Long noncoding RNA DLX6-AS1 targets miR-124-3p/CDK4 to accelerate Ewing's sarcoma.
ABSTRACT: Ewing's sarcoma is one of leading cause of malignancy occurred in the children and adolescents worldwide. Given the emerging critical role of long noncoding RNA (lncRNA) in the human cancer, as well as Ewing's sarcoma, we aim to identify the biological role of DLX6-AS1 in the tumorigenesis. Results unveil that DLX6-AS1 expression was increased in the tissue sample and cells. Functionally, the silencing of DLX6-AS1 could repress the proliferation and accelerate the apoptosis of Ewing's sarcoma cells. Mechanically, DLX6-AS1 functioned as the sponge of miR-124-3p, and then miR-124-3p targeted the 3'-UTR of CDK4 mRNA, forming the DLX6-AS1/miR-124-3p/CDK4 regulatory pathway. In conclusion, the critical role of DLX6-AS1 might unveil a potential therapeutic target for Ewing's sarcoma.
Project description:In the present study, we investigated the role of lncRNA mus <i>distal-less homeobox 6 antisense 1 (DLX6-AS1)</i> during cerebral impairment induced by stroke. DLX6-AS1 levels were upregulated during ischemia/reperfusion (I/R) and downregulation of DLX6-AS1 reduced acute injury and ameliorated long-term neurological impairments induced by cerebral I/R in mice. Additionally, silencing of DLX6-AS1 significantly decreased the neuronal apoptosis in vivo and in vitro. Furthermore, inhibition of miRNA-149-3p led to enhance the apoptosis, which confirmed that DLX6-AS1 could sponge miR-149-3p. Finally, BOK was predicted to be the target of miR-149-3p using TargetScanVert software. And the silencing of DLX6-AS1 inhibited BOK expression both <i>in vivo</i> and <i>in vitro</i>, which was reversed by a miR-149-3p inhibitor. At meantime, BOK promoted OGD/R induced apoptosis in N2a cells. Therefore, this suggests that miR-149-3p sponging by DLX6-AS1 may lead to cerebral neuron I/R-induced impairments through upregulation of apoptotic BOK activity, which offers a new approach to the treatment of stroke impairment.
Project description:Background:Neuroblastoma (NB) is a common malignant tumor of the sympathetic nervous system, mainly disturbing children. Long non-coding RNAs (lncRNAs) serving as promising cancer biomarkers have been well recognized. Our study intends to explore the functions of lncRNA X-inactive specific transcript (DLX6-AS1) in NB and provide a potential action mechanism. Methods:The expression of DLX6-AS1, miR-506-3p and signal transducer and activator of transcription 2 (STAT2) was measured by quantitative real-time polymerase chain reaction (qRT-PCR). Cell proliferation was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and colony formation assay. Cell cycle distribution was determined by flow cytometry assay. The protein level of cell cycle-related markers and STAT2 was detected by Western blot. Glycolysis progress was evaluated according to glucose consumption, lactate production and ATP level. The target genes were predicted by the online database Starbase3.0 and verified by dual-luciferase reporter assay. Results:DLX6-AS1 expression was highly elevated in NB tissues and cells. DLX6-AS1 deficiency inhibited NB cell proliferation, cell cycle and glycolysis in vitro. MiR-506-3p was a target of DLX6-AS1, and miR-506-3p absence partly reversed the effects of DLX6-AS1 deficiency. Besides, STAT2 was targeted by miR-506-3p, and its expression was regulated by DLX6-AS1 through miR-506-3p. MiR-506-3p restoration also inhibited NB cell malignant behaviors, and STAT2 overexpression partially abolished the role of miR-506-3p restoration. Moreover, DLX6-AS1 deficiency weakened tumor growth in vivo. Conclusion:DLX6-AS1 regulated cell proliferation, cell cycle and glycolysis in vitro and tumor growth in vivo to promote the development of NB by upregulating STAT2 via targeting miR-506-3p.
