Knockdown Of Antisense Non-coding RNA In The INK4 Locus Research Articles

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 long non‑coding RNA ANRIL inhibits the proliferation and promotes the apoptosis of Burkitt lymphoma cells through the TGF‑β1 signaling pathway.

Burkitt lymphoma (BL) has a high mortality rate and its treatment is currently limited to chemotherapy combined with immunotherapy. The long non-coding RNA antisense non-coding RNA in the INK4 locus (ANRIL) has been identified as an oncogene that can regulate cell proliferation and apoptosis in multiple types of cancer. However, the function of ANRIL in BL remains unknown. The present study aimed to determine the effect of ANRIL on cell proliferation and apoptosis in BL. Reverse transcription-quantitative PCR was used to analyze the expression levels of ANRIL in BL cells. The effect of ANRIL knockdown on BL cells was determined using Cell Counting Kit-8, flow cytometric, western blotting, immunofluorescence staining and Hoechst staining assays. The results revealed that ANRIL silencing inhibited the proliferation and promoted the apoptosis of BL cells. In addition, the expression levels of cyclin D1, E2F transcription factor 1 and Bcl-2 were downregulated, while the expression levels of cyclin-dependent kinase inhibitor 1A, Bcl-2-associated X protein, cleaved-caspase-9/pro-caspase-9 and cleaved-caspase-3/pro-caspase-3 were upregulated. Furthermore, the knockdown of ANRIL activated the TGF-β1 signaling pathway, as evidenced by the upregulated expression levels of TGF-β1, phosphorylated (p)-SMAD2/3/SMAD2/3, p-SMAD1/SMAD1 and sphingosine-1-phosphate receptor 2. Moreover, the protective effect of ANRIL silencing in BL could be inhibited by the TGF-β receptor type I/II dual inhibitor, LY2109761. In conclusion, the findings of the present study suggested that the knockdown of ANRIL may inhibit cell proliferation and promote cell apoptosis in BL by regulating the TGF-β1 signaling pathway, which may provide a novel target for the treatment of BL.

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.

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.

LncRNA ANRIL knockdown ameliorates retinopathy in diabetic rats by inhibiting the NF-κB pathway.

To evaluate the impacts of long non-coding ribonucleic acid (lncRNA) antisense non-coding RNA in the INK4 locus (ANRIL) on diabetic retinopathy (DR) in rats and the underlying mechanism. A total of 60 adult male Sprague Dawley (SD) rats were randomly divided into 3 groups: the Sham group (n=20), DR group (n=20) and DR + lncRNA ANRIL knockdown group [DR + lncRNA ANRIL small interfering RNA (siRNA) group, n=20]. DR model in rats was established by intraperitoneal injection of streptozocin (STZ; 60 mg/kg). Meanwhile, a certain dose of lncRNA ANRIL siRNA was added dropwise into rat eyes of DR + lncRNA ANRIL siRNA group during model induction to downregulate lncRNA ANRIL expression in the retina. 16 weeks later, rat retinal tissues were taken to extract total RNA and protein. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) was applied to detect the expression levels of lncRNA ANRIL, interleukin-1 (IL-1), IL-6 and monocyte chemotactic protein-1 (MCP-1) in each group of the rat retina. Pathological structure of rat retinal tissues in each group was observed via hematoxylin and eosin (H&E) staining. Immunohistochemistry was adopted to measure the expression levels of B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax) and P65 in each group of retinal tissues. In addition, the retinal vascular permeability in each group of rats was detected by fluorescent staining. Finally, Western blotting was utilized to determine the expressions of genes in the P65 signaling pathway. Compared with rats in the Sham group, lncRNA ANRIL was upregulated in rat retinal tissues harvested from the DR + lncRNA ANRIL siRNA group (p<0.05). After knockdown of lncRNA ANRIL in the retinal tissues of DR rats, pathologic damage was alleviated notably, and the levels of inflammatory markers (IL-1, IL-10 and MCP-1) were lowered markedly (p<0.05). The protein expressions of Bax and P65 decreased evidently, while the protein level of Bcl-2 increased markedly (p<0.05) in DR rats with ANRIL knockdown. Furthermore, Western blotting results revealed that inhibition of lncRNA ANRIL could prominently repress the phosphorylation level of P65 in the retinal tissues of DR rats (p<0.05). LncRNA ANRIL knockdown can significantly ameliorate the retinopathy in diabetic rats by blocking the nuclear factor-kappa B (NF-κB) signaling pathway.

