lncRNA Antisense Non-coding RNA In The INK4 Locus Research Articles

Association of ANRIL Gene Single-Nucleotide Polymorphisms With Allergic Rhinitis in Kurdish Population From Kermanshah, Iran.

Allergic rhinitis (AR) is the most common inflammatory disorder of the upper airway caused by aberrant immune responses to allergens in genetically predisposed individuals. Recently, the long noncoding RNA (lncRNA) antisense noncoding RNA in the INK4 locus (ANRIL) has been identified as a novel genetic factor associated with increased AR risk. This study aimed to evaluate the potential correlation of ANRIL gene single nucleotide polymorphisms (SNPs) with AR risk in the Kurdish population of Kermanshah, Iran. In this case-control study, 130 AR patients and 130 healthy controls were recruited to genotype for two SNPs of the ANRIL gene (rs1333048 and rs10757278) using the Tetra-primer amplification refractory mutation system polymerase chain reaction (T-ARMS-PCR) method. Our results showed no significant difference for the alleles and genotypes frequency distribution of lncRNA ANRIL SNPs (rs1333048 and rs10757278) between AR patients and healthy controls (p > 0.05). Additionally, the dominant, additive and recessive genetic models of both SNPs were not associated with altered susceptibility to AR risk (p > 0.05). The results demonstrated that the ANRIL gene rs1333048 and rs10757278 polymorphisms might not be associated with susceptibility to AR in the Kurdish population of Kermanshah, Iran.

LncRNA ANRIL accelerates wound healing in diabetic foot ulcers via modulating HIF1A/VEGFA signaling through interacting with FUS.

Diabetic foot ulcer (DFU) is a frequently diagnosed complication of diabetes, and remains a heathcare burden worldwide. However, the pathogenesis of DFU is still largely unclear. The objective of this study is to delineate the function and underlying mechanism of lncRNA antisense non coding RNA in the INK4 locus (ANRIL) in endothelial progenitor cells (EPCs) and DFU mice. The DFU mouse model was established, and EPCs were subjected to high glucose (HG) treatment to mimic diabetes. qRT-PCR or western blot was employed to detected the expression of ANRIL, HIF1A, FUS and VEGFA. CCK-8 and Annexin V/PI staining were used to monitor cell proliferation and apoptosis. Wound healing, Transwell invasion and tube formation assays were conducted to assess cell migration, invasion and angiogenesis, respectively. The association between ANRIL and FUS was verified by RNA pull-down and RIP assays. Luciferase and ChIP assays were employed to investigate HIF1A-mediated transcriptional regulation of VEGFA and ANRIL. The histological alterations of DFU wound healing were observed by H&E and Masson staining. ANRIL was downregulated in peripheral blood samples of DFU patients, DFU mice and HG-treated EPCs. Mechanistically, ANRIL regulated HIFA mRNA stability via recruiting FUS. VEGFA and ANRIL were transcriptionally regulated by HIF1A. Functional experiments revealed that HG suppressed EPC proliferation, migration, invasion and tube formation, but promoted apoptosis via ANRIL/HIF1A axis. ANRIL accelerated DFU wound healing via modulating HIF1A expression in vivo. ANRIL accelerated wound healing in DFU via modulating HIF1A/VEGFA signaling in a FUS-dependent manner.

LncRNA ANRIL Facilitates Vascular Smooth Muscle Cell Proliferation and Suppresses Apoptosis via Modulation of miR-7/FGF2 Pathway in Intracranial Aneurysms.

Proliferation and apoptosis of vascular smooth muscle cells (VSMCs) are linked to intracranial aneurysm (IA) formation and progression. Long antisense noncoding RNA in the INK4 locus (ANRIL) has been reported to regulate VSMC functions in several cardiovascular diseases. However, little is known about how ANRIL influences VSMC proliferation and apoptosis during IA pathogenesis. The expression level of ANRIL in the plasma and arterial wall tissues of patients with IA was detected by real-time quantitative polymerase chain reaction. The functional role of ANRIL in the regulation of VSMC proliferation and apoptosis and its downstream regulatory mechanism were determined using Cell Counting Kit 8, immunofluorescence, terminal-deoxynucleotidyl transferase-mediated UTP nick end labeling, western blotting, luciferase reporter assay, and RNA immunoprecipitation assay. ANRIL was downregulated in the plasma and arterial wall tissues of patients with IA, when compared with control groups. Overexpression of ANRIL significantly promoted VSMC proliferation and blocked cell apoptosis. Mechanistic studies demonstrated that ANRIL directly bound to microRNA-7 (miR-7) and that overexpression of miR-7 overturned the increased cell proliferation and decreased cell apoptosis, which was induced by ANRIL restoration. Besides, further study showed that ANRIL positively regulated fibroblast growth factor 2 (FGF2) expression via targeting miR-7. These results suggested that ANRIL affects VSMC proliferation and apoptosis via regulation of the miR-7/FGF2 pathway in IA, which provided a potential novel strategy for the treatment of IA.

