To investigate the effect of glucose transporter-1 (GLUT1) inhibition on diabetic retinopathy, we divided forty-eight mice into scrambled siRNA, diabetic scrambled siRNA, and GLUT1 siRNA (intravitreally injected) groups. Twenty-one weeks after diabetes induction, we calculated retinal glucose concentrations, used electroretinography (ERG) and histochemical methods to assess photoreceptor degeneration, and conducted immunoblotting, leukostasis and vascular leakage assays to estimate microangiopathy. The diabetic scrambled siRNA and GLUT1 siRNA exhibited higher glucose concentrations than scrambled siRNA, but GLUT1 siRNA group concentrations were only 50.05% of diabetic scrambled siRNA due to downregulated GLUT1 expression. The diabetic scrambled siRNA and GLUT1 siRNA had lower ERG amplitudes and ONL thicknesses than scrambled siRNA. However, compared with diabetic scrambled siRNA, GLUT1 siRNA group amplitudes and thicknesses were higher. Diabetic scrambled siRNA cones were more loosely arranged and had shorter outer segments than GLUT1 siRNA cones. ICAM-1 and TNF-α expression levels, adherent leukocyte numbers, fluorescence leakage areas and extravasated Evans blue in diabetic scrambled siRNA were higher than those in scrambled siRNA. However, these parameters in the GLUT1 siRNA were lower than diabetic scrambled siRNA. Together, these results demonstrate that GLUT1 siRNA restricted glucose transport by inhibiting GLUT1 expression, which decreased retinal glucose concentrations and ameliorated diabetic retinopathy.

Aberrant expression of the zinc finger protein (ZIC) family has been extensively reported to contribute to progression and metastasis in multiple human cancers. However, the functional roles and underlying mechanisms of ZIC2 in non-small cell lung cancer (NSCLC) are largely unknown. In this study, ZIC2 expression was evaluated using qRT-PCR, western blot, and immunohistochemistry, respectively. Animal experiments in vivo and functional assays in vitro were performed to investigate the role of ZIC2 in NSCLC. Luciferase assays and chromatin immunoprecipitation (ChIP) were carried out to explore the underlying target involved in the roles of ZIC2 in NSCLC. Here, we reported that ZIC2 was upregulated in NSCLC tissues, and high expression of ZIC2 predicted worse overall and progression-free survival of NSCLC patients. Silencing ZIC2 repressed tumorigenesis and reduced the anoikis resistance of NSCLC cells. Mechanical investigation further revealed that silencing ZIC2 transcriptionally inhibited Src expression and inactivated steroid receptor coactivator/focal adhesion kinase signaling, which further attenuated the anoikis resistance of NSCLC cells. Importantly, our results showed that the number of circulating tumor cells (CTCs) was positively correlated with ZIC2 expression in NSCLC patients. Collectively, our findings unravel a novel mechanism implicating ZIC2 in NSCLC, which will facilitate the development of anti-tumor strategies in NSCLC.

Background: MiR-182-5p, a cancer-related microRNA (miRNA), modulates tumorigenesis and patient outcomes in various human malignances. This study interroted the clinicopathological significance and molecular mechanisms of miR-182-5p in non-small cell lung cancer (NSCLC). Methods: The clinical significance of miR-182-5p in NSCLC subtypes was determined based on an analysis of 124 samples (lung adenocarcinomas [LUADs], n = 101; lung squamous cell carcinomas [LUSCs], n = 23) obtained from NSCLC patients and paired noncancer tissues and an analysis of data obtained from public miRNA-seq database, miRNA-chip database, and the scientific literature. The NSCLC samples (n = 124) were analyzed using the real-time quantitative polymerase chain reaction (RT-qPCR). Potential targets of miR-182-5p were identified using lists generated by miRWalk v.2.0, a comprehensive atlas of predicted and validated targets of miRNA-target interactions. Molecular events of miR-182-5p in NSCLC were unveiled based on a functional analysis of candidate targets. The association of miR-182-5p with one of the candidate target genes, homeobox A9 (HOXA9), was validated using in-house RT-qPCR and dual-luciferase reporter assays. Results: The results of the in-house RT-qPCR assays analysis of data obtained from public miRNA-seq databases, miRNA-chip databases, and the scientific literature all supported upregulation of the expression level of miR-182-5p level in NSCLC. Moreover, the in-house RT-qPCR data supported the influence of upregulated miR-182-5p on malignant progression of NSCLC. In total, 774 prospective targets of miR-182-5p were identified. These targets were mainly clustered in pathways associated with biological processes, such as axonogenesis, axonal development, and Ras protein signal transduction, as well as pathways involved in axonal guidance, melanogenesis, and longevity regulation, in multiple species. Correlation analysis of the in-house RT-qPCR data and dual-luciferase reporter assays confirmed that HOXA9 was a direct target of miR-182-5p in NSCLC. Conclusions: The miR-182-5p expression level was upregulated in NSCLC tissues. MiR-182-5p may exert oncogenic influence on NSCLC through regulating target genes such as HOXA9.

