Nc-RNA-mediated low expression of AZIN1 correlated with unfavorable prognosis in kidney renal clear cell carcinoma.

OBJECTIVE: Kidney renal clear cell carcinoma (KIRC, ccRCC) is the most common type of renal cancer with high recurrence and mortality. It has long been recognized that Antizyme inhibitor 1 (AZIN1) serves as a pro-oncogenic molecule in multiple cancers. However, the clinicopathological features of AZIN1 in KIRC remain unexplored. MATERIALS AND METHODS: The Cancer Genome Atlas (TCGA, TIMER, and GEPIA) were employed for pan-cancer expression and survival analysis of AZIN1, indicating the unique anti-tumor role of AZIN1 in KIRC. The expression and clinical characteristics of AZIN1 in KIRC were further proven via Human Protein Atlas and TCGA. single-sample GSEA was employed to investigate the immune infiltration of AZIN1. Then the downstream pathways were illustrated via the LinkedOmics, Metascape, and Cytoscape databases. The possible upper regulating noncoding RNAs (ncRNAs) were analyzed from five programs-TargetScan, StarBase, miRanda, PITA, and miRmap. RESULTS: AZIN1 is downregulated in KIRC patients. Lower levels of AZIN1 were linked with unfavorable outcomes in KIRC patients. The AZIN1 expression was positively related to immune cell infiltration in KIRC. We also elucidated a possible upstream regulatory ncRNA of AZIN1 in KIRC namely STK4-AS1/AC068338.2-miR-106b-5p-AZIN1 axis as well as the downstream signaling pathways. CONCLUSION: This study illustrated the unique anti-tumor role of AZIN1 in KIRC and provided potential value for guiding immunotherapy and targeted therapy.

Ame-miR-1-3p of bee venom reduced cell viability through the AZIN1/OAZ1-ODC1-polyamines pathway and enhanced the defense ability of honeybee (Apis mellifera L.).

Bee venom serves as an essential defensive weapon for bees and also finds application as a medicinal drug. MicroRNAs (miRNAs) serve as critical regulators and have been demonstrated to perform a variety of biological functions. However, the presence of miRNAs in bee venom needs to be confirmed. Therefore, we conducted small RNA sequencing and identified 158 known miRNAs, 15 conserved miRNAs and 4 novel miRNAs. It is noteworthy that ame-miR-1-3p, the most abundant among them, accounted for over a quarter of all miRNA reads. To validate the function of ame-miR-1-3p, we screened 28 candidate target genes using transcriptome sequencing and three target gene prediction software (miRanda, PITA and TargetScan) for ame-miR-1-3p. Subsequently, we employed real-time quantitative reverse transcription PCR (qRT-PCR), Western blot and other technologies to confirm that ame-miR-1-3p inhibits the relative expression of antizyme inhibitor 1 (AZIN1) by targeting the 3' untranslated region (UTR) of AZIN1. This, in turn, caused ODC antizyme 1 (OAZ1) to bind to ornithine decarboxylase 1 (ODC1) and mark ODC1 for proteolytic destruction. The reduction in functional ODC1 ultimately resulted in a decrease in polyamine biosynthesis. Furthermore, we determined that ame-miR-1-3p accelerates cell death through the AZIN1/OAZ1-ODC1-polyamines pathway. Our studies demonstrate that ame-miR-1-3p diminishes cell viability and it may collaborate with sPLA2 to enhance the defence capabilities of honeybees (Apis mellifera L.). Collectively, these data further elucidate the defence mechanism of bee venom and expand the potential applications of bee venom in medical treatment.

Extracellular vesicle­mediated RNA editing may underlie the heterogeneity and spread of hepatocellular carcinoma in human tissue and in vitro.