LncRNA ANRIL promotes NLRP3 inflammasome activation in uric acid nephropathy through miR-122-5p/BRCC3 axis

This study is designed to explore the mechanism by which long non-coding RNA (lncRNA) antisense non-coding RNA in the INK4 locus (ANRIL) plays a pathogenic role in uric acid nephropathy (UAN). The expressions of ANRIL, miR-122-5p, BRCA1-BRCA2-containing complex subunit 3 (BRCC3) and NOD-like receptor protein 3 (NLRP3) were determined in UAN patients and uric acid-treated HK-2 cells by qRT-PCR. Protein levels of BRCC3 and NLRP3 were examined by western blot. The levels of inflammatory cytokines were quantified by ELISA. CCK-8 assay was used to assess cell viability. Apoptosis was detected by Annexin V-FITC/PI double-labeled flow cytometry and TUNEL assay. The interaction between ANRIL, miR-122-5p and BRCC3 were studied using luciferase reporter assay. The role of ANRIL in renal injury was evaluated in experimental rats. ANRIL and BRCC3 were highly expressed while miR-122-5p was down-regulated in serum of UAN patients and uric acid-treated tubular epithelial cells. Luciferase reporter assay and in vitro rescue experiment confirmed that ANRIL promoted NLRP3 inflammasome activation by up-regulating BRCC3 expression via sponging miR-122-5p. Furthermore, in vivo experiment validated that knockdown of ANRIL alleviated renal injury of UAN rats. ANRIL exerted pathogenic effect in UAN to promote NLRP3 inflammasome activation via miR-122-5p/BRCC3 axis.

Knockdown of long non-coding RNA ANRIL inhibits tumorigenesis in human gastric cancer cells via microRNA-99a-mediated down-regulation of BMI1.

Long non-coding RNA antisense non-coding RNA in the INK4 locus (ANRIL) has been reported to promote tumorigenesis via regulating microRNA (miR)-99a in gastric cancer cells. However, the role of each component involved in it is still not well understood. This study aimed to verify the role of ANRIL in gastric cancer as well as the underlying mechanisms. ANRIL levels in clinical gastric cancer tissues and cell lines were tested by qPCR. Effects of ANRIL silence on cell viability, migration and invasion, apoptosis, and miR-99a expression in MKN-45 and SGC-7901 cells were measured using CCK-8, Transwell assay, flow cytometry, and qPCR assays, respectively. Then, effects of miR-99a inhibition on ANRIL-silenced cells were evaluated. B-lymphoma Mo-MLV insertion region 1 (BMI1) expression, after abnormal expression of ANRIL and miR-99a, was determined. Finally, expression of key proteins in the apoptotic, Notch, and mTOR pathways was assessed. ANRIL level was elevated in gastric cancer tissues and cell lines. Knockdown of ANRIL suppressed cell viability, migration, and invasion, and increased apoptosis through up-regulating miR-99a. Furthermore, ANRIL silence down-regulated BMI1 via up-regulating miR-99a. BMI1 silence down-regulated Bcl-2 and key kinases in the Notch and mTOR pathways and up-regulated p16 and cleaved caspases. We verified the tumor suppressive effects of ANRIL knockdown in gastric cancer cells via crosstalk with miR-99a. Together, we provided a novel regulatory mechanism for ANRIL in gastric cancer, in which ANRIL silence down-regulated BMI1 via miR-99a, along with activation of the apoptotic pathway and inhibition of the Notch and mTOR pathways.

Knockdown of LncRNA ANRIL suppresses cell proliferation, metastasis, and invasion via regulating miR-122-5p expression in hepatocellular carcinoma.

Previous studies reported that lncRNA antisense non-coding RNA in the INK4 locus (ANRIL) was upregulated in hepatocellular carcinoma (HCC) tissues and decreased expression of ANRIL could suppress cell proliferation, metastasis, and invasion and induce apoptosis of HCC cells. However, the molecular mechanism of ANRIL involved in HCC tumorigenesis is still unknown. The expressions of ANRIL and miR-122-5p in HCC tissues and cells were quantified by qRT-PCR. MTT assay, colony formation assay, wound healing assay, and transwell invasion assay were performed to evaluate cell growth, metastasis, and invasion, respectively. RNA immunoprecipitation (RIP) assay and luciferase reporter assay were performed to determine whether ANRIL could directly bind to miR-122-5p in HCC cells. Xenograft tumor experiment was conducted to confirm the biological role and underlying mechanism of ANRIL in vivo. The results showed that ANRIL was upregulated and miR-122-5p was downregulated in HCC tissues and cells. ANRIL was negatively correlated with miR-122-5p expression in HCC tissues. Knockdown of ANRIL or miR-122-5p overexpression suppressed HCC cell viability, colony formation ability, metastasis, and invasion. ANRIL was demonstrated to directly bind to miR-122-5p and inhibit its expression. Forced expression of ANRIL abolished the inhibitory effect of miR-122-5p overexpression on HCC progression. In vivo experiment demonstrated that ANRIL knockdown impeded tumor growth in vivo and increased miR-122-5p expression. Our finding suggested that knockdown of ANRIL suppressed cell proliferation, metastasis and invasion via regulating miR-122-5p expression in HCC, illustrating the underlying mechanism of the oncogenic role of ANRIL in HCC.