Project description:In this study, we aim at investigating the expression and regulation role of long non-coding RNA (lncRNA) DLX6-AS1 in bladder cancer (BC). DLX6-AS1 was highly expressed in BC tissues and significant negative correlation with the 5-year survival in the BC patients. The results showed that the proliferation, migration and invasion activities of BC cells were promoted by DLX6-AS1 overexpression, while cell apoptosis was repressed. However, knockdown DLX6-AS1 presented an pposite regulatory effect, and DLX6-AS1 knockdown delayed tumor in vivo. The potential target of DLX6-AS1 in BC was predicted and verified by RIP, RNA pull-down, and dual-luciferase reporter assays as miR-195-5p. The results showed that miR-195-5p was down-regulated in BC tissues, the expression of which was significantly negative correlated with DLX6-AS1 expression. In addition, the results also showed that miR-195-5p targeted and down-regulated the VEGFA. Knockdown of DLX6-AS1 up-regulated miR-195-5p expression and down-regulated VEGFA expression. Moreover, down-regulation of VEGFA expression caused by DLX6-AS1 inhibited phosphorylation of Raf-1, MEK1/2, and ERK1/2, while miR-195-5p inhibitors abolished the effect of silencing DLX6-AS1 expression. Our study demonstrated that DLX6-AS1 played an oncogenic role in BC through miR-195-5p-mediated VEGFA/Ras/Raf/MEK/ERK pathway.
Project description:Background:Long noncoding RNAs (lncRNAs) play essential roles in tumor progression. However, the functions and targets of lncRNAs in neuroblastoma (NB) progression still remain to be determined. In this study, we aimed to investigate the effect of lncRNA DLX6 antisense RNA 1 (DLX6-AS1) on NB and the underlying mechanism involved. Methods:Through mining of public microarray datasets, we identify aberrantly expressed lncRNAs in NB. The gene expression levels were determined by quantitative real-time PCR, and protein expression levels were determined by western blot assay. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, colony formation assay, wound-healing assay, transwell invasion assays and flow cytometry analysis were utilized to examine cell proliferation, migration, invasion and apoptosis. Luciferase reporter assay was performed to confirm the interaction between DLX6-AS1and its potential targets. Tumor xenograft assay was used to verify the role of DLX6-AS1 in NB in vivo. Results:We identified DLX6-AS1 was upregulated in NB by using a public microarray dataset. The expression of DLX6-AS1 was increased in NB tissues and derived cell lines, and high expression of DLX6-AS1 was positively correlated with advanced TNM stage and poor differentiation. Knockdown of DLX6-AS1 induced neuronal differentiation, apoptosis and inhibited the growth, invasion, and metastasis of NB cells in vitro and impaired tumor growth in vivo. MiR-107 was the downstream target of DLX6-AS1. MiR-107 was found to target brain-derived neurotrophic factor (BDNF) which is an oncogene in NB. Knockdown of miR-107 or overexpression of BDNF reversed the suppression of NB progression caused by DLX6-AS1 silence. Conclusion:Overall, our finding supports that DLX6-AS1 promotes NB progression by regulating miR-107/BDNF pathway, acting as a novel therapeutic target for NB.
Project description:OBJECTIVE:To investigate the expression of long-chain noncoding growth stasis specific protein 6 antisense RNA1 (lncRNA DLX6-AS1) in nasopharyngeal carcinoma (NPC) tissues and cells, and its regulatory effect on malignant phenotypes of NPC cells. METHODS:The expressions of DLX6-AS1, miR-199a-5p, and HIF-1? mRNA in NPC issues and cells were detected by qRT-PCR. The proliferation, metastasis, and invasion of cells were monitored via MTT and transwell assay. The interactions between DLX6-AS1 and miR-199a-5p, miR-199a-5p and HIF-1? were verified by luciferase activity assay. Western blot was performed to determine the regulatory effect of DLX6-AS1 and miR-199a-5p on HIF-1? protein. RESULTS:The expression of lncRNA DLX6-AS1 was up-regulated in NPC tissues and cells. The proliferation, migration, and invasion of NPC were enhanced by overexpressed DLX6-AS1 but inhibited by DLX6-AS1 knockdown. In addition, DLX6-AS1 can be used as a kind of ceRNA to regulate miR-199a-5p and, thereby modulating the expression of HIF-1?. CONCLUSION:We found that DLX6-AS1 was a cancer-promoting lncRNA to facilitate the progression of NPC, and its underlying mechanism was suppressing miR-199a-5p expression. This study can provide novel clues for the treatment of NPC.