Extracellular vesicles (EVs) produced by various cells, including tumor cells, carry biomolecules to neighboring cells. In hepatocellular carcinoma (HCC), adenosine to inosine RNA editing of antizyme inhibitor 1 (AZIN1), specifically regulated by adenosine deaminase acting on RNA­1 (ADAR1), promotes carcinogenesis. The present study examined if EVs and ADAR1 in the EVs released from HCC cells are transferred to neighboring cells in co­culture systems and reporter assay. Distribution of the ADAR1 expression in human tissues were examined by immunohistochemistry. EVs released from HCC cells containing ADAR1 were delivered to neighboring HCC cells and non­cancerous hepatocytes. The increased ADAR1 protein levels resulted in serine to glycine substitution at residue 367 of AZIN1, which augmented transformation potential and increased aggressive behavior of cancer cells. In clinically resected samples, ADAR1 distribution was highly heterogeneous within the tumor specimen and denser in non­cancerous tissue surrounding the HCC tissue. These observations suggested that ADAR1 protein may be delivered from HCC cells to neighboring cells via EVs and that EV­mediated RNA editing may serve a pivotal role in determining HCC heterogeneity and spread.

ADAR1 and AZIN1 RNA editing function as an oncogene and contributes to immortalization in endometrial cancer.

OBJECTIVE: Adenosine-to-inosine (A-to-I) RNA editing is a recently described epigenetic modification, which is believed to constitute a key oncogenic mechanism in human cancers. However, its functional role and clinical significance in endometrial cancer (EC) remain unclear. METHODS: Adenosine Deaminase family Acting on RNA1 (ADAR1) expression and Antizyme inhibitor 1 (AZIN1) RNA editing were examined to clarify the correlation with clinicopathological parameters and prognosis in EC patients. The biological functions and inhibitory effects of ADAR1 knockdown were investigated in JHUCS-1 and TU-ECS-1 EC cell lines. RESULTS: ADAR1 showed significant association with worse histology (P = 0.006), and lymph vascular space involvement (P = 0.049) in EC. The level of AZIN1 RNA editing was also significantly associated with worse histology (P = 0.012). ADAR1 expression was significantly correlated with AZIN1 RNA editing level (R = 0.729, R2 = 0.547, P < 0.001). Multivariate analysis indicated that higher ADAR1 expression along with AZIN1 RNA editing is an independent predictor of prognosis in EC patients (P = 0.015). Knockdown of ADAR1 led to increased MDA-5, RIG-I, PKR, and IRF-7 expression, which in turn resulted in increased levels of Bak and apoptosis in EC cells. CONCLUSIONS: High ADAR1 expression along with AZIN1 RNA editing could be a predictor of worse prognosis in EC. ADAR1 could be a potential therapeutic target in EC patients.

LncRNA AZIN1-AS1 ameliorates myocardial ischemia-reperfusion injury by targeting miR-6838-5p/WNT3A axis to activate Wnt-ß/catenin signaling pathway.

Myocardial reperfusion, the effective therapy for acute myocardial infarction (AMI), commonly leads to myocardial ischemia/reperfusion (I/R) injury. The effects and functional mechanisms of LncRNA AZIN1-AS1 on myocardial I/R injury in vivo and vitro are not uncovered. In our present study, we established myocardial I/R injury model of mice and H/R injury model of cardiomyocytes and we discovered AZIN1-AS1 was decreased but miR-6838-5p was increased significantly in myocardial tissues injured by I/R treatment and H9c2 cells injured by hypoxia/reoxygenation (H/R) treatment. Silencing AZIN1-AS1 down-regulated cell viability but up-regulated apoptosis rate and CK-MB in addition LDH release of cardiomyocyte under H/R injury. However, overexpression of AZIN1-AS1 recovered abovementioned effects. Additionally, miR-6838-5p was found to be the direct target of AZIN1-AS1 and exhibited negative correlation with AZIN1-AS1. Moreover, miR-6838-5p inhibitor effectively eliminated the effects of AZIN1-AS1 knockdown on H/R-injured myocardial cells. Further experiments showed that WNT3A was the target of miR-6838-5p axis and overexpression of WNT3A also counteracted the roles of AZIN1-AS1 knockdown. Furthermore, knockdown of AZIN1-AS1 dramatically inhibited the activity of WNT-ß/catenin signaling pathway, which was recovered effectively by plasmid with overexpressing WNT3A. Therefore, this study firstly revealed that LncRNA AZIN1-AS1/miR-6838 axis inhibited apoptosis by activating WNT/ß-catenin pathway to protect mice or H9c2 cell from I/R-induced or H/R-induced injury respectively, which advised that AZIN1-AS1 could be regarded as a potential target for treating patients with AMI.

