1 group was reduced (1.06±0.09 vs 0.53±0.05, P<0.05), the number of cell clone formation was reduced (114.00±8.03 vs 57.00±4.13, P<0.05), while the apoptosis rate [(7.93±0.68)% vs (25.43±1.94)%] and the protein level of cleaved-caspase-3 were increased (P<0.05). Compared with the miR-con group, the cell viability of the miR-200b-5p group was decreased (1.05±0.08 vs 0.57±0.05, P<0.05), the number of cell clone formation was decreased (118.00±10.02 vs 64.00±5.13, P<0.05), while the apoptosis rate [(7.89±0.71)% vs (23.15±1.62)%] and the protein level of cleaved-caspase-3 were increased (P<0.05). lncRNA ADPGK-AS1 could target the expression of miR-200b-5p. Compared with the si-lncRNA ADPGK-AS1+ anti-miR-con group, cell viability of the si-lncRNA ADPGK-AS1+ anti-miR-200b-5p group was increased (0.53±0.04 vs 1.25±0.10, P<0.05), and the number of cell clones was increased (54.00±4.39 vs 125.00±10.03, P<0.05), while the rate of apoptosis [(25.38±1.53)% vs (9.76±0.71)%] and the protein level of cleaved-caspase-3 were decreased (P<0.05). Conclusion: Interfering with the expression of lncRNA ADPGK-AS1 could inhibit the proliferation and clone formation and induce apoptosis of retinoblastoma cells by targeting the expression of miR-200b-5p.

A novel MYCN-YTHDF1 cascade contributes to retinoblastoma tumor growth by eliciting m6A -dependent activation of multiple oncogenes.

Retinoblastoma, the most prevalent primary intraocular tumor in children, leads to vision impairment, disability and even death. In addition to RB1 inactivation, MYCN activation has been documented as another common oncogenic alteration in retinoblastoma and represents one of the high-risk molecular subtypes of retinoblastoma. However, how MYCN contributes to the progression of retinoblastoma is still incompletely understood. Here, we report that MYCN upregulates YTHDF1, which encodes one of the reader proteins for N6-methyladenosine (m6A) RNA modification, in retinoblastoma. We further found that this MYCN-upregulated m6A reader functions to promote retinoblastoma cell proliferation and tumor growth in an m6A binding-dependent manner. Mechanistically, YTHDF1 promotes the expression of multiple oncogenes by binding to their mRNAs and enhancing mRNA stability and translation in retinoblastoma cells. Taken together, our findings reveal a novel MYCN-YTHDF1 regulatory cascade in controlling retinoblastoma cell proliferation and tumor growth, pinpointing an unprecedented mechanism for MYCN amplification and/or activation to promote retinoblastoma progression.

Effects of long non-coding RNA MALAT1-targeting miR-570-3p and miR-34a on the invasion, proliferation, and apoptosis of human retinoblastoma cells

Purpose: To investigate the expression of lncRNA MALAT1-targeting miR-570-3p and miR-34a and its effects on the invasion, proliferation, and apoptosis of human retinoblastoma cells.&#x0D; Methods: MiR-34a, miR-570-3p, and IncRNA MALAT1 in a human normal retinal vascular endothelial cell line (ACBRI-181), human retinoblastoma cell line (SO-Rb50), human normal retinal tissue and human retinoblastoma tissue were determined. Luciferase assay was used to verify the targeting relationship between LncRNA MALAT1 and miR-570-3p and miR-34a. while cell invasion, cell apoptosis and cell proliferation were assessed by Transwell assay, flow cytometry, and tumor pellet-forming assay, respectively.&#x0D; Results: LncRNA MALAT1 in SO-Rb50 cell line and human retinoblastoma tissue line were significantly up-regulated, while the expression levels of miR-34a and miR-570-3p were significantly down-regulated (p &lt; 0.05). Luciferase assay results showed that lncRNA MALAT1 targeted miR-570-3p and miR-34a. The invasion and proliferation of SO-Rb50 cells in the miR-570-3p inhibitor and miR-34a inhibitor groups were significantly increased, while the apoptosis of SO-Rb50 cells was significantly decreased (p &lt; 0.05). However, the invasion and proliferation of SO-Rb50 cells in sh-MALAT1 group were significantly decreased, while apoptosis significantly increased. However, compared with sh-MALAT1 group alone, the invasion and proliferation of SO-Rb50 cells in co-transfected sh-MALAT1+miR-570-3p inhibitor + miR-34a inhibitor group w significantly increased, but apoptosis significantly decreased (p &lt; 0.05).&#x0D; Conclusion: LncRNA MALAT1 negatively regulates miR-570-3p and miR-34a to promote the invasion and proliferation of human retinoblastoma SO-Rb50 cells and inhibit apoptosis. These findings may be of significance in developing suitable therapies for human retinoblastoma

Retraction: MiR-486-3p inhibits the proliferation, migration and invasion of retinoblastoma cells by targeting ECM1.

This article is being retracted from Bioscience Reports at the request of the Editor-in-Chief and the Editorial Board following receipt of a notification from a reader, alerting the Editorial Board to concerns regarding the western blots and flow-cytometry plots of this manuscript. The western blots have concerning features such as the shapes of the bands and absence of any detectable background, and the flow-cytometry plots of Figure 6E share strong similarities with those found in other papers by unrelated authors, including in Figures 2E and 4F of Li et al. 2019 (https://doi.org/10.1080/15384101.2019.1674071). The authors have been contacted with regards to these concerns and subsequently the retraction, but they have not responded to the Journal's queries or the concerns raised. Given the extent of the issues raised, the Editorial Board stand by the decision to retract the article.