RNA binding protein ELAVL1-mediated USP33 stabilizes HIF1A to promote pathological proliferation, migration and angiogenesis of RECs.

BACKGROUND: Dysfunction of retinal vascularization plays pathogenic roles in retinopathy of prematurity (ROP). Hypoxia-inducible factor 1 alpha (HIF1A) is activated by hypoxia and contributes to ROP progression. Herein, we clarified the mechanism underlying HIF1A activation in human retinal vascular endothelial cells (HRECs) under hypoxia. METHODS: Protein expression was assayed by immunoblot analysis. Cell migration, microtubule formation, invasion, proliferation, and viability were detected by wound-healing, tube formation, transwell, EdU, and CCK-8 assays, respectively. Bioinformatics was used to predict the deubiquitinase-HIF1A interactions and RNA binding proteins (RBPs) bound to USP33. The impact of USP33 on HIF1A deubiquitination was validated by immunoprecipitation (IP) assay. RNA stability analysis was performed with actinomycin D (Act D) treatment. The ELAVL1/USP33 interaction was assessed by RNA immunoprecipitation experiment. RESULTS: In hypoxia-exposed HRECs, HIF1A and USP33 protein levels were upregulated. Deficiency of HIF1A or USP33 suppressed cell migration, proliferation and microtubule formation of hypoxia-exposed HRECs. Mechanistically, USP33 deficiency led to an elevation in HIF1A ubiquitination and degradation. USP33 deficiency reduced HIF1A protein levels to suppress the proliferation and microtubule formation of hypoxia-induced HRECs. Moreover, the RBP ELAVL1 stabilized USP33 mRNA to increase USP33 protein levels. ELAVL1 decrease repressed the proliferation and microtubule formation of hypoxia-induced HRECs by reducing USP33. CONCLUSION: Our study identifies a novel ELAVL1/USP33/HIF1A regulatory cascade with the ability to affect hypoxia-induced pathological proliferation, angiogenesis, and migration in HRECs.

Hsa_circ_0000527 Downregulation Suppresses the Development of Retinoblastoma by Modulating the miR-27a-3p/HDAC9 Pathway.

BACKGROUND: Accumulating evidence indicates that the progression of retinoblastoma (RB) may involve circRNA dysfunction. We aimed to disclose the role of hsa_circ_0000527 and its potential functional mechanism in RB. METHODS: The expression of hsa_circ_0000527, miR-27a-3p and histone deacetylase 9 (HDAC9) mRNA was monitored using quantitative real-time polymerase chain reaction (qPCR). Functional assays, including cell proliferation and apoptosis, were investigated using cell counting kit-8 (CCK-8) assay, colony formation assay and flow cytometry assay. The expression of apoptosis-associated proteins and HDAC9 protein was detected by western blot. The targeting relationship between miR-27a-3p and hsa_circ_0000527 or HDAC9 was verified by dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. Besides, Xenograft models were constructed to confirm the effect of hsa_circ_0000527 in vivo. RESULTS: Hsa_circ_0000527 and HDAC9 were upregulated, while miR-27a-3p was downregulated in RB tissues and cells. Hsa_circ_0000527 downregulation repressed RB cell proliferation and induced RB cell apoptosis. MiR-27a-3p was a target of hsa_circ_0000527, and hsa_circ_0000527 suppressed the expression of miR-27a-3p. MiR-27a-3p inhibition reversed the role of hsa_circ_0000527 downregulation. In addition, HDAC9 was a target of miR-27a-3p, and hsa_circ_0000527 indirectly regulated HDAC9 expression by targeting miR-27a-3p. MiR-27a-3p restoration inhibited RB cell proliferation and promoted apoptosis, which was reversed by HDAC9 overexpression. Hsa_circ_0000527 downregulation could inactivate the PI3K/AKT pathway. Moreover, hsa_circ_0000527 downregulation blocked tumor growth rate in vivo. CONCLUSION: hsa_circ_0000527 downregulation blocked the progression of RB by regulating the miR-27a-3p/HDAC9 pathway, which might be associated with the inactivation of the PI3K/AKT pathway.