Long non-coding RNA MALAT1 aggravated liver ischemia-reperfusion injury via targeting miR-150-5p/AZIN1.

Long non-coding RNA (lncRNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) plays a crucial role in the process of renal ischemia-reperfusion (IR) injury and myocardial IR injury. However, its mechanism in liver IR injury is not clear. IR and hypoxia/reoxygenation (H/R) model were built on C57BL/6 mice. Blood samples were obtained from the inferior vena cava of the model mice. MALAT1 expression was detected in IR model and H/R model. Supported by experimental results, the impacts of MALAT1 on viability, apoptosis, and inflammation of H/R model cells were detected. The correlation between MALAT1 and downstream genes was analyzed by mechanism assays. MALAT1 was detected to be upregulated in IR model and H/R model. MALAT1 knockdown had inhibitory effects on apoptosis and inflammatory reaction while promoting liver cell viability in H/R condition. Meanwhile, MALAT1 targeted miR-150-5p to regulate antizyme inhibitor 1 (AZIN1) in liver cells. Finally, MALAT1 regulated viability, apoptosis, and inflammatory reaction of liver cells by targeting miR-150-5p and AZIN1. To conclude, MALAT1 targeted miR-150-5p/AZIN1 to accelerate liver IR injury, suggesting that MALAT1 might be a novel target for liver IR injury.

Targeting RNA editing of antizyme inhibitor 1: A potential oligonucleotide-based antisense therapy for cancer.

Dysregulated adenosine-to-inosine (A-to-I) RNA editing is implicated in various cancers. However, no available RNA editing inhibitors have so far been developed to inhibit cancer-associated RNA editing events. Here, we decipher the RNA secondary structure of antizyme inhibitor 1 (AZIN1), one of the best-studied A-to-I editing targets in cancer, by locating its editing site complementary sequence (ECS) at the 3' end of exon 12. Chemically modified antisense oligonucleotides (ASOs) that target the editing region of AZIN1 caused a substantial exon 11 skipping, whereas ECS-targeting ASOs effectively abolished AZIN1 editing without affecting splicing and translation. We demonstrate that complete 2'-O-methyl (2'-O-Me) sugar ring modification in combination with partial phosphorothioate (PS) backbone modification may be an optimal chemistry for editing inhibition. ASO3.2, which targets the ECS, specifically inhibits cancer cell viability in vitro and tumor incidence and growth in xenograft models. Our results demonstrate that this AZIN1-targeting, ASO-based therapeutics may be applicable to a wide range of tumor types.

AZIN1-AS1, A Novel Oncogenic LncRNA, Promotes the Progression of Non-Small Cell Lung Cancer by Regulating MiR-513b-5p and DUSP11.