Differential non-coding RNAs expression profiles of invasive and non-invasive pituitary adenomas.

BackgroundPituitary adenoma (PA) accounts for 10–15% of all intracranial neoplasms. Despite their benign nature, PA often shows invasive growth. MicroRNAs (miRNAs) and long non-coding RNAs (lncRNAs) are a class of non-coding RNAs that play important roles in PA initiation and progression.AimThe aim of this study was to find specific profiles of miR-200a and long non-coding RNA (lncRNA) antisense non-coding RNA in the INK4 locus (ANRIL) in PA based on a comparative study using Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) analyses of tumor tissue and plasma.MethodsPlasma and PA tissue samples were obtained from two groups of included patients (15 invasive and 15 non-invasive PA). In addition, plasma samples from patients with invasive PA have collected pre- and post-operation. Plasma and tissue samples subjected to qRT-PCR analyses for the expression levels of miR-200a and lncRNA ANRIL.ResultsThe expression levels of miR-200a and lncRNA ANRIL were increased in tissue samples patients with invasive PA than in the patients with non-invasive PA. In addition, the expression levels of circulating miR-200a and lncRNA ANRIL were increased in patients with invasive PA than in patients with non-invasive PA in the pre-operation period. However, the expression level of plasma circulating miR-200a and lncRNA ANRIL was decreased in patients with invasive PA in the post-operation period. Our results depicted a miR-200a and lncRNA ANRIL expression in tissue and plasma samples in the patients with invasive PA. In addition, Receiver Operating Characteristic (ROC) curve was used to evaluate the diagnostic value of these circulating miR-200a and lncRNA ANRIL.ConclusionThe expression of these tumor-associated ncRNAs has been elevated in the PAs. Therefore, miR-200a and lncRNA ANRIL represents as biomarkers for diagnosis and potential targets for novel invasive PA treatment strategies.

Overexpression of long non-coding RNA ANRIL in B-acute lymphoblastic leukemia

Background: Dysregulation of LncRNA antisense non-coding RNA in the INK4 locus (ANRIL) expression is implicated in pathogenesis and disease progression of a variety of cancer types. However, the expression level of ANRIL in pediatric patients with B-cell precursor acute lymphoblastic leukemia (BCP-ALL) has not been elucidated, yet. The present study is an attempt to evaluate the expression level of ANRIL at different clinical stages in pediatric patients with BCP-ALL. Materials and Methods: This case-control study was conducted in Tehran, Iran on peripheral blood samples obtained at diagnosis, complete remission, and relapse phases from a total of 50 pediatric BCP-ALL patients who were admitted to Mahak Hospital and Rehabilitation Complex, and Rasul Akram Hospital. The ANRIL expression analysis was performed by the quantitative real-time polymerase chain reaction (qRT-PCR) method. To test the statistical significance, a nonparametric Mann-Whitney U test was used. Results: The mean fold-changes of ANRIL gene expression in newly diagnosed patients were [31.51 (18.28 to 44.75)] compared to the control group [1.06 (0.73 to 1.38)] indicating significant overexpression (P<0.001). ANRIL fold-changes significantly declined following achievement of complete remission [1.24 (0.80 to 1.69)] compared to the newly diagnosed patients (p<0.001) and increased as the patients experienced relapse [285.4 (269.70 to 301) (P<0.001)]. Conclusions: LncRNA ANRIL may contribute to BCP-ALL pathogenesis and disease progression; casting new light on the application of ANRIL as a potential biomarker or therapeutic target in BCP-ALL.

Critical role of SIRT1 upregulation on the protective effect of lncRNA ANRIL against hypoxia/reoxygenation injury in H9c2 cardiomyocytes.