Project description:Gastric cancer (GC) is the second most prevalent carcinoma resulting in cancer-related deaths in the world, with differences among geographic areas. Although the incidence and mortality rates of GC in Asia are decreasing, the search for diverse and effective therapies of GC is still needed to be fully inquired. The present research explored the expression pattern, functional role and underlying mechanism of DLX6-AS1 in GC. Firstly, we measured DLX6-AS1 expression in GC and then found the elevated level of DLX6-AS1. To further inspect the function role of DLX6-AS1 involved in GC, we performed lost-of-function assays. The silencing of DLX6-AS1 suppressed cell proliferation, migration and EMT process of GC cells. Subsequently, we uncovered that MAP4K1 was also up-regulated in GC and could be positively regulated by DLX6-AS1. Moreover, MAP4K1 down-regulation similarly inhibited GC progression. In addition, DLX6-AS1 stabilized MAP4K1 via modulating FUS. In summary, DLX6-AS1 modulated GC progression through FUS-regulated MAP4K1. Our paper exposed the role and regulatory mechanism of DLX6-AS1 in GC, which suggested a novel and valid therapy for GC patients.
Project description:Distal-less homeobox 6 antisense RNA 1 (DLX6-AS1) is upregulated in various solid tumors and serves a critical role in the tumorigenesis of cancer. However, to the best of our knowledge, the expression of circulating DLX6-AS1 and its role in the diagnosis of non-small cell lung cancer (NSCLC) have not been previously clarified. The aim of the present study was to investigate the expression and clinical significance of circulating DLX6-AS1 using reverse transcription-quantitative PCR in serum and exosomes derived from patients with NSCLC and healthy donors. The diagnostic value of circulating DLX6-AS1 was identified by receiver operating characteristic curve (ROC) analysis. First, it was revealed that the expression levels of DLX6-AS1 were significantly increased in tumor tissues compared with in adjacent normal tissues. In addition, DLX6-AS1 was highly expressed in NSCLC cell lines compared with in BEAS-2B cells. DLX6-AS1-knockdown inhibited cell proliferation and migration in vitro. It was subsequently demonstrated that the serum DLX6-AS1 level was significantly higher in patients with NSCLC compared with in healthy controls. Additionally, the higher DLX6-AS1 expression was associated with advanced disease stage, positive lymph node metastasis and poor tumor differentiation of NSCLC. ROC analysis demonstrated that the sensitivity and specificity of DLX6-AS1 were higher than those of CYFRA21-1, which is a serum marker for NSCLC. Finally, exosomal DLX6-AS1 expression was increased in patients with NSCLC compared with in healthy controls. The present data implied that circulating DLX6-AS1 was mainly incorporated into exosomes, providing a novel potential diagnostic marker for NSCLC.
Project description:<h4>Background</h4>Bladder cancer is the most common human urological malignancies with poor prognosis, and the pathophysiology of bladder cancer involves multi-linkages of regulatory networks in the bladder cancer cells. Recently, the long noncoding RNAs (lncRNAs) have been extensively studied for their role on bladder cancer progression. In this study, we evaluated the expression of DLX6 Antisense RNA 1 (DLX6-AS1) in the cancerous bladder tissues and studied the possible mechanisms of DLX6-AS1 in regulating bladder cancer progression.<h4>Methods</h4>Gene expression was determined by qRT-PCR; protein expression levels were evaluated by western blot assay; in vitro functional assays were used to determine cell proliferation, invasion and migration; nude mice were used to establish the tumor xenograft model.<h4>Results</h4>Our results showed the up-regulation of DLX6-AS1 in cancerous bladder cancer tissues and bladder cell lines, and high expression of DLX6-AS1 was correlated with advance TNM stage, lymphatic node metastasis and distant metastasis. The in vitro experimental data showed that DLX6-AS1 overexpression promoted bladder cancer cell growth, proliferation, invasion, migration and epithelial-to-mesenchymal transition (EMT); while DLX6-AS1 inhibition exerted tumor suppressive actions on bladder cancer cells. Further results showed that DLX6-AS1 overexpression increased the activity of Wnt/?-catenin signaling, and the oncogenic role of DLX6-AS1 in bladder cancer cells was abolished by the presence of XAV939. On the other hand, DLX6-AS1 knockdown suppressed the activity of Wnt/?-catenin signaling, and the tumor-suppressive effects of DLX6-AS1 knockdown partially attenuated by lithium chloride and SB-216763 pretreatment. The in vivo tumor growth study showed that DLX6-AS1 knockdown suppressed tumor growth of T24 cells and suppressed EMT and Wnt/?-catenin signaling in the tumor tissues.<h4>Conclusion</h4>Collectively, the present study for the first time identified the up-regulation of DLX6-AS1 in clinical bladder cancer tissues and in bladder cancer cell lines. The results from in vitro and in vivo assays implied that DLX6-AS1 exerted enhanced effects on bladder cancer cell proliferation, invasion and migration partly via modulating EMT and the activity of Wnt/?-catenin signaling pathway.