BACKGROUND: Emerging researches have demonstrated that aberrantly expressed long non-coding RNAs (lncRNAs) have great significance in non-small cell lung cancer (NSCLC) progression. The aim of this study was to explore the role of lncRNA AZIN1 antisense RNA 1 (AZIN1-AS1) in NSCLC and the related mechanism. METHODS: Expressions of AZIN1-AS1 and miR-513b-5p in NSCLC samples were detected by qRT-PCR. NSCLC cell lines (H1299 and HCC827) were used in vitro assays. CCK-8 assay, EdU assay, wound healing test and Transwell assay were carried out to test the biological influence of AZIN1-AS1 on NSCLC cells. Subcutaneous xenotransplanted tumor model and tail vein injection model were established to test the role of AZIN1-AS1 in vivo. Interactions between AZIN1-AS1 and miR-513b-5p, miR-513b-5p and dual-specificity phosphatase 11 (DUSP11) were determined by bioinformatic analysis, qRT-PCR, Western blot, and luciferase reporter assay. RESULTS: AZIN1-AS1 was up-regulated in NSCLC cells and tissues, while miR-513b-5p was significantly down-regulated. Silencing of AZIN1-AS1 or overexpression of miR-513b-5p markedly inhibited proliferation, migration and invasion of NSCLC cells, while overexpression of AZIN1-AS1 or inhibition of miR-513b-5p functioned oppositely. Importantly, AZIN1-AS1 mediated the promotion of malignancy of NSCLC cells was reversed by miR-513b-5p mimics. What's more, AZIN1-AS1 could down-regulate miR-513b-5p via sponging it, and there existed a negative correlation between AZIN1-AS1 expression and miR-513b-5p expression in NSCLC samples. AZIN1-AS1 also enhanced the expression levels of DUSP11, which was proved as a target gene of miR-513b-5p. Further in vivo experiments showed that silencing of AZIN1-AS1 decreased tumor growth and metastasis, which was accompanied by overexpression of miR-513b-5p and inhibition of DUSP11 in tumor tissues. CONCLUSION: AZIN1-AS1 acts as a tumor promoter in NSCLC, which is ascribed to the regulation of miR-513b-5p and DUSP11.

Activation of AZIN1 RNA editing is a novel mechanism that promotes invasive potential of cancer-associated fibroblasts in colorectal cancer.

Adenosine-to-inosine (A-to-I) RNA editing is a recently described epigenetic modification, which is believed to constitute a key oncogenic mechanism in human cancers. However, its functional role in cancer-associated fibroblasts (CAFs) within the tumor microenvironment (TME) and its clinical significance remains unclear. Herein, we systematically analyzed a large cohort of 627 colorectal cancer (CRC) specimens, and investigated the expression pattern of ADAR1 and its biological significance on the antizyme inhibitor 1 (AZIN1) RNA editing levels. Both ADAR1 expression and AZIN1 RNA editing levels were significantly elevated in CRC tissues vs. normal mucosa, and these findings correlated with the increased expression of mesenchymal markers, Vimentin (ρ = 0.44) and Fibroblast activation protein (ρ = 0.38). Intriguingly, ADAR1 expression was specifically upregulated in both cancer cells and fibroblasts from cancerous lesions. Conditioned medium from cancer cells led to induction of ADAR1 expression and activation of AZIN1 RNA editing in fibroblasts (p < 0.05). Additionally, edited AZIN1 enhanced the invasive potential of fibroblasts. In conclusion, we provide novel evidence that hyper-editing of AZIN1 enhances the invasive potential of CAFs within the TME in colon and is an important predictor of tumor invasiveness in CRC.

Enhanced AZIN1 RNA editing and overexpression of its regulatory enzyme ADAR1 are important prognostic biomarkers in gastric cancer.

BACKGROUND: Adenosine-to-inosine (A-to-I) RNA editing is catalyzed by adenosine deaminases acting on RNA (ADAR) enzymes. Recent evidence suggests that RNA editing of antizyme inhibitor 1 (AZIN1) RNA is emerging as a key epigenetic alteration underlying cancer pathogenesis. METHODS: We evaluated AZIN1 RNA editing levels, and the expression of its regulator, ADAR1, in 280 gastric tissues from 140 patients, using a RNA editing site-specific quantitative polymerase chain reaction assays. We also analyzed the clinical significance of these results as disease biomarkers in gastric cancer (GC) patients. RESULTS: Both AZIN1 RNA editing levels and ADAR1 expression were significantly elevated in GC tissues compared with matched normal mucosa (P < 0.0001, 0.0008, respectively); and AZIN1 RNA editing was positively correlated with ADAR1 expression. Elevated expression of ADAR1 significantly correlated with poor overall survival (P = 0.034), while hyper-edited AZIN1 emerged as an independent prognostic factor for OS and disease-free survival in GC patients [odds ratio (OR):1.98, 95% CI 1.17-3.35, P = 0.011, OR: 4.55, 95% CI 2.12-9.78, P = 0.0001, respectively]. Increased AZIN1 RNA editing and ADAR1 over-expression were significantly correlated with key clinicopathological factors, such as advanced T stage, presence of lymph node metastasis, distant metastasis, and higher TNM stages in GC patients. Logistic regression analysis revealed that hyper-editing status of AZIN1 RNA was an independent risk factor for lymph node metastasis in GC patients [hazard ratio (HR):3.03, 95% CI 1.19-7.71, P = 0.02]. CONCLUSIONS: AZIN1 RNA editing levels may be an important prognostic biomarker in GC patients, and may serve as a key clinical decision-making tool for determining preoperative treatment strategies in GC patients.

