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Backgrounds: Retinal vascular dysfunction is an important factor to the progression of diabetic retinopathy(DR). Multiple abnormal microRNAs (miRNAs) contributes to the pathogenesis of vascular dysfunction. However, the role and underlying mechanism of miR-424 in retinal vascular endothelial cells dysfunction under hyperglycemia stress remain obscure. Methods: Rhesus macaque choroid retinal endothelial cell line (RF/6A) cells were cultured under normal glucose (NG) and high glucose (HG) condition. qPCR was used to quantify the mRNA expression of miR-424 and Cyclin D1 (CCND1) and western blot was applied to detect the protein amount of CCND1. RF/6A cells were transfected with miR-424 mimics, miR-424 inhibitor, miR-424 inhibitor+ siRNA-CCND1 or with vehicle molecules. The cell proliferation, wound healing, tube formation ability and cell cycle were evaluated by MTT assay, scratch wound healing assay, tube formation assay and flow cytometric analysis, respectively. Interaction between miR-424 and CCND1 was predicted with bioinformatics and confirmed by Dual Luciferase Reporter Analysis.Results: Compared with NG, miR-424 was up-regulated and cell phenotype such as proliferation, wound healing and tube formation were inhibited in HG. The phenotypes can be reversed by inverting miR-424 expression under different conditions. CCND1 was confirmed as one of target genes of miR-424 and it can be modulated at transcriptional or translation level. Manipulation of silencing CCND1 can reverse the influences, such as promotion in proliferation, scratch wound tube formation and cell cycle, induced by transfecting miR-424 inhibitor into RF/6A cells under HG. Conclusions: Overexpression of miR-424 in RF/6A cells under HG stress significantly inhibited the cell function such as cell proliferation, wound healing, tube formation through suppressing CCND1 and blocking cell cycle.

The lack of axonal regeneration is the major cause of vision loss after optic nerve injury in adult mammals. Activating the PI3K/AKT/mTOR signaling pathway has been shown to enhance the intrinsic growth capacity of neurons and to facilitate axonal regeneration in the central nervous system after injury. The deletion of the mTOR negative regulator phosphatase and tensin homolog (PTEN) enhances regeneration of adult corticospinal neurons and ganglion cells. In the present study, we used a tyrosine-mutated (Y444F) AAV2 vector to efficiently express a short hairpin RNA (shRNA) for silencing PTEN expression in retinal ganglion cells. We evaluated cell survival and axonal regeneration in a rat model of optic nerve axotomy. The rats received an intravitreal injection of wildtype AAV2 or Y444F mutant AAV2 (both carrying shRNA to PTEN) 4 weeks before optic nerve axotomy. Compared with the wildtype AAV2 vector, the Y444F mutant AAV2 vector enhanced retinal ganglia cell survival and stimulated axonal regeneration to a greater extent 6 weeks after axotomy. Moreover, post-axotomy injection of the Y444F AAV2 vector expressing the shRNA to PTEN rescued ~19% of retinal ganglion cells and induced axons to regenerate near to the optic chiasm. Taken together, our results demonstrate that PTEN knockdown with the Y444F AAV2 vector promotes retinal ganglion cell survival and stimulates long-distance axonal regeneration after optic nerve axotomy. Therefore, the Y444F AAV2 vector might be a promising gene therapy tool for treating optic nerve injury.

Activating PI3K/AKT/mTOR signaling pathway via deleting phosphatase and tensin homolog (PTEN) has been confirmed to enhance intrinsic growth capacity of neurons to facilitate the axons regeneration of central nervous system after injury. Considering conditional gene deletion is currently not available in clinical practice, we exploited capsid residue tyrosine 444 to phenylalanine mutated single-stranded adeno-associated virus serotype 2 (AAV2) as a vector delivering short hairpin RNA to silence PTEN to promote retinal ganglion cells (RGCs) survival and axons regeneration in adult rat optic nerve axotomy paradigm. We found that mutant AAV2 displayed higher infection efficiency to RGCs and Müller cells by intravitreal injection, mediated PTEN suppression, resulted in much more RGCs survival and more robust axons regeneration compared with wild type AAV2, due to the different extent of the mTOR complex-1 activation and glutamate aspartate transporter (GLAST) regulation. These results suggest that high efficiency AAV2-mediated PTEN knockdown represents a practicable therapeutic strategy for optic neuropathy.

Age-related macular degeneration (AMD), the leading cause of visual loss after the age of 60 years, is a degenerative retinal disease involving a variety of environmental and hereditary factors. Although it has been implicated that immune system is involved in the disease progression, the exact role that microglia has is still unclear. Here we demonstrated that knockout of Ccr2 gene could alleviate photoreceptor cell death in mice retinas exposed to chronic blue light. In Ccr2−/− mice, a damaged microglia recruitment was shown in retina and this could protect the visual function in electroretinogram and alleviate the photoreceptor apoptosis, which thus helped attenuate the blue light-induced retinopathy. We further found an increased co-location of NLRP3, Iba-1, and IL-1β in fluorescence and a concomitant increased protein expression of NLRP3, caspase-1, and IL-1β in western blotting in chronic blue light-induced retinopathy. Moreover, the activation of microglia and their cellular NLRP3 inflammasomes occurred as an earlier step before the structural and functional damage of the mice retinas, which collectively supported that microglial NLRP3 inflammasome might be the key to the chronic blue light-induced retinopathy.

Recently, long non-coding RNAs (lncRNAs) are identified as new crucial regulators of diverse cellular processes, including cell proliferation, differentiation and cancer cells metastasis. Accumulating evidence has revealed that aberrant lncRNA expression plays important roles in carcinogenesis and tumor progression. However, the expression pattern and biological function of lncRNAs in non-small-cell lung cancer (NSCLC) remain largely unknown. In this study, we performed comprehensive analysis of lncRNA expression in human NSCLC samples by using microarray data from Gene Expression Omnibus. After validation in a cohort of 80 pairs of NSCLC tissues, we identified a differentially expressed novel oncogenic lncRNA termed as AGAP2-AS1. The AGAP2-AS1 expression level was significantly upregulated in NSCLC tissues and negatively correlated with poor prognostic outcomes in patients. In vitro loss- and gain-of-function assays revealed that AGAP2-AS1 knockdown inhibited cell proliferation, migration and invasion, and induced cell apoptosis. In vivo assays also confirmed the ability of AGAP2-AS1 to promote tumor growth. Furthermore, mechanistic investigation showed that AGAP2-AS1 could bind with enhancer of zeste homolog 2 and lysine (K)-specific demethylase 1A, and recruit them to KLF2 and LATS2 promoter regions to repress their transcription. Taken together, our findings indicate that AGAP2-AS1 may act as an oncogene by repressing tumor-suppressor LATS2 and KLF2 transcription. By clarifying the AGAP2-AS1 mechanisms underlying NSCLC development and progression, these findings might promote the development of novel therapeutic strategies for this disease.