Dysregulation of long non-coding RNA (IncRNA) antisense non-coding RNA in the INK4 locus (ANRIL) is associated with the risk of myocardial infarction (MI). Therefore, the present study aimed to determine the mechanisms underlying this association, which is currently poorly understood, to the best of our knowledge. The current study used an in vitro myocardial ischemia and reperfusion (MI/R) model, in which H9c2 cardiomyocytes were exposed to hypoxia/reoxygenation (H/R), which demonstrated that ANRIL expression was downregulated and that ANRIL positively regulated sirtuin 1 (SIRT1) expression following H/R injury. Subsequently, it was demonstrated that ANRIL upregulated SIRT1 expression by sponging microRNA-181a (miR-181a). In addition, ANRIL overexpression reduced lactate dehydrogenase release and apoptosis of H9c2 cardiomyocytes exposed to H/R, indicating that ANRIL prevented H/R-induced cardiomyocyte injury. Moreover, both miR-181a overexpression and SIRT1 knockdown significantly decreased the protective effects of ANRIL on H/R-induced cardiomyocyte injury, thus demonstrating that SIRT1 upregulation via sponging miR-181a is a critical mechanism that mediates the function of ANRIL. These results provided a novel mechanistic insight into the role of ANRIL in H/R-injured cardiomyocytes and suggested that the ANRIL/miR-181a/SIRT1 axis may be a therapeutic target for reducing MI/R injury.

Association between lncRNA ANRIL genetic variants with the susceptibility to ischemic stroke: From a case-control study to meta-analysis.

Background:Recent studies have reported that lncRNA (long noncoding RNAs) antisense non-coding RNA in the INK4 locus (ANRIL) plays important roles in the development of atherosclerosis through regulating cell apoptosis, proliferation, and adhesion. GWAS (genome-wide association studies) identified common genetic variants within ANRIL could confer risk of ischemic stroke (IS) in southern Sweden.Methods:We performed a case-control study, including 567 IS patients and 552 healthy controls from unrelated northern Chinese Han population, aiming to explore the association between lncRNA ANRIL rs2383207, rs4977574 polymorphisms and the risk of IS. Subsequently we implemented a meta-analysis to further assess the relationship of these variants and the disease.Results:In our case-control study, no significant associations were observed in all models between above 2 polymorphisms and IS. Next in our subgroup analysis, we detected significant association between GA genotype of rs4977574 and the increased risk of LAA-IS (large-artery atherosclerotic ischemic stroke), similar elevated risk also appeared in the GG + GA genotype under the dominant model (P = .048, OR = 1.385, 95% CIs 1.002–1.914; P = .040, OR = 1.378, 95% CIs 1.015–1.872, respectively). As for rs2383207, negative results were obtained under all models and subgroups. Our meta-analysis showed a significant association between rs4977574 polymorphism and IS risk in allele model (G vs A P = .002, OR = 1.137, 95% CIs 1.048–1.234); with respect to rs2383207 polymorphism, no significant association between that and the risk of IS was detected under the dominant model (GA + AA vs GG, P = .061, OR = 0.923, 95% CIs 0.849–1.004), or recessive model (AA vs GA + GG, P = .656, OR = 0.972, 95% CIs 0.858–1.101), or allele model (A vs G, P = .326, OR = 0.952, 95% CIs 0.863–1.050). Likewise, no significant association between rs2383207 and IS was found in different stoke subtypes (P > .05).Conclusions:Our findings indicated G allele of lncRNA ANRIL rs4977574 could increase the risk of IS, and the variant may be associated with susceptibility to LAA-IS in Chinese Han population.

Upregulating the Expression of LncRNA ANRIL Promotes Osteogenesis via the miR-7-5p/IGF-1R Axis in the Inflamed Periodontal Ligament Stem Cells.

BackgroundLong non-coding RNA (lncRNA) antisense non-coding RNA in the INK4 locus (ANRIL) is a base length of about 3.8 kb lncRNA, which plays an important role in several biological functions including cell proliferation, migration, and senescence. This study ascertained the role of lncRNA ANRIL in the senescence and osteogenic differentiation of inflamed periodontal ligament stem cells (iPDLSCs).MethodsHealthy periodontal ligament stem cells (hPDLSCs) and iPDLSCs were isolated from healthy/inflamed periodontal ligament tissues, respectively. The proliferation abilities were determined by CCK-8, EdU assay, and flow cytometry (FCM). The methods of Western blot assay (WB), quantitative real-time polymerase chain reaction (qRT-PCR), alizarin red staining, alkaline phosphatase (ALP) staining, ALP activity detection, and immunofluorescence staining were described to determine the biological influences of lncRNA ANRIL on iPDLSCs. Senescence-associated (SA)-β-galactosidase (gal) staining, Western blot analysis, and qRT-PCR were performed to determine cell senescence. Dual-luciferase reporter assays were conducted to confirm the binding of lncRNA ANRIL and miR-7-5-p, as well as miR-7-5p and insulin-like growth factor receptor (IGF-1R).ResultsHPDLSCs and iPDLSCs were isolated and cultured successfully. LncRNA ANRIL and IGF-1R were declined, while miR-7-5p was upregulated in iPDLSCs compared with hPDLSCs. Overexpression of ANRIL enhanced the osteogenic protein expressions of OSX, RUNX2, ALP, and knocked down the aging protein expressions of p16, p21, p53. LncRNA ANRIL could promote the committed differentiation of iPDLSCs by sponging miR-7-5p. Upregulating miR-7-5p inhibited the osteogenic differentiation of iPDLSCs. Further analysis identified IGF-1R as a direct target of miR-7-5p. The direct binding of lncRNA ANRIL and miR-7-5p, miR-7-5p and the 3′-UTR of IGF-1R were verified by dual-luciferase reporter assay. Besides, rescue experiments showed that knockdown of miR-7-5p reversed the inhibitory effect of lncRNA ANRIL deficiency on osteogenesis of iPDLSCs.ConclusionThis study disclosed that lncRNA ANRIL promotes osteogenic differentiation of iPDLSCs by regulating the miR-7-5p/IGF-1R axis.

