RBX1 regulates uveal melanoma immune-related genes via STAT1

Objective·To investigate the role of RBX1 (ring-box protein 1) in the regulation of immune-related genes in uveal melanoma (UVM) tumor cells.Methods·The Cancer Genome Atlas (TCGA) was searched to analyze the expression levels of RBX1 in tumors and the correlation with clinical stages and survival prognosis. RBX1 was transiently knocked down in the UVM cell lines, i.e., 92.1, OMM2.3 and MEL290 by using small interfering RNAs (siRNAs) targeting RBX1. RNA sequencing was performed on the 92.1 cells with transient knockdown of RBX1, and the differentially expressed genes between the siRBX1-transfected cells and control cells were analyzed by gene set enrichment analysis (GSEA) to investigate the relationship between RBX1 and tumor immune-related genes. Based on the results of the analysis, signal transducer and activator of transcription 1 (STAT1) and its downstream CXC chemokine ligand 9 (CXCL9) and CXCL10 mRNA expression levels were detected by real-time fluorescence quantitative PCR (qPCR) in 92.1, OMM2.3 and MEL290 cells with transient knockdown of RBX1, respectively. The protein expression levels of STAT1 and p-STAT1 in 92.1 cells were detected by Western blotting. The cell lines OMM2.3 and MEL290, in which RBX1 was transiently knocked down, were treated with 5 nmol/L or 10 nmol/L STAT1 inhibitor fludarabine for 48 h, and the mRNA expression levels of CXCL9 and CXCL10 were detected by qPCR.Results·TCGA database analysis showed that RBX1 was highly expressed in a variety of tumors compared to the normal tissues, particularly in adrenocortical carcinoma and UVM. In addition, the patients with late stage of these two kinds of tumors had higher expression level of RBX1, and the patients with higher expression level of RBX1 had shorter overall survival time (P<0.05). RNA sequencing of 92.1 cells with transiently knocked down RBX1 and control cells revealed differential genes, and GSEA showed that RBX1 was involved in the regulation of tumor immune-related pathways. Heat map analysis showed an increase in STAT1 expression after RBX1 knockdown. In the 92.1, OMM2.3 and MEL290 cell lines, qPCR showed increases in STAT1, CXCL9 and CXCL10 mRNA expression after transient knockdown of RBX1, and Western blotting showed that STAT1 and p-STAT1 expression increased after knockdown of RBX1 in 92.1 cell lines. The increases of CXCL9 and CXCL10 mRNA in OMM2.3 and MEL290 cell lines were suppressed by STAT1 inhibitors.Conclusion·RBX1 may regulate CXCL9 and CXCL10 expression via STAT1 in UVM cells and is involved in tumor immune regulation

Electrospun chitosan-graft-poly (ɛ-caprolactone)/poly (ɛ-caprolactone) nanofibrous scaffolds for retinal tissue engineering

A promising therapy for retinal diseases is to employ biodegradable scaffolds to deliver retinal progenitor cells (RPCs) for repairing damaged or diseased retinal tissue. In the present study, cationic chitosan-graft-poly(ɛ-caprolactone)/polycaprolactone (CS-PCL/PCL) hybrid scaffolds were successfully prepared by electrospinning. Characterization of the obtained nanofibrous scaffolds indicated that zeta-potential, fiber diameter, and the content of amino groups on their surface were closely correlated with the amount of CS-PCL in CS-PCL/PCL scaffolds. To assess the cell–scaffold interaction, mice RPCs (mRPCs) were cultured on the electrospun scaffolds for 7 days. In-vitro proliferation assays revealed that mRPCs proliferated faster on the CS-PCL/PCL (20/80) scaffolds than the other electrospun scaffolds. Scanning electron microscopy and the real-time quantitative polymerase chain reaction results showed that mRPCs grown on CS-PCL/PCL (20/80) scaffolds were more likely to differentiate towards retinal neurons than those on PCL scaffolds. Taken together, these results suggest that CS-PCL/PCL(20/80) scaffolds have potential application in retinal tissue engineering