Long noncoding RNAs (lncRNAs) regulate multiple biological effects in cancers. Recently, RNA methylation has been found to modify not only coding RNAs but also some noncoding RNAs. How RNA methylation affects lncRNAs to affect colorectal cancer (CRC) progression remains elusive. The expression of LINC01559 was explored through RNA sequencing, quantitative real-time PCR (qRT-PCR) and in situ hybridization (ISH). The preliminary exploration of its function was performed using Western blotting (WB) and immunohistochemistry (IHC). Functional experiments in vitro and in vivo were conducted to explore the biological functions of LINC01559 in CRC. The LINC01559/miR-106-5p/PTEN axis was verified through fluorescence in situ hybridization (FISH), luciferase assays, and rescue experiments. RIP-sequencing, m6A RNA immunoprecipitation (MeRIP) assays and bioinformatic analysis were conducted to determine the upstream mechanism of LINC01559. The results showed that LINC01559 was downregulated in CRC compared with normal controls. Lower expression of LINC01559 in CRC patients predicted a poor prognosis. In addition, PTEN was found to be positively correlated with LINC01559, and miR-106b-5p could be the link between LINC01559 and PTEN. Then, silencing LINC01559 restored the malignant phenotype of CRC cells, while cotransfection of miR-106b-5p inhibitor neutralized this effect. Mechanistically, we found abundant m6A modification sites on LINC01559. Then, we uncovered these sites as potential targets of METTL3 through experiments in vivo. The results revealed a negative functional regulation of the LINC01559/miR-106b-5p/PTEN axis in CRC progression and explored a new mechanism of METTL3-mediated m6A modification on LINC01559. These results elucidate a novel potential therapeutic target for CRC treatment.

To observe the effect of inhibiting long non-coding RNA (lncRNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) on diabetic neurodegeneration. Thirty-six 8-week-old C57BL/6 mice were randomly divided into normal control, diabetic control, diabetic scrambled small interfering RNAs (siRNAs) and diabetic MALAT1-siRNA groups. After diabetic induction with streptozocin intraperitoneally-injection, the diabetic MALAT1-siRNA group was intravitreally injected with 1 µL 20 µmol/L MALAT1 siRNA, and the diabetic scrambled siRNA group was injected with the same amount of scrambled siRNA. Electroretinography was performed to examine photoreceptor functions 16wk after diabetes induction. MALAT1 expression was detected via real time polymerase chain reaction. Cone morphological changes were examined using immunofluorescence. Rod morphological changes were examined by determining outer nuclear layer (ONL) thickness. The upregulation of retinal MALAT1 expression was detected in the diabetic control mice, while MALAT1 expression in the diabetic MALAT1-siRNA mice was decreased by 91.48% compared to diabetic control mice. The diabetic MALAT1-siRNA and diabetic control mice showed lower a-wave and b-wave amplitudes than did the normal control mice in scotopic and photopic electroretinogram, while the diabetic MALAT1-siRNA mice showed higher amplitudes than diabetic control mice. Morphological examination revealed that ONL thickness in the diabetic MALAT1-siRNA and diabetic control mice was lower than normal control mice. However, ONL thickness was greater in the diabetic MALAT1-siRNA mice than diabetic control mice. Moreover, the diabetic control mice performed a sparser cone cell arrangement and shorter outer segment morphology than diabetic MALAT1-siRNA mice. Inhibiting retinal MALAT1 results in mitigative effects on the retinal photoreceptors, thus alleviating diabetic neurodegeneration.

Purpose: Intricate signaling networks and transcriptional regulators translate pathogen recognition into defense responses. The aim of this study was to identify the weighted genes involved in diabetic retinopathy (DR) in different rodent models of diabetes. Methods: We performed a gene coexpression analysis of publicly available microarray data, namely, the GSE19122 dataset from the Gene Expression Omnibus database. We conducted gene coexpression analysis on the microarray data to identify modules of functionally related coexpressed genes that are differentially expressed in different rodent models. We leveraged a richly curated expression dataset and used weighted gene coexpression network analysis to construct an undirected network. We screened 30 genes in the most closely related module. A protein-protein interaction network was constructed for the genes in the most related module using the Search Tool for the Retrieval of Interacting Genes. Gene Ontology enrichment analysis and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis were performed for the 30 genes. Results: Five visual perception-related genes (Pde6g, Guca1a, Rho, Sag, and Prph2) were significantly upregulated. Based on the competing endogenous RNA hypothesis, a link between the long noncoding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) and visual perception-related mRNAs was constructed using bioinformatics tools. Six potential microRNAs (miR-155-5p, miR-1a-3p, miR-122-5p, miR-223-3p, miR-125b-5p, and miR-124-3p) were also screened. Conclusions: MALAT1 might play important roles in DR by regulating Sag and Guca1a through miR-124-3p and regulating Pde6g through miR-125b-5p.