Polyamine Control of Translation Elongation Regulates Start Site Selection on Antizyme Inhibitor mRNA via Ribosome Queuing.

Translation initiation is typically restricted to AUG codons, and scanning eukaryotic ribosomes inefficiently recognize near-cognate codons. We show that queuing of scanning ribosomes behind a paused elongating ribosome promotes initiation at upstream weak start sites. Ribosomal profiling reveals polyamine-dependent pausing of elongating ribosomes on a conserved Pro-Pro-Trp (PPW) motif in an inhibitory non-AUG-initiated upstream conserved coding region (uCC) of the antizyme inhibitor 1 (AZIN1) mRNA, encoding a regulator of cellular polyamine synthesis. Mutation of the PPW motif impairs initiation at the uCC's upstream near-cognate AUU start site and derepresses AZIN1 synthesis, whereas substitution of alternate elongation pause sequences restores uCC translation. Impairing ribosome loading reduces uCC translation and paradoxically derepresses AZIN1 synthesis. Finally, we identify the translation factor eIF5A as a sensor and effector for polyamine control of uCC translation. We propose that stalling of elongating ribosomes triggers queuing of scanning ribosomes and promotes initiation by positioning a ribosome near the start codon.

Crucial Role of miR-433 in Regulating Cardiac Fibrosis.

Dysregulation of microRNAs has been implicated in many cardiovascular diseases including fibrosis. Here we report that miR-433 was consistently elevated in three models of heart disease with prominent cardiac fibrosis, and was enriched in fibroblasts compared to cardiomyocytes. Forced expression of miR-433 in neonatal rat cardiac fibroblasts increased proliferation and their differentiation into myofibroblasts as determined by EdU incorporation, α-SMA staining, and expression levels of fibrosis-associated genes. Conversely, inhibition of miR-433 exhibited opposite results. AZIN1 and JNK1 were identified as two target genes of miR-433. Decreased level of AZIN1 activated TGF-ß1 while down-regulation of JNK1 resulted in activation of ERK and p38 kinase leading to Smad3 activation and ultimately cardiac fibrosis. Importantly, systemic neutralization of miR-433 or adeno-associated virus 9 (AAV9)-mediated cardiac transfer of a miR-433 sponge attenuated cardiac fibrosis and ventricular dysfunction following myocardial infarction. Thus, our work suggests that miR-433 is a potential target for amelioration of cardiac fibrosis.

Hepatocellular carcinoma: transcriptome diversity regulated by RNA editing.

Hepatocellular carcinoma (HCC) can be envisioned as a prolonged multi-stage process accumulating genetic and epigenetic changes. In the past years, DNA alterations lent us important clues to the comprehension of molecular pathways involved in HCC. However, as an increasing number of RNAs were identified to be subject to A-to-I modifications, it has become apparent that RNA editing might be the causal basis of various human diseases. Recent evidence has strengthened this notion by correlating hyper-edited AZIN1 (antizyme inhibitor 1) protein with HCC onset and the mechanisms that regulate cell transformation. As we continue to demystify it, RNA editing astonishes us with its diverse substrates, esoteric functions, elaborate machinery and complex interaction with HBV/HCV viral infection. In this review, we examine the contribution of A-to-I RNA editing to caner onset/progression and explore its potential implications for cancer treatment advances.