FBXO38 regulates ocular melanoma proliferation through the PI3K-Akt signaling pathway

Objective·To investigate the effect of F-box only protein 38 (FBXO38) on the ocular melanoma proliferation and the potential regulatory pathway.Methods·Human skin cutaneous melanoma A375 and human uveal melanoma OMM2.3 cell lines with FBXO38 knockdown and overexpression were constructed by FBXO38 short hairpin RNA (shRNA) and FBXO38 overexpression plasmids respectively. Knockdown and overexpression efficiency of FBXO38 at transcription and protein levels were verified by using quantitative real-time PCR (qRT-PCR) and Western blotting. The effects of FBXO38 on melanoma cell proliferation were detected through clonal formation assay, BrdU immunofluorescence staining and CCK8 cell proliferation assay. By using The Cancer Genome Atlas (TCGA) database, differentially expressed genes were analyzed in the high and low expression groups of FBXO38. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment was performed to reveal the signaling pathways associated with FBXO38. CCK8 cell proliferation assay was used to detect the inhibition rates of the signaling pathway inhibitors on cells with different FBXO38 expression levels. qRT-PCR and Western blotting were used to detect whether the signaling pathway was activated after knocking down FBXO38.Results·qRT-PCR and Western blotting verified that mRNA and protein expression levels of FBXO38 in FBXO38 knockdown A375 and OMM2.3 cell lines decreased compared with the control group, while the expression levels of FBXO38 in the overexpression cell lines increased compared with wild type group (P<0.05). Clonal formation assay, BrdU immunofluorescence staining and CCK8 cell proliferation assay showed that FBXO38 knockdown significantly enhanced the proliferation of A375 and OMM2.3 cells (P<0.05), while overexpression of FBXO38 inhibited melanoma cell proliferation (P<0.05). Enrichment analysis showed that in skin cutaneous melanoma and uveal melanoma, FBXO38 expression influenced the phosphoinositide 3-kinase/protein kinase B (PI3K-Akt) pathway activation. Compared with those in the control group, the inhibition rates of PI3K inhibitor LY294002 and mTOR1 inhibitor Everolimus in the FBXO38 knockdown group significantly improved (P<0.05), while their inhibition rates of the overexpression group significantly decreased compared with those of control cells (P<0.05). Western blotting results showed that after knocking down FBXO38, expression levels of PTEN, P21 and P53 proteins decreased, while expression level of MDM2 protein increased. The qRT-PCR results showed a significant decrease in P53 transcription level (P<0.05) and a significant increase in MDM2 transcription level in FBXO38 knockdown cells (P<0.05).Conclusion·FBXO38 plays a role in regulating the proliferation of ocular melanoma, and this regulatory effect is related to the PI3K-Akt signaling pathway

Comparison of Intra-Arterial Chemotherapy Efficacy Delivered Through the Ophthalmic Artery or External Carotid Artery in a Cohort of Retinoblastoma Patients

Purpose: To evaluate the efficacy of an external carotid artery (ECA) alternative route in intra-arterial chemotherapy (IAC) for treatment of retinoblastoma.Methods: In this retrospective, single-centre, case-control study, 98 retinoblastoma patients who received successful IAC were included. The drug delivery routes were the primary ophthalmic artery (OA) route and the ECA route when OA catheterization was not feasible.Results: A total of 337 successful IAC procedures were performed in our study, of which 32 (9.5%) procedures were performed through the ECA route. Eighteen eyes (18.4%) accepted at least one IAC through branches of the ECA. Statistical analysis showed that there was no significant difference in ocular clinical results (enucleation, death, recurrence and event-free) between the ECA and OA routes. No significant association was found between the route of drug delivery and the ocular survival time (p = 0.69). The use of ECA catheterization in at least one IAC cycle was not a predictor of enucleation (HR: 1.58; 95% CI: 0.56–4.46, p = 0.39). The increasing number of procedures through the ECA route did not increase the risk of enucleation (HR: 1.64; 95% CI: 0.42–6.39, p = 0.48).Conclusion: The ECA alternative route did not affect the efficacy of IAC in retinoblastoma. When the standard OA approach is not feasible, ECA system catheterization should be considered