Partial lamellar keratoplasty for peripheral corneal disease using a graft from the glycerin-preserved corneoscleral rim.

BACKGROUND: To evaluate the feasibility of partial lamellar keratoplasty (LK) for treatment of peripheral corneal disease (PCD) using a graft from the corneoscleral rim preserved in glycerin. METHODS: Patients who underwent LK for PCD at Shandong Eye Hospital from January 2006 to December 2012 were included. Corneoscleral rims of donor grafts, out of which the corneal buttons had been punched and used for penetrating keratoplasty, were preserved in glycerin and used for LK procedures. A trephine of 7.5-8.0 mm was used to mark the central cornea. An excision was made according to the size of the lesions, after which a partial ring-shaped corneoscleral graft was transplanted to repair the peripheral cornea. Visual acuity, refractive astigmatism, and complications were monitored. RESULTS: There were 26 patients (28 eyes), including 15 patients with Terrien's marginal degeneration, eight with Mooren's ulcer, two with Wegener's granulomatosis, and one with peripheral corneal ulcer. The mean follow-up was 15 months (range, 6-36 months). Uncorrected visual acuity was improved by a mean of 3.17 Snellen chart lines (P = 0.010), and best spectacle-corrected visual acuity was improved by a mean of 0.85 Snellen chart lines (P = 0.045) at 6 months after surgery. The refractive astigmatism decreased by 5.1 ± 8.6 diopters (P = 0.003). Two patients suffering graft ulcers (recurrence) were successfully treated by a second partial LK. No graft rejection occurred. CONCLUSIONS: Partial LK using a graft from the corneoscleral rim appears to be effective in the treatment of patients with PCD. This approach saves the donor graft, which is important in the countries or regions with scarcity of donor tissue.

[Comparison of Pentacam and OrbscanII systems for the diagnosis of keratoconus suspects].

OBJECTIVE: To compare the sensitivity of Pentacam and OrbscanII and to analyze the morphology characteristic of topography in normal eyes and different stages of keratoconus, to provide the basis for early screening of keratoconus suspects. METHODS: One hundred and ninety six normal eyes, 50 eyes with keratoconus suspect and 73 eyes with clinical keratoconus were enrolled. The changes of corneal anterior/posterior curvature, best fitting sphere (BFS). Inferior-Superior value (I-S) (3 mm and 5 mm), classification of elevation maps and corneal thickness were measured. RESULTS: There were significant differences including posterior curvature, anterior/posterior BFS, elevation value and corneal thickness in normal eyes between the data measured by these two different instruments, values obtained by Pentacam were less than those by Orbscan. Concerning the elevation maps pattern, the progressively decreasing pattern was dominant in Pentacam with normal eyes and keratoconus suspects, with the progressively increasing pattern accounting for 14.4% (28/196) and 20.0% (10/50) respectively in anterior surface, and 2.0% (41/96) and 26.0% (13/50) in posterior surface. The progressively increasing pattern was in dominant with clinical keratoconus, accounting for 91.7% (67/73) and 94.5% (69/73) respectively in anterior and posterior surface. Progressively increasing pattern in Orbscan was presented in 80.4% (165/196) normal eyes and suspected or clinical stage keratoconus. As to the receiver-operating-characteristic (ROC) curves and cutoff value, Orbscan I-S value (3 mm and 5 mm) of anterior and posterior surface was sensitive for the diagnosis of keratoconus suspects, with the cutoff value of anterior and posterior 5 mm I-S at 1.15 D and 0.65 D, respectively. In addition to the sensitivity of I-S values in Pentacam, the elevation value of anterior and posterior surface also displayed important diagnostic meaning. The cutoff of anterior, posterior elevation values was 4.5 µm and 7.5 µm, respectively. CONCLUSIONS: Numerous measured indicators revealed obviously difference between the two instruments in normal eyes, with slightly smaller values in Pentacam. In addition to I-S values, pattern changes and elevation values in elevation map of Pentacam represent remarkable advantages for the screening of keratoconus suspects. There is an incremental risk of corneal ectasia in the increasing pattern of elevation map.