Project description:BACKGROUND:Liver cancer stem cells (LCSCs) are a small subset of cells characterized by unlimited self-renewal, cell differentiation, and uncontrollable cellular growth. LCSCs are also resistant to conventional therapies and are thus believed to be held responsible for causing treatment failure of hepatocellular carcinoma (HCC). It has been recently found that long non-coding RNAs (lncRNAs) are important regulators in HCC. This present study aims to explore the underlying mechanism of how lncRNA DLX6-AS1 influences the development of LCSCs and HCC. METHODS:A microarray-based analysis was performed to initially screen differentially expressed lncRNAs associated with HCC. We then analyzed the lncRNA DLX6-AS1 levels as well as CADM1 promoter methylation. The mRNA and protein expression of CADM1, STAT3, CD133, CD13, OCT-4, SOX2, and Nanog were then detected. We quantified our results by evaluating the spheroid formation, proliferation, and tumor formation abilities, as well as the proportion of tumor stem cells, and the recruitment of DNA methyltransferase (DNMT) in LCSCs when lncRNA DLX6-AS1 was either overexpressed or silenced. RESULTS:LncRNA DLX6-AS1 was upregulated in HCC. The silencing of lncRNA DLX6-AS1 was shown to reduce and inhibit spheroid formation, colony formation, proliferation, and tumor formation abilities, as well as attenuate CD133, CD13, OCT-4, SOX2, and Nanog expression in LCSCs. Furthermore, downregulation of lncRNA DLX6-AS1 contributed to a reduction in CADM1 promoter methylation via suppression of DNMT1, DNMT3a, and DNMT3b in LCSCs and inactivating the STAT3 signaling pathway. CONCLUSION:This study demonstrated that down-regulated lncRNA DLX6-AS1 may inhibit the stem cell properties of LCSCs through upregulation of CADM1 by suppressing the methylation of the CADM1 promoter and inactivation of the STAT3 signaling pathway.
Project description:EWS-FLI1 is a chromosome translocation-derived chimeric transcription factor that has a central and rate-limiting role in the pathogenesis of Ewing's sarcoma. Although the EWS-FLI1 transcriptomic signature has been extensively characterized on the mRNA level, information on its impact on non-coding RNA expression is lacking. We have performed a genome-wide analysis of microRNAs affected by RNAi-mediated silencing of EWS-FLI1 in Ewing's sarcoma cell lines, and differentially expressed between primary Ewing's sarcoma and mesenchymal progenitor cells. Here, we report on the identification of hsa-mir-145 as the top EWS-FLI1-repressed microRNA. Upon knockdown of EWS-FLI1, hsa-mir-145 expression dramatically increases in all Ewing's sarcoma cell lines tested. Vice versa, ectopic expression of the microRNA in Ewing's sarcoma cell lines strongly reduced EWS-FLI1 protein, whereas transfection of an anti-mir to hsa-mir-145 increased the EWS-FLI1 levels. Reporter gene assays revealed that this modulation of EWS-FLI1 protein was mediated by the microRNA targeting the FLI1 3'-untranslated region. Mutual regulations of EWS-FLI1 and hsa-mir-145 were mirrored by an inverse correlation between their expression levels in four of the Ewing's sarcoma cell lines tested. Consistent with the role of EWS-FLI1 in Ewing's sarcoma growth regulation, forced hsa-mir-145 expression halted Ewing's sarcoma cell line growth. These results identify feedback regulation between EWS-FLI1 and hsa-mir-145 as an important component of the EWS-FLI1-mediated Ewing's sarcomagenesis that may open a new avenue to future microRNA-mediated therapy of this devastating malignant disease.