Intrachromosomal Looping Is Required for Activation of Endogenous Pluripotency Genes during Reprogramming

SummaryGeneration of induced pluripotent stem cells (iPSCs) by defined factors is an extremely inefficient process, because there is a strong epigenetic block preventing cells from achieving pluripotency. Here we report that virally expressed factors bound to the promoters of their target genes to the same extent in both iPSCs and unreprogrammed cells (URCs). However, expression of endogenous pluripotentcy genes was observed only in iPSCs. Comparison of local chromatin structure of the OCT4 locus revealed that there was a cohesin-complex-mediated intrachromosomal loop that juxtaposes a downstream enhancer to the gene’s promoter, enabling activation of endogenous stemness genes. None of these long-range interactions were observed in URCs. Knockdown of the cohesin-complex gene SMC1 by RNAi abolished the intrachromosomal interaction and affected pluripotency. These findings highlight the importance of the SMC1-orchestrated intrachromosomal loop as a critical epigenetic barrier to the induction of pluripotency

An inherited FGFR2 mutation increased osteogenesis gene expression and result in Crouzon syndrome

Abstract Background FGFR2 encodes a fibroblast growth factor receptor whose mutations are responsible for the Crouzon syndrome, involving craniosynostosis and facial dysostosis with shallow orbits. However, few reports are available quantifying the orbital volume of Crouzon syndrome and there was little direct evidence to show FGFR2 mutation actually influencing orbital morphology. Methods Ten Crouzon syndrome patients underwent a standard ophthalmologic assessment. Morphology study was carried out based on 3-dimensional computed tomography scan to calculate orbital volume. Genomic DNA was extracted from peripheral blood leukocytes of the patients and genomic screening of FGFR2. A three-dimensional computer model was used to analyse the structural positioning of the mutation site that was predicted possible impact on functional of FGFR2 protein. Real-time PCR was performed to analyse the expression of bone maker gene. Results We describe a FGFR2 mutation (p.G338R, c.1012G > C) in a Chinese family with Crouzon syndrome. Computational analysis showed the mutate protein obviously changes in the local spatial structure compared with wild-type FGFR2. The expression of osteocalcin and alkaline phosphatase two osteoblast specific genes significantly increased in orbital bone directly from patient compared to normal individual, which may lead to facial dysostosis. This is compatible with the shallow and round orbits in our Crouzon syndrome patient. Conclusions Our study further identified G338R FGFR2 mutation (c1012G > C) lead to inherited Crouzon syndrome. Thus, early intervention, both medically and surgically, as well as disciplined by a multiple interdisciplinary teams are crucial to the management of this disorder

Novel Insights into the Role of Long Noncoding RNA in Ocular Diseases

Recent advances have suggested that long noncoding RNAs (lncRNAs) are differentially expressed in ocular tissues and play a critical role in the pathogenesis of different types of eye diseases. Here, we summarize the functions and mechanisms of known aberrantly-expressed lncRNAs and present a brief overview of relevant reports about lncRNAs in such ocular diseases as glaucoma, proliferative vitreoretinopathy (PVR), diabeticretinopathy (DR), and ocular tumors. We intend to highlight comprehensive studies that provide detailed data about the mechanisms of lncRNAs, their applications as diagnostic or prognostic biomarkers, and their potential therapeutic targets. Although our understanding of lncRNAs is still in its infancy, these examples may provide helpful insights into the methods by which lncRNAs interfere with ocular diseases