LncRNA ANRIL negatively regulated chitooligosaccharide-induced radiosensitivity in colon cancer cells by sponging miR-181a-5p.

The radiosensitivity of colon cancer cells can be regulated by noncoding RNAs. In this study, the lncRNA antisense non-coding RNA in the INK4 locus (ANRIL) was selected to analyze its regulatory role in chitooligosaccharides (COS)-related radiosensitivity in colon cancer cells. The ANRIL expression in colon cancer cell lines was examined using real-time quantitative polymerase chain reaction (RT-qPCR), based on which we selected the cell line that presented the highest expression of ANRIL for radiosensitivity research. The cells were exposed to X-rays (0 Gy, 2 Gy, 4 Gy, and 6 Gy) and evaluated for changes in ANRIL and miR-181a-5p expression using RT-qPCR. Cell viability was evaluated using the CCK8 method, while apoptosis was detected with flow cytometry assays. Dual luciferase assays validated the binding between ANRIL and miR-181a-5p. The cell survival rates after differential COS treatments (0 mg/mL, 1.0 mg/mL, 2.0 mg/mL, 3.0 mg/mL, 4.0 mg/mL, and 5.0 mg/mL) were rated using CCK8 assay. The cells with the strongest dosage of COS (5.0 mg/mL) were selected to further investigate the role of ANRIL/miR-181a-5p in modulating the radiosensitivity observed with CCK-8 and flow cytometry assays. The ANRIL was highly expressed in colon cancer cells lines, especially in the SW480 cell line. Irradiation significantly decreased cell viability and ANRIL expression in a dose-dependent manner. The overexpression of ANRIL reduced the cell apoptosis rate after irradiation. MiR-181a-5p directly bound to ANRIL and was upregulated by irradiation in a dose-dependent manner. The suppression of miR-181a-5p decreased cell apoptosis. The COS treatment notably downregulated cell survival and promoted apoptosis in cells exposed to irradiation. The overexpression of ANRIL partially reversed COS-induced apoptosis and the inhibition rate; the upregulation of miR-181a-5p could counteract the impact of ANRIL regulation in cells. The ANRIL negatively regulated radiosensitivity induced by COS in colon cancer cells by sponging miR-181a-5p.

LncRNA ANRIL promotes multiple myeloma progression and bortezomib resistance by EZH2-mediated epigenetically silencing of PTEN.

Multiple myeloma (MM) is a plasma cell malignancy of bone marrow. In the present study, we aimed to study the function and potential mechanism of the antisense non-coding RNA in the INK4 Locus (ANRIL) in MM. The expression levels of ANRIL in MM patients and healthy donors were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). The effects and mechanisms of ANRIL in MM were evaluated by cell viability assay, BrdU incorporation assay, tumor xenograft model, flow cytometry, western blot, RNA immunoprecipitation (RIP), transcriptome RNA sequencing, and chromatin immunoprecipitation (ChIP). We found that ANRIL was upregulated in MM patients and cell lines, and associated with advanced international staging system (ISS) stage and poor overall survival. Enforced ANRIL expression promoted proliferation and tumor xenograft growth of MM cells, while knockdown of ANRIL exhibited opposite effects. Moreover, ANRIL overexpression increased the half-maximal inhibitory concentration (IC50) of bortezomib and reduced bortezomib-induced apoptosis in MM cells. ANRIL was found to accumulate in the nuclei of MM cells, and interact with EZH2 by RIP assay. Transcriptome RNA sequencing identified PTEN as a target of ANRIL in MM cells. In the ChIP assay, knockdown of ANRIL reduced EZH2 occupancy and H3K27me3 binding to the promoter region of PTEN. Furthermore, EZH2 knockout or PTEN restoration abrogated the effects caused by ANRIL overexpression in MM cells. Our results indicated that ANRIL exerted oncogenic functions and conferred chemoresistance of MM cells by EZH2-mediated epigenetically silencing of PTEN.

