Transfer RNA derived fragment, tRF-Glu-CTC, aggravates the development of neovascular age-related macular degeneration.

Rationale: Angiogenesis expedites tissue impairment in many diseases, including age-related macular degeneration (AMD), a leading cause of irreversible blindness in elderly. A substantial proportion of neovascular AMD patients, characterized by aberrant choroidal neovascularization (CNV), exhibit poor responses or adverse reactions to anti-VEGF therapy. Herein, we aimed to unveil the function of newly identified transfer RNA-derived small RNA, tRF-Glu-CTC, in the pathology of CNV and determine its potential in inhibiting angiogenesis. Methods: Small non-coding RNA sequencing and quantitative polymerase chain reaction were conducted to detect expression pattern of tRF-Glu-CTC in CNV development. Immunofluorescence staining, fundus fluorescein angiography and ex vivo choroidal sprouting assays were employed for the evaluation of tRF-Glu-CTC's function in CNV development. The role of tRF-Glu-CTC in endothelial cells were determined by in vitro endothelial cell proliferation, migration and tube formation assays. Transcriptome sequencing, dual-luciferase reporter assay and in vitro experiments were conducted to investigate downstream mechanism of tRF-Glu-CTC mediated pathology. Results: tRF-Glu-CTC exhibited substantial up-regulation in AMD patients, laser-induced CNV model, and endothelial cells under hypoxia condition, which is a hallmark of CNV. Inhibiting tRF-Glu-CTC reduced angiogenesis and hypoxia stress in the neovascular region without neuroretina toxicity in laser-induced CNV model, showing an anti-angiogenic effect comparable to bevacizumab, while overexpression of tRF-Glu-CTC significantly augmented CNV. Mechanically, under hypoxia condition, angiogenin was involved in the production of tRF-Glu-CTC, which in turn triggered endothelial cell tubulogenesis, migration and promoted the secretion of inflammatory factors via the suppression of vasohibin 1 (VASH1). When downregulating VASH1 expression, the inhibition of tRF-Glu-CTC showed minimal suppression on angiogenesis. Conclusions: This study demonstrated the important role of tRF-Glu-CTC in the progression of angiogenesis. Targeting of tRF-Glu-CTC may be an alternative to current anti-VEGF therapy for CNV in AMD and other conditions with angiogenesis.

Identification and characterization of differentially expressed circular RNAs in extraocular muscle of oculomotor nerve palsy.

BACKGROUND: Oculomotor nerve palsy (ONP) is a neuroparalytic disorder resulting in dysfunction of innervating extraocular muscles (EOMs), of which the pathological characteristics remain underexplored. METHODS: In this study, medial rectus muscle tissue samples from four ONP patients and four constant exotropia (CXT) patients were collected for RNA sequencing. Differentially expressed circular RNAs (circRNAs) were identified and included in functional enrichment analysis, followed by interaction analysis with microRNAs and mRNAs as well as RNA binding proteins. Furthermore, RT-qPCR was used to validate the expression level of the differentially expressed circRNAs. RESULTS: A total of 84 differentially expressed circRNAs were identified from 10,504 predicted circRNAs. Functional enrichment analysis indicated that the differentially expressed circRNAs significantly correlated with skeletal muscle contraction. In addition, interaction analyses showed that up-regulated circRNA_03628 was significantly interacted with RNA binding protein AGO2 and EIF4A3 as well as microRNA hsa-miR-188-5p and hsa-miR-4529-5p. The up-regulation of circRNA_03628 was validated by RT-qPCR, followed by further elaboration of the expression, location and clinical significance of circRNA_03628 in EOMs of ONP. CONCLUSIONS: Our study may shed light on the role of differentially expressed circRNAs, especially circRNA_03628, in the pathological changes of EOMs in ONP.

The profiles and clinical significance of extraocular muscle-expressed lncRNAs and mRNAs in oculomotor nerve palsy.

Introduction: Oculomotor nerve palsy (ONP) arises from primary abnormalities in the central neural pathways that control the extraocular muscles (EOMs). Long non-coding RNAs (lncRNAs) have been found to be involved in the pathogenesis of various neuroparalytic diseases. However, little is known about the role of lncRNAs in ONP. Methods: We collected medial rectus muscle tissue from ONP and constant exotropia (CXT) patients during strabismus surgeries for RNA sequencing analysis. Differentially expressed mRNAs and lncRNAs were revealed and included in the functional enrichment analysis. Co-expression analysis was conducted between these differentially expressed mRNAs and lncRNAs, followed by target gene prediction of differentially expressed lncRNAs. In addition, lncRNA-microRNA and lncRNA-transcription factor-mRNA interaction networks were constructed to further elaborate the pathological changes in medial rectus muscle of ONP. Furthermore, RT-qPCR was applied to further validate the expression levels of important lncRNAs and mRNAs, whose clinical significance was examined by receiver operating characteristic (ROC) curve analysis. Results: A total of 618 differentially expressed lncRNAs and 322 differentially expressed mRNAs were identified. The up-regulated mRNAs were significantly related to cholinergic synaptic transmission (such as CHRM3 and CHRND) and the components and metabolism of extracellular matrix (such as CHI3L1 and COL19A1), while the down-regulated mRNAs were significantly correlated with the composition (such as MYH7 and MYL3) and contraction force (such as MYH7 and TNNT1) of muscle fibers. Co-expression analysis and target gene prediction revealed the strong correlation between MYH7 and NR_126491.1 as well as MYOD1 and ENST00000524479. Moreover, the differential expressions of lncRNAs (XR_001739409.1, NR_024160.1 and XR_001738373.1) and mRNAs (CDKN1A, MYOG, MYOD1, MYBPH, TMEM64, STATH, and MYL3) were validated by RT-qPCR. ROC curve analysis showed that lncRNAs (XR_001739409.1, NR_024160.1, and NR_002766.2) and mRNAs (CDKN1A, MYOG, MYOD1, MYBPH, TMEM64, and STATH) might be promising biomarkers of ONP. Conclusions: These results may shed light on the molecular biology of EOMs of ONP, as well as the possible correlation of lncRNAs and mRNAs with clinical practice.