Downregulation of lncRNA ANRIL represses tumorigenicity and enhances cisplatin-induced cytotoxicity via regulating microRNA let-7a in nasopharyngeal carcinoma.

Many studies have demonstrated that upregulation of long non-coding RNA (lncRNA) antisense non-coding RNA in the INK4 locus (ANRIL) plays an oncogenic role in various tumors, including nasopharyngeal carcinoma (NPC). The aim of this study is to explore the effect of ANRIL in NPC progression and cisplatin (DDP)-induced cytotoxicity. The results showed that ANRIL was highly expressed and let-7a was downregulated in NPC tissues and cells. Luciferase assay revealed that ANRIL could negatively regulate miR-let-7a expression. ANRIL knockdown inhibited NPC cell proliferation and induced apoptosis, while anti-let-7a reversed these effects. Combination treatment of si-ANRIL and DDP led to a lower viability, a more DNA strand breaks damage and a higher comet tail length compared with any single treatment, whereas let-7a inhibitor abolished these effects. Furthermore, depletion of ANRIL exacerbated DDP-induced cytotoxicity in NPC cells in vivo. Taken together, these data indicated that knockdown of ANRIL represses tumorigenicity and enhances DDP-induced cytotoxicity via regulating microRNA let-7a in NPC cells, providing a promising therapeutic strategy for NPC patients.

Silencing of long non-coding RNA ANRIL inhibits the development of multidrug resistance in gastric cancer cells

The development of multidrug resistance (MDR) is a crucial cause of therapy failure in gastric cancer, which results in disease recurrence and metastasis. Long non-coding RNAs (lncRNAs) have been proven to be critical in carcinogenesis and metastasis of gastric cancer. However, little is known about the roles of ANRIL (antisense non-coding RNA in the INK4 locus) in gastric cancer MDR. The aim of our study is to identify the biological function of ANRIL in gastric cancer MDR. In our results, ANRIL was highly expressed in gastric cancer tissues of cisplatin-resistant and 5-fluorouracil (5-FU)-resistant patients, and the same upregulation trends were observed in cisplatin-resistant cells (BGC823/DDP) and 5-FU-resistant cells (BGC823/5-FU). In addition, BGC823/DDP and BGC823/5-FU cells transfected with ANRIL siRNA and treated with cisplatin or 5-FU, respectively, exhibited significant lower survival rate, decreased invasion capability, and high percentage of apoptotic tumor cells. The influence of ANRIL knockdown on MDR was assessed by measuring IC50 of BGC823/DDP and BGC823/5-FU cells to cisplatin and 5-FU, the result showed that silencing ANRIL decreased the IC50 values in gastric cancer cells. Moreover, qRT-PCR and western blotting revealed that ANRIL knockdown decreased the expression of MDR1 and MRP1, both of which are MDR related genes; regression analysis showed that the expression of ANRIL positively correlated with the expression of MDR1 and MRP1, resprectively In summary, knockdown of lncRNA ANRIL in gastric cancer cells inhibits the development of MDR, suggesting an efficacious target for reversing MDR in gastric cancer therapy.

Long non-coding RNA ANRIL is up-regulated in bladder cancer and regulates bladder cancer cell proliferation and apoptosis through the intrinsic pathway

Antisense non-coding RNA in the INK4 locus (ANRIL) is a member of long non-coding RNAs and has been reported to be dysregulated in several human cancers. However, the role of ANRIL in bladder cancer remains unclear. This present study aimed to investigate whether and how ANRIL involved in bladder cancer. Our results showed up-regulation of ANRIL in bladder cancer tissues versus the corresponding adjacent non-tumor tissues. To explore the specific mechanisms, ANRIL was silenced by small interfering RNA or short hairpin RNA transfection in human bladder cancer T24 and EJ cells. Knockdown of ANRIL repressed cell proliferation and increased cell apoptosis, along with decreased expression of Bcl-2 and increased expressions of Bax, cytoplasmic cytochrome c and Smac and cleaved caspase-9, caspase-3 and PARP. However, no change of cleaved caspase-8 level was observed. Furthermore, in vivo experiment confirmed that knockdown of ANRIL inhibited tumorigenic ability of EJ cells in nude mice. Meanwhile, in accordance with in vitro study, knockdown of ANRIL inhibited expression of Bcl-2 and up-regulated expressions of Bax and cleaved caspase-9, but did not affect cleaved caspase-8 level. In conclusion, we first report that ANRIL possibly serves as an oncogene in bladder cancer and regulates bladder cancer cell proliferation and apoptosis through the intrinsic apoptosis pathway.