Knockdown of lncRNA ANRIL suppresses the production of inflammatory cytokines and mucin 5AC in nasal epithelial cells via the miR-15a-5p/JAK2 axis.

The incidence of allergic rhinitis (AR) is increasing worldwide. Human nasal epithelial cells (HNECs) are the key cells in the occurrence of AR. Antisense non-coding RNA in the INK4 locus (ANRIL) was discovered to be involved in the progression of AR. However, the mechanism by which ANRIL mediates the progression of AR remains to be determined. The present study aimed to further explore the mechanism by which ANRIL regulates AR. Thereby, HNECs were treated with IL-13 to mimic AR in vitro. The mRNA expression levels of ANRIL, microRNA (miR)-15a-5p, JAK2, mucin 5AC (MUC5AC), granulocyte-macrophage colony-stimulating factor (GM-CSF) and eotaxin-1, and protein expression levels of JAK2, STAT3 and phosphorylated-STAT3 in HNECs were analyzed using reverse transcription-quantitative PCR and western blotting, respectively. ELISAs were used to detect the secretory levels of inflammatory cytokines and mucin in cell supernatants. In addition, a dual luciferase reporter assay was used to confirm the downstream target of ANRIL and the target gene of miR-15a-5p. The results revealed that the secretory levels of eotaxin-1, GM-CSF and MUC5AC were significantly upregulated by IL-13 in the supernatant of HNECs. The expression levels of ANRIL and JAK2 were also upregulated in IL-13-induced HNECs, while the expression levels of miR-15a-5p were downregulated. In addition, ANRIL was identified to bind to miR-15a-5p. The IL-13-induced upregulation of eotaxin-1, GM-CSF and MUC5AC mRNA expression and secretory levels was significantly inhibited by the genetic knockdown of ANRIL, while the miR-15a-5p inhibitor effectively reversed this effect. JAK2 was also discovered to be directly targeted by miR-15a-5p. The overexpression of JAK2 significantly suppressed the therapeutic effect of miR-15a-5p mimics on IL-13-induced inflammation in vitro. In conclusion, the findings of the present study suggested that the genetic knockdown of ANRIL may suppress the production of inflammatory cytokines and mucin in IL-13-treated HNECs via regulation of the miR-15a-5p/JAK2 axis. Thus, ANRIL may serve as a novel target for AR treatment.

SiRNA-mediated lncRNA ANRIL knockdown enhances the sensitivity of gastric cancer cells to doxorubicin

Background: Long non-coding RNAs (lncRNAs) play pivotal roles in carcinogenesis and the development of drug resistance in various malignancies. Objectives: The current study aimed to explore the impact of antisense non-coding RNA in the INK4 locus (ANRIL) silencing on proliferation and the sensitivity of KATO III gastric cells to the doxorubicin as a common chemotherapeutic agent. Methods: The KATO III cells were transfected with ANRIL siRNA (si-ANRIL) using Lipofectamine™2000 reagent. Following that, the relative ANRIL levels were determined by quantitative Real-Time PCR. Trypan blue assay was conducted to observe the tumor cell proliferation following the transfection. Moreover, the MTT assay was performed to identify the cytotoxic effects of doxorubicin and si-ANRIL alone or in combination on KATO III cells. The effects of si-ANRIL/doxorubicin on KATO III cells migration and apoptosis were assessed by wound healing assay and ELISA cell death method, respectively. Results: The results showed that the si-ANRIL significantly diminished ANRIL expression level in a time-dependent manner contributing to the distinct repression of cell growth and enhanced apoptosis. Furthermore, the si-ANRIL synergistically elevated the cytotoxic impacts of doxorubicin. Additionally, the ANRIL down-regulation dramatically promoted its induction of apoptosis. Moreover, KATO III cells transfected with si-ANRIL and exposed with doxorubicin revealed significantly reduced invasion capability and enhanced apoptosis rate. Conclusion: These results demonstrated that the knock-down of lncRNA ANRIL could be a potential therapeutic strategy to trigger apoptosis and circumvent doxorubicin-resistance.

The lncRNA ANRIL regulates endothelial dysfunction by targeting the let-7b/TGF-βR1 signalling pathway.