CD47 expression in gastric cancer clinical correlates and association with macrophage infiltration.

BACKGROUND: CD47 has been identified as an innate immune checkpoint and found to be associated with inferior survival in various types of cancer. However, the critical role of CD47 in gastric cancer and its association with tumor associated macrophages remain unclear. METHODS: Tumor tissues of gastric cancer from Zhongshan Hospital and data from GSE62254, GSE84437 and TCGA datasets were analyzed. Immunohistochemistry was performed to detect the expression of CD47, CD11c, CD163 and CD68 in gastric cancer tissues. Kaplan-Meier curves and Cox model were used for comparing the clinical outcomes of patients belonging to different subgroups. RESULTS: Gastric cancer patients with high CD47 expression exhibited poor prognosis and inferior therapeutic responsiveness to fluorouracil-based adjuvant chemotherapy (ACT). A positive correlation was found between M1-polarized macrophage infiltration and CD47 expression in gastric cancer; however, the prognostic value of M1-polarized macrophages was attenuated in CD47-high gastric cancer patients. Moreover, we found that CD47 mRNA level was enriched in microsatellite-instable (MSI) subtype of gastric cancer and associated with ARID1A mutation and FGFR2 signaling pathway activation. CONCLUSIONS: Aberrant CD47 expression represented an independent predictor for adverse survival outcome and ACT resistance in gastric cancer. Targeting CD47 might be a promising strategy for gastric cancer patients.

Lymphocyte-activation gene 3 expression associates with poor prognosis and immunoevasive contexture in Epstein-Barr virus-positive and MLH1-defective gastric cancer patients.

Lymphocyte activation gene 3 (LAG-3) is a transmembrane immune checkpoint that facilitates immune escape via suppressing T-cell-mediated anti-tumor immunity. The role of LAG-3 in gastric cancer is little known. Consequently, we assessed the clinical significance of LAG-3 in gastric cancer. In our study, patients with gastric cancer from Zhongshan Hospital (n = 464) and data from the Asian Cancer Research Group (n = 300) were analyzed. LAG-3+ cell infiltration and other immune contexture in gastric cancer were detected by immunohistochemistry. Kaplan-Meier curves and log-rank test were used for survival analyses. Intratumoral LAG-3+ cells mainly accumulated in Epstein-Barr virus (EBV)-positive (EBV subtype) and MLH1-defective (dMLH1 subtype) gastric cancer. Furthermore, LAG-3+ cell infiltration was strongly associated with inferior clinical outcomes in patients with these two subtypes of gastric cancer. Moreover, we found intratumoral LAG-3+ cell high infiltration was associated with an immunoevasive contexture featured by decreased IFN-γ+ cells and perforin-1+ cells, but increased regulatory T cells and M2-like macrophages in EBV/dMLH1 subtype of gastric cancer. LAG-3 was a poor prognostic factor and might be a potential immunotherapeutic target in EBV-positive and MLH1-defective gastric cancer.

Tumor-infiltrating podoplanin+ cells in gastric cancer: clinical outcomes and association with immune contexture.

Podoplanin (PDPN) has been proved to have significant immunoregulatory effects in several types of malignancies and is considered to be a novel immune checkpoint molecule. However, the clinical significance of PDPN and its potential influence on immune contexture in gastric cancer remain obscure. Here, we aimed to investigate the clinical outcomes and immunoregulatory role of tumor-infiltrating PDPN+ cells (tPDPNs) in gastric cancer. A total of 454 tumor tissue microarray specimens and 68 fresh tumor tissues of gastric cancer patients from Zhongshan Hospital, and transcriptional data of 293 gastric cancer patients from The Cancer Genome Atlas were included. We demonstrated that tPDPNs high subgroup experienced worse overall survival and disease-free survival, and indicated inferior therapeutic responsiveness to fluorouracil-based adjuvant chemotherapy (ACT) in gastric cancer. The abundance of tPDPNs was correlated with an immunoevasive contexture characterized by pro-tumor macrophage and dysfunctional CD8+ T cell infiltration. Moreover, dysfunctional CD8+ T cells in tPDPNs high subgroup exhibited decreased interferon-γ, granzyme B and perforin-1 expression yet elevated programmed cell death-1 (PD-1) and T-cell immunoglobulin and mucin-domain containing-3 (TIM-3) expression. Stratification of gastric cancer patients into different risk groups based on tPDPNs and CD8+ T cells showed distinct prognosis, responsiveness to ACT and molecular characteristics. This study revealed that the abundance of tPDPNs could identify an immunoevasive contexture and might be applied as an independent predictor for poor prognosis and suboptimal ACT responsiveness. Thus, we recommended tPDPNs as a promising therapeutic target in gastric cancer.