Retinal microglia protect against vascular damage in a mouse model of retinopathy of prematurity.

Retinopathy of prematurity (ROP) is a common cause of blindness in preterm babies. As a hypoxia-induced eye disease characterized by neovascularization, its association with retinal microglia has been noted but not well documented. We performed a comprehensive analysis of retinal microglia and retinal vessels in mouse oxygen-induced retinopathy (OIR), an animal model of ROP. In combination with a pharmacological inhibitory strategy, the role of retinal microglia in vascular network maintenance was investigated. Postnatal day (P) 7 C57BL/6J mouse pups with their nursing mother were exposed to 75% oxygen for 5 days to induce OIR. Age-matched room air-treated pups served as controls. On P12, P17, P21, P25, and P30, retinal microglia and vessels were visualized and quantified based on their location and activation status. Their relationship with retinal vessels was also analyzed. On P5 or P12, retinal microglia inhibition was achieved by intravitreal injection of liposomes containing clodronate (CLD); retinal vasculature and microglia were examined in P12 and P17 OIR retinae. The number of retinal microglia was increased in the superficial areas of OIR retinae on P12, P17, P21, P25, and P30, and most of them displayed an amoeboid (activated) morphology. The increased retinal microglia were associated with increased superficial retinal vessels in OIR retinae. The number of retinal microglia in deep retinal areas of OIR retinae also increased from P17 to P30 with a ramified morphology, which was not associated with reduced retinal vessels. Intravitreal injection of liposomes-CLD caused a significant reduction in retinal microglia. Loss of retinal microglia before hyperoxia treatment resulted in increased vessel obliteration on P12 and subsequent neovascularization on P17 in OIR retinae. Meanwhile, loss of retinal microglia immediately after hyperoxia treatment on P12 also led to more neovascularization in P17 OIR retinae. Our data showed that activated microglia were strongly associated with vascular abnormalities upon OIR. Retinal microglial activation continued throughout OIR and lasted until after retinal vessel recovery. Pharmacological inhibition of retinal microglia in either hyperoxic or hypoxic stage of OIR exacerbated retinal vascular consequences. These results suggested that retinal microglia may play a protective role in retinal vasculature maintenance in the OIR process.

CircRTN4 promotes pancreatic cancer progression through a novel CircRNA-miRNA-lncRNA pathway and stabilizing epithelial-mesenchymal transition protein.

BACKGROUND: Circular RNAs (circRNAs) play important roles in many biological processes. However, the detailed mechanism underlying the critical roles of circRNAs in cancer remains largely unexplored. We aim to explore the molecular mechanisms of circRTN4 with critical roles in pancreatic ductal adenocarcinoma (PDAC). METHODS: CircRTN4 expression level was examined in PDAC primary tumors. The oncogenic roles of circRTN4 in PDAC tumor growth and metastasis were studied in mouse tumor models. Bioinformatics analysis, luciferase assay and miRNA pulldown assay were performed to study the novel circRTN4-miRNA-lncRNA pathway. To identify circRTN4-interacting proteins, we performed circRNA-pulldown and mass spectrometry in PDAC cells. Protein stability assay and 3-Dimensional structure modeling were performed to reveal the role of circRTN4 in stabilizing RAB11FIP1. RESULTS: CircRTN4 was significantly upregulated in primary tumors from PDAC patients. In vitro and in vivo functional studies revealed that circRTN4 promoted PDAC tumor growth and liver metastasis. Mechanistically, circRTN4 interacted with tumor suppressor miR-497-5p in PDAC cells. CircRTN4 knockdown upregulated miR-497-5p to inhibit the oncogenic lncRNA HOTTIP expression. Furthermore, we identified critical circRTN4-intercting proteins by circRNA-pulldown in PDAC cells. CircRTN4 interacted with important epithelial-mesenchymal transition (EMT)- driver RAB11FIP1 to block its ubiquitination site. We found that circRTN4 knockdown promoted the degradation of RAB11FIP1 by increasing its ubiquitination. Also, circRTN4 knockdown inhibited the expression of RAB11FIP1-regulating EMT-markers Slug, Snai1, Twist, Zeb1 and N-cadherin in PDAC. CONCLUSION: The upregulated circRTN4 promotes tumor growth and liver metastasis in PDAC through the novel circRTN4-miR-497-5p-HOTTIP pathway. Also, circRTN4 stabilizes RAB11FIP1 to contribute EMT.

Autophagic Upregulation Is Cytoprotective in Ischemia/Reperfusion-Injured Retina and Retinal Progenitor Cells.