The long noncoding RNA antisense noncoding RNA in the INK4 locus (ANRIL) plays a critical role in the development of atherosclerosis. However, the precise effect of ANRIL on endothelial dysfunction remains unclear. In this study, we investigated ANRIL expression in patients with coronary artery disease and elucidated the molecular mechanism underlying its effect. ANRIL expression was detected in the blood plasma of 111 patients. We analysed the correlation between ANRIL and endothelial dysfunction markers. We also examined the effect of ANRIL on the regulation of endothelial dysfunction. ANRIL levels were increased in patients with acute coronary syndrome. The expression of ANRIL is associated with the inflammatory cytokines monocyte chemoattractant protein-1 and interleukin-10, which are secreted in response to endothelial dysfunction. Knockdown of ANRIL significantly promoted cell proliferation and tubule formation and inhibited inflammatory activation and apoptosis of human umbilical vein endothelial cells (HUVEC). ANRIL-mediated inhibition of let-7b regulates HUVEC dysfunction by targeting the TGF-βR1/Smad signalling pathway. This study highlights a new therapeutic strategy for preventing endothelial dysfunction associated with cardiovascular disease.

The correlation of serum long non-coding RNA ANRIL with risk factors, functional outcome, and prognosis in atrial fibrillation patients with ischemic stroke.

BackgroundThis study aimed to evaluate the predictive value of long non‐coding RNA (lncRNA) antisense non‐coding RNA in the INK4 locus (ANRIL) for atrial fibrillation (AF) patients with ischemic stroke and investigate its correlation with risk factors, functional outcome, and prognosis.MethodsA total of 386 consecutive AF patients were recruited. AF patients were followed up for 24‐48 months by outpatient follow‐up, telephone follow‐up, and medical record. The time of ischemic stroke in patients with AF was recorded, and follow‐up was continued for 6 months. LncRNA ANRIL expression from serum was detected by quantitative real‐time polymerase chain reaction (qRT‐PCR).ResultsCompared with the AF with ischemic stroke group (14.3 ± 2.3), patients in the AF without ischemic stroke group (11.9 ± 1.8) had significantly lower serum lncRNA ANRIL levels (P < .05). The sensitivity and specificity of lncRNA ANRIL for identifying AF with ischemic stroke were 76.6% and 81.4%, respectively. Spearman correlation analysis results shown that lncRNA ANRIL was significantly correlated with the NIHSS score (r Spearman = .490, P < .001) and the mRS score (r Spearman = .466, P < .001). Compared with the lncRNA ANRIL high‐expression group, the recurrence‐free survival (RFS) of the lncRNA ANRIL low‐expression group was significantly higher (χ2 = 11.009, log‐rank P < .001). Cox proportional regression model analysis indicated that the serum lncRNA ANRIL level (P = .004), NIHSS score (P = .001), infarct volume (P = .035), and smoking (P < .001) were the risk factors for AF with ischemic stroke.ConclusionSerum lncRNA ANRIL exerts a good predictive value for AF with ischemic stroke, and its increased expression is correlated with worse RFS for patients.

The lncRNA ANRIL is down-regulated in peripheral blood of patients with periodontitis.

Long non-coding RNAs (lncRNAs) have crucial roles in lncRNAs in periodontal development and disorders of this tissue. A number of lncRNAs especially those regulating immune responses contribute in the pathophysiology of periodontitis. In the current case-control study, we assessed expression levels of two immune response-related lncRNAs namely the antisense non-coding RNA in the INK4 locus (ANRIL) and metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) in gingival tissues and blood samples of patients with periodontitis and healthy subjects. Expression of ANRIL was significantly lower in peripheral blood of patients compared with controls (Posterior Beta RE = -1.734, P value = 0.035). However, when diving study participants based on their gender, no significant difference was found between patients and sex-matched controls. Expression of this lncRNA was not different between periodontitis tissues and normal tissues. Expression of MALAT1 was not different between samples obtained from cases and controls. Tissue or blood expressions of ANRIL or MALAT1 were not correlated with age of either patients or controls. There were significant correlations between expression levels of ANRIL and MALAT1 in gingival tissues both in cases (r = 0.62, P < 0.0001) and in controls (r = 0.37, P < 0.0001). However, blood levels of these lncRNAs were not correlated with each other either in cases or in controls. Most notably, there was no significant correlation between expression levels of these lncRNAs in gingival tissues and in the blood of study participants. The current study indicates dysregulation of ANRIL in the peripheral blood of patients with periodontitis in spite of its normal levels in gingival tissues which might reflect disturbance in systemic immune responses in these patients.

Hypoxia-induced lncRNA ANRIL promotes cisplatin resistance in retinoblastoma cells through regulating ABCG2 expression.

