Protective effect of DLX6-AS1 silencing against cerebral ischemia/reperfusion induced impairments.
ABSTRACT: In the present study, we investigated the role of lncRNA mus distal-less homeobox 6 antisense 1 (DLX6-AS1) 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 in vivo and in vitro, 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: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:Background:Colorectal cancer (CRC) is considered as the second common death-induced cancer. More recently, association of long non-coding RNAs (lncRNAs) with CRC has been extensively investigated. Therefore, the present study was performed to determine whether lncRNA MAF BZIP Transcription Factor G Antisense RNA 1 (MAFG-AS1) could regulate biological activities of CRC cells and unravel the underlying mechanisms. Methods:CRC and corresponding adjacent tissues were collected to determine the expression of lncRNA MAFG-AS1, microRNA-149-3p (miR-149-3p) and homeobox B8 (HOXB8) by RT-qPCR. Dual luciferase reporter gene assay was used to explore the targeting relationship between miR-149-3p and lncRNA MAFG-AS1 and between miR-149-3p and HOXB8, followed by RNA immunoprecipitation for verification. Migration, proliferation, invasion, and apoptosis of HCT116 and LoVo cells were examined when lncRNA MAFG-AS1 was silenced or miR-149-3p was overexpressed. Furthermore, tumorigenicity of HCT116 and LoVo cells was measured in vivo by tumor xenograft in nude mice. Results:LncRNA MAFG-AS1 and HOXB8 were found to be highly expressed in CRC tissues and cells, while miR-149-3p was under-expressed. LncRNA MAFG-AS1 negatively regulated miR-149-3p while miR-149-3p downregulated HOXB8. In addition, lncRNA MAFG-AS1 silencing by shRNA or miR-149-3p upregulation by mimic suppressed the migration, proliferation, invasion and tumorigenesis but promoted the apoptosis of HCT116 and LoVo cells. Conclusion:Taken together, lncRNA MAFG-AS1 downregulation inhibits the malignant behaviors of CRC cells by upregulating miR-149-3p and downregulating HOXB8, providing a potential therapeutic target for CRC treatment.
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:Background:Currently, cancer-related competing endogenous RNA (ceRNA) networks are attracting significant interest. As long noncoding RNA ZEB1-AS1 has been reported to function as an oncogene due to sponging microRNAs (miRNAs) in several cancers, we hypothesized that it could interact with specific miRNAs to form regulatory networks and facilitate the growth of gastric cancer (GC). Methods:MiRNAs interacting with ZEB1-AS1 were screened for and selected by bioinformatics analysis. Overexpression or repression of ZEB1-AS1 was performed to determine whether it could regulate selected miRNAs. Quantitative real-time polymerase chain reactions (qPCR) validated the expression profiles of ZEB1-AS1 and miR-149-3p in GC cell lines and tissue. Statistical analysis determined the clinical significance of ZEB1-AS1 in relation to miR-149-3p. Cell counting, wound healing and transwell assays were performed to assess cell proliferation, migration and invasion. A luciferase reporter assay was utilized to confirm the putative miR-149-3p-binding sites in ZEB1-AS1. Results:Briefly, bioinformatics analysis inferred that ZEB1-AS1 interacts with miR-204, miR-610, and miR-149. Gain- or loss-of function assays suggested that ZEB1-AS1 negatively regulates miR-149-3p, miR-204-5p and miR-610 in GC cells. Validated by qPCR, ZEB1-AS1 was up-regulated and miR-149-3p down-regulated in GC cells and tissue. Data analyses indicated that ZEB1-AS1 and miR-149-3p are associated with the independent diagnosis and prognosis of GC. Functional assays support the theory that miR-149-3p hinders GC proliferation, migration and invasion, whereas its overexpression abrogates the corresponding effects induced by ZEB1-AS1. Lastly, dissection of the molecular mechanisms involved indicated that ZEB1-AS1 can regulate GC partly via a ZEB1-AS1/miR-149-3p axis. Conclusions:ZEB1-AS1 can interact with specific miRNAs, forming a miRNA-mediated ceRNA network and promoting GC progress, partly through a ZEB1-AS1/miR-149-3p axis.