The cytoprotective versus cytotoxic role of macroautophagy in ocular ischemia/reperfusion injuries remains controversial and its effects under hyperglycemia are unclear. We investigated the involvement of autophagy in in vitro and in vivo normoglycemic and hyperglycemic models of retinal ischemia/reperfusion injury. Retinal ischemia (2 h) and reperfusion (2 or 22 h) was induced in wild-type and type I diabetic Ins2Akita/+ mice using a middle cerebral artery occlusion model. R28 retinal precursor cells were subjected to CoCl2-induced hypoxia with or without autophagic inhibitor NH4Cl. Autophagic regulation during ischemia/reperfusion was assessed through immunohistochemical detection and Western blotting of microtubule-associated protein 1A/1B-light chain 3 (LC3) and lysosomal associated membrane protein 1 (LAMP1). Effect of autophagic inhibition on cell viability and morphology under hypoxic conditions was also evaluated. Upregulation of autophagic markers in the inner retinae was seen after two hours reperfusion, with tapering of the response following 22 h of reperfusion in vivo. LC3-II turnover assays confirmed an increase in autophagic flux in our hypoxic in vitro model. Pharmacological autophagic inhibition under hypoxic conditions decreased cell survival and induced structural changes not demonstrated with autophagic inhibition alone. Yet no statistically significant different autophagic responses in ischemia/reperfusion injuries were seen between the two glycemic states.

LLGL1 Regulates Gemcitabine Resistance by Modulating the ERK-SP1-OSMR Pathway in Pancreatic Ductal Adenocarcinoma.

BACKGROUND & AIMS: Gemcitabine resistance is rapidly acquired by pancreatic ductal adenocarcinoma (PDAC) patients. Novel approaches that predict the gemcitabine response of patients and enhance gemcitabine chemosensitivity are important to improve patient survival. We aimed to identify genes as novel biomarkers to predict the gemcitabine response and the therapeutic targets to attenuate chemoresistance in PDAC cells. METHODS: Genome-wide RNA interference screening was conducted to identify genes that regulated gemcitabine chemoresistance. A cell proliferation assay and a tumor formation assay were conducted to study the role of lethal giant larvae homolog 1 (LLGL1) in gemcitabine chemoresistance. Levels of LLGL1 and its regulating targets were measured by immunohistochemical staining in tumor tissues obtained from patients who received gemcitabine as a single therapeutic agent. A gene-expression microarray was conducted to identify the targets regulated by LLGL1. RESULTS: Silencing of LLGL1 markedly reduced the gemcitabine chemosensitivity in PDAC cells. Patients had significantly shorter survival (6 months) if they bore tumors expressing low LLGL1 level than tumors with high LLGL1 level (20 months) (hazard ratio, 0.1567; 95% CI, 0.05966-0.4117). Loss of LLGL1 promoted cytokine receptor oncostatin M receptor (OSMR) expression in PDAC cells that led to gemcitabine resistance, while knockdown of OSMR effectively rescued the chemoresistance phenotype. The LLGL1-OSMR regulatory pathway showed great clinical importance because low LLGL1 and high OSMR expressions were observed frequently in PDAC tissues. Silencing of LLGL1 induced phosphorylation of extracellular signal-regulated kinase 2 and specificity protein 1 (Sp1), promoted Sp1 (pThr453) binding at the OSMR promoter, and enhanced OSMR transcription. CONCLUSIONS: LLGL1 possessed a tumor-suppressor role as an inhibitor of chemoresistance by regulating OSMR-extracellular signal-regulated kinase 2/Sp1 signaling. The data sets generated and analyzed during the current study are available in the Gene Expression Omnibus repository (ID: GSE64681).

Prevalence of occult hepatitis B infection in a highly endemic area for chronic hepatitis B: a study of a large blood donor population.

BACKGROUND AND AIMS: The aim of the present study was to determine the population prevalence of occult hepatitis B (OHB) infection and its clinical profile in a highly endemic area of chronic hepatitis B virus disease. METHODS: OHB was first identified by individual sample testing for hepatitis B surface antigen (HBsAg) followed by nucleic acid testing (NAT) and vice versa for 3044 (cohort 1, stored sera from donation within 1 year) and 9990 (cohort 2, prospective study) blood donors, respectively. OHB was confirmed meticulously by ≥2 out of 3 tests with detectable hepatitis B virus (HBV) DNA using a sensitive standardised assay. Detailed serology and viral load in the serum and liver were studied. RESULTS: The prevalence of OHB was 0.13% (4/3044) and 0.11% (11/9967) for cohort 1 and 2, respectively. In cohort 2, 10 out of 11 OHB samples were positive for anti-HBc (hepatitis B core antigen) antibody (all were immunoglobulin G). Seven had detectable anti-HBs. The serum HBV DNA levels were extremely low (highest 14.1 IU/ml). Of the six donors who underwent liver biopsies, all had normal liver biochemistry, extremely low liver HBV DNA (highest 6.21 copies/cell) and nearly normal liver histology. For those with viral sequence generation, none had the common HBsAg mutant G145R. CONCLUSIONS: The prevalence of OHB in a highly endemic area of chronic HBV was very low, thus implying a low impact on transfusion services. To implement universal screening, the high cost of NAT should be taken into account. OHB blood donors had very low HBV replication, and normal liver biochemistry and histology, conferring a favourable prognosis.