Cisplatin (DDP) resistance limits its efficacy for retinoblastoma (Rb). Hypoxia-inducible factor-1α (HIF-1α) has been shown to contribute to chemotherapy resistance in tumours under hypoxic conditions. This study was designed to explore the role and mechanism of long non-coding RNA (lncRNA) antisense non-coding RNA in the INK4 locus (ANRIL) in regulating DDP resistance in Rb cells under hypoxia and to validate whether HIF-1α was involved in this process. The interaction between HIF-1α and the promoter of ANRIL was analyzed using ChIP assay. Cell proliferation and apoptosis, as well as protein levels of drug resistance-related proteins (ABCG2 and MDR1) were examined to evaluate DDP resistance in Rb cells. The interactions between miR-328 and ANRIL as well as miR-328 and ABCG2 were analyzed using dual-luciferase reporter assays. Upon hypoxia, HIF-1α directly bound to the ANRIL promoter region to transcriptionally activate ANRIL. The hypoxia-induced ANRIL promoted Rb cell resistance to DDP, as evidenced by facilitation of cell proliferation, inhibition of cell apoptosis and upregulation of ABCG2 and MDR1. Mechanistically, ANRIL promoted Rb cell resistance to DDP by acting as a sponge of miR-328 to upregulate expression of ABCG2, which was confirmed as a direct target of miR-328. Collectively, hypoxia-induced ANRIL promotes DDP resistance in Rb cells by sponging miR-328 to upregulate ABCG2 expression.

Epigenetic silencing of LncRNA ANRIL enhances liver fibrosis and HSC activation through activating AMPK pathway

Long non‐coding RNAs (LncRNAs) and DNA methylation are important epigenetic mark play a key role in liver fibrosis. Currently, how DNA methylation and LncRNAs control the hepatic stellate cell (HSC) activation and fibrosis has not yet been fully characterized. Here, we explored the role of antisense non‐coding RNA in the INK4 locus (ANRIL) and DNA methylation in HSC activation and fibrosis. The expression levels of DNA methyltransferases 3A (DNMT3A), ANRIL, α‐Smooth muscle actin (α‐SMA), Type I collagen (Col1A1), adenosine monophosphate‐activated protein kinase (AMPK) and p‐AMPK in rat and human liver fibrosis were detected by immunohistochemistry, qRT‐PCR and Western blotting. Liver tissue histomorphology was examined by haematoxylin and eosin (H&E), Sirius red and Masson staining. HSC was transfected with DNMT3A‐siRNA, over‐expressing ANRIL and down‐regulating ANRIL. Moreover, cell proliferation ability was examined by CCK‐8, MTT and cell cycle assay. Here, our study demonstrated that ANRIL was significantly decreased in activated HSC and liver fibrosis tissues, while Col1A1, α‐SMA and DNMT3A were significantly increased in activated HSC and liver fibrosis tissues. Further, we found that down‐regulating DNMT3A expression leads to inhibition of HSC activation. Reduction in DNMT3A elevated ANRIL expression in activated HSC. Furthermore, we performed the over expression ANRIL suppresses HSC activation and AMPK signalling pathways. In sum, our study found that epigenetic DNMT3A silencing of ANRIL enhances liver fibrosis and HSC activation through activating AMPK pathway. Targeting epigenetic modulators DNMT3A and ANRIL, and offer a novel approach for liver fibrosis therapy.

LncRNA ANRIL regulates cell proliferation and migration via sponging miR-339-5p and regulating FRS2 expression in atherosclerosis.

Atherosclerosis (AS), with high risk of stroke or cerebrovascular disease, is one of the most common causes of death worldwide. Increasing evidence shows that long non-coding RNA (lncRNA) antisense non-coding RNA in the INK4 locus (ANRIL) is related to atherothrombotic stroke susceptibility and contributes to AS progression. However, the underlying mechanism was not explained yet. Human aorta vascular smooth muscle cells (HA-VSMCs) and human umbilical vein endothelial cells (HUVECs) were treated with oxidized Low Density Lipoprotein (ox-LDL) and considered as AS cell models. Quantitative reverse transcriptase PCR (qRT-PCR) and Western blotting were employed to investigate the mRNA and protein expression level, respectively. Microscopic examination through fluorescent in situ hybridization (FISH) was used to determine the location of ANRIL. Cell proliferation and migration assays were demonstrated to evaluate the functional role of ANRIL in AS. Potential target of ANRIL was determined using Luciferase reporter assay and RNA immunoprecipitation (RIP). ANRIL was upregulated and miR-399-5p was down-regulated in both human atherosclerotic plaques and ox-LDL-induced cells. ANRIL was located in cytoplasm and promoted cell proliferation and migration by sponging miR-399-5p. Further analysis identified fibroblast growth factor receptor substrate 2 (FRS2) as a direct target of miR-399-5p. Finally, RAS/RAF/ERK signal pathway was proved to be involved in the regulation of ANRIL on the progression of AS. These results revealed the underlying mechanism that ANRIL promoted AS progression by sponging miR-399-5p and regulating RAS/RAF/ERK signal pathway, suggesting that ANRIL might be a potential target for the therapeutic strategy of AS.