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: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: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:Pancreatic ductal adenocarcinoma (PDAC) remains a leading cause of cancer-related death due to the failure of traditional therapies. In the present study, we attempted to construct a lncRNA-miRNA-mRNA network which may modulate PDAC cell proliferation and Gemcitabine-induced cell apoptosis starting from CDK14, a new member of the CDK family and an oncogene in many cancers. Based on TCGA data, a significant positive correlation was observed between lncRNA MSC-AS1 and CDK14. Moreover, MSC-AS1 expression was upregulated in PDAC tissues. Higher MSC-AS1 expression was correlated with poorer prognosis in patients with PDAC. MSC-AS1 knockdown in Panc-1 and BxPC-3 cells significantly inhibited the cell proliferation. Moreover, miR-29b-3p, which has been reported to act as a tumor suppressor, was predicted to bind to both MSC-AS1 and CDK14. Contrary to MSC-AS1, higher miR-29b-3p expression was correlated to better prognosis in patients with PDAC. In both PDAC cell lines, miR-29b-3p negatively regulated MSC-AS1 and CDK14. As confirmed using luciferase reporter gene and RIP assays, MSC-AS1 served as a ceRNA for miR-29b-3p to counteract miR-29b-mediated CDK14 repression. MSC-AS1 knockdown inhibited CDK14 protein levels and PDAC proliferation and enhanced gemcitabine-induced cell death and apoptosis while miR-29b-3p inhibition exerted an opposing effect; the effect of MSC-AS1 knockdown was partially attenuated by miR-29b-3p inhibition. Taken together, we demonstrated that MSC-AS1/miR-29b-3p axis modulates the cell proliferation and GEM-induced cell apoptosis in PDAC cell lines through CDK14. We provided a novel experimental basis for PDAC treatment from the perspective of lncRNA-miRNA-mRNA network.
Project description:Background:Multiple myeloma (MM) is a prevalent hematological malignancy. Long noncoding RNAs are correlated with the development of MM. In this project, the function of lncRNA opa interacting protein 5-antisense 1 (OIP5-AS1) in MM and the potential mechanistic pathway were explored. Methods:The expression of OIP5-AS1, microRNA (miR)-27a-3p and tuberous sclerosis 1 (TSC1) was analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) assay. Cell proliferation was assessed by Cell Counting Kit-8 (CCK-8) assay, colony formation assay and Bromodeoxyuridine (BrdU) staining. And cell apoptosis was evaluated by flow cytometry assay. Cell metastasis was assessed utilizing transwell assay. Western blot analysis was employed to detect protein level. The target relation between miR-27a-3p and OIP5-AS1 or TSC1 was confirmed via dual-luciferase reporter assay and RNA immunoprecipitation assay. Tumor xenograft assay was conducted to measure the function of OIP5-AS1 in vivo. Results:The expression levels of OIP5-AS1 and TSC1 were decreased in MM, whereas miR-27a-3p was upregulated. High level of OIP5-AS1 could predict favourable prognosis of MM patients. Overexpression of OIP5-AS1 inhibited cell viability, colony formation ability, migration and invasion, induced cell cycle arrest in G1 phase and apoptosis of MM cells in vitro as well as repressed tumorigenesis in vivo. MiR-27a-3p was a target of OIP5-AS1, and reversed the impact of OIP5-AS1 on MM cells. MiR-27a-3p directly targeted TSC1. Silencing of miR-27a-3p repressed MM progression by elevating TSC1 expression. OIP5-AS1 upregulated TSC1 by sponging miR-27a-3p. Conclusion:OIP5-AS1 repressed multiple myeloma progression by regulating miR-27a-3p/TSC1 axis.
Project description:AIM:To explore the mechanism underlying the protective effect of adipose-derived mesenchymal stem cells (ADMSCs) against ischemic stroke by focusing on miR-21-3p/MAT2B axis. METHODS:Ischemic brain injury was induced in 126 rats by middle cerebral artery occlusion (MCAO). The effect of ADMSC administration on blood-brain barrier (BBB) condition, apoptosis, inflammation, and the activity of miR-21-3p/MAT2B axis was assessed. The role of miR-21-3p inhibition in the function of ADMSCs was further validated in in vitro neural cells. RESULTS:ADMSCs administration improved BBB condition, inhibited apoptosis, and suppressed inflammation. It also reduced the abnormally high level of miR-21-3p in MCAO rats. Dual luciferase assays showed that miR-21-3p directly inhibited the MAT2B expression in neural cells, and miR-21-3p inhibition by inhibitor or ADMSC-derived exosomes in neurons attenuated hypoxia/reoxygenation-induced impairments similarly to that of ADMSCs in vivo. CONCLUSION:This study confirmed the protective effect of ADMSCs against ischemic brain injury exerted by suppressing miR-21-3p level and up-regulating MAT2B level.