LncRNA ANRIL promotes cell proliferation, migration and invasion during acute myeloid leukemia pathogenesis via negatively regulating miR-34a.

LncRNA ANRIL (antisense non-coding RNA in the INK4 locus) was highly expressed in acute myeloid leukemia (AML) patients to promote AML pathogenesis. In this study, we aimed to investigate the roles and molecular events of ANRIL associated with AML progression. Expression patterns of ANRIL and miR-34a in the bone marrow (BM) samples and cell lines were determined using qRT-PCR. Cell proliferation, apoptosis, migration and invasion of cells with ANRIL knockdown or miR-34a overexpression were assessed by CCK-8, EdU staining, flow cytometry and Transwell assays, respectively. The dual-luciferase reporter assay was employed to validate the relationship between miR-34a and Histone deacetylase 1 (HDAC1). The binding of E2 F1 (E2 F transcription factor 1) with gene promoter was analyzed by ChIP. Furthermore, the tumorigenicity of AML was determined by xenograft transplantation in nude mice. ANRIL was up-regulated both in the BM samples from AML patients and cell lines (HL-60 and THP-1), of which expression was negatively correlated with miR-34a expression. ANRIL knockdown inhibited cell proliferation, migration and invasion but promoted apoptosis of AML cells, while overexpression of miR-34a exerted opposite effects. miR-34a was verified as a downstream gene targeted by ANRIL. Moreover, HDAC1 was a direct target of miR-34a, and HDAC1 overexpression impaired the recruitment of E2 F1 to ASPP2 (apoptosis stimulating proteins of p53) gene promoter. ANRIL knockdown significantly inhibited the tumorigenesis of AML. ANRIL promotes AML development through HDAC1-mediated epigenetic suppression of ASPP2 via negatively regulating miR-34a, which might serve as a therapeutic target for AML treatment.

Influence of lncRNA ANRIL on neuronal apoptosis in rats with cerebral infarction by regulating the NF-κB signaling pathway.

The aim of this study was to evaluate the influence of long non-coding ribonucleic acid (lncRNA) antisense non-coding RNA in the INK4 locus (ANRIL) on neuronal apoptosis in rats with cerebral infarction (CI), and to further explore the underlying mechanism of lncRNA ANRIL in the occurrence and development of CI. A total of 60 adult male Wistar rats were randomly divided into three groups using a random number table, including sham group (n=20), CI group (n=20) and CI + lncRNA ANRIL knockdown group [CI + lncRNA ANRIL small-interfering RNA (siRNA) group, n=20]. Focal CI was constructed by suture occlusion. After successful modeling, lncRNA ANRIL siRNA was stereotactically injected into the lateral ventricle of the rats. 24 h after operation, the neurological function of the rats in each group was evaluated by the modified neurological severity score (mNSS). Meanwhile, the infarction area of brain tissues was evaluated using the triphenyl tetrazolium chloride (TTC) method. The protein expression levels of apoptosis-related genes, including B-cell lymphoma-2 (Bcl-2) and Bcl-2 associated X protein (Bax), were detected via Western blotting. Subsequently, immunofluorescence staining was performed to detect the expression and location of Caspase-3 in brain tissues. Moreover, the apoptosis level of rats in each group was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. Furthermore, the expressions of nuclear factor-κB (NF-κB) signaling pathway-related proteins were detected via Western blotting. Polymerase Chain Reaction (PCR) results revealed that the expression level of lncRNA ANRIL in the CI group was significantly increased when compared with that of the sham group (p<0.05). The results of mNSS and TTC staining manifested that knockdown of lncRNA ANRIL could significantly reduce CI-induced neurological deficits and CI area (p<0.05). At the same time, knockdown of lncRNA ANRIL markedly decreased the level of Bax, whereas increased the expression of Bcl-2 (p<0.05). Besides, the number of apoptotic cells in the CI + lncRNA ANRIL siRNA group was remarkably decreased (p<0.05). In addition, lncRNA ANRIL down-regulation remarkably inhibited the phosphorylation of p65 (p<0.05). The inhibitory effect of lncRNA ANRIL knockdown on neuronal apoptosis in CI rats may be probably related to its inhibition of the NF-κB signaling pathway. Furthermore, lncRNA ANRIL inhibitor is expected to become a targeted drug in the clinical treatment of CI.