A short peptide encoded by long non-coding RNA small nucleolar RNA host gene 6 promotes cell migration and epithelial-mesenchymal transition by activating transforming growth factor-beta/SMAD signaling pathway in human endometrial cells.

BACKGROUND: Endometrial dysfunction is closely correlated with the development of multiple severe gynecological disorders including intrauterine adhesion. Accumulating evidence supports that some long non-coding RNAs (lncRNAs) have peptide-coding potential. In this text, the peptide-coding ability of lncRNA SNHG6 was examined. Also, the effects of an SNHG6-encoded peptide on the viability and migration of human endometrial stromal cells (hESCs) and human endometrial epithelial cells (hEECs) and related molecular mechanisms were explored. METHODS: The peptide-encoding potential of SNHG6 was predicted by FuncPEP and getorf databases and validated by western blot assay. Cell viability was tested by cell counting kit-8 assay. Cell migratory ability was examined by wound healing and transwell migration assays. Protein levels of genes were measured by western blot assay. RESULTS: Prediction analysis suggested that SNHG6 had the potential peptide-coding ability and multiple open-reading frames (ORFs). Western blot validated that SNHG6 ORF#1 and ORF#2 could translate into short peptides. SNHG6 ORF#2 overexpression facilitated cell migration and epithelial-mesenchymal transition (EMT) in hESCs and hEECs, while these effects were abrogated by transforming growth factor-beta (TGF-ß)/SMAD signaling inhibitor GW788388. Moreover, GW788388 inhibited the increase of p-SMAD2 and p-SMAD3 levels induced by SNHG6 ORF#2 in hESCs. SNHG6 ORF#2-encoded peptide did not influence endometrial stromal and epithelial cell viability. CONCLUSIONS: LncRNA SNHG6 ORF#1 and ORF#2 could translate into small peptides and SNHG6 ORF#2 overexpression promoted cell migration and EMT by activating the TGF-ß/SMAD pathway in hESCs and hEECs, suggesting the potential roles of SNHG6-encoded peptides in the development of endometrial stromal and epithelial cells and related gynecological diseases.

Mechanisms for hypoxia-induced long non-coding small nucleolar RNA host gene 14 in promoting temozolomide resistance of glioma. / ä½Žæ°§è¯±å¯¼é•¿é“¾éžç¼–ç æ ¸å† å°RNAå®¿ä¸»åŸºå› 14促进胶质瘤替莫唑胺耐药的机制.

OBJECTIVES: This study aims to investigate the role of hypoxia-induced long non-coding small nucleolar RNA host gene 14 (lncRNA SNHG14) in glioma temozolomide (TMZ) resistance and underlying mechanisms. METHODS: According to different treatments, the experiment was divided into a normoxia group and a hypoxia group, a control group and a TMZ group. The lncRNA SNHG14 and O6-methylguanine DNA methyltransferase (MGMT) levels in glioma SNB19 and U251 cell line were detected by real-time PCR and Western blotting, respectively, and the association of lncRNA SNHG14 level with hypoxia and TMZ treatment was analyzed. siRNA was used to knockdown the lncRNA SNHG14 expression in glioma cells, and the transfected glioma cells were divided into a negative control group (si-NC group) and a si-SNHG14 group. The interference efficiency was examined by real-time PCR, the key factor MGMT of lncRNA SNHG14 sensitivity regulation was detected by Western blotting, and the cell apoptosis was detected by cytometry. In addition, MTT method was used to detect the cell viability of gliomas in the different groups under the different TMZ concentrations, and the effect of lncRNA SNHG14 on TMZ sensitivity of gliomas was analyzed. Online tools were used to predict miRNAs that could specifically bind to lncRNAs SNHG14 and MGMT. A si-NC group, a si-SNHG14 group, a normoxia group and a hypoxia group were set up, and the changes of miR-143 abundance in different environments were observed by real-time PCR. miR-143 mimics and inhibitor were used to change the level of miR-143 in glioma cells. A NC inhibitor group, a miR-143 inhibitor group, a NC mimics group and a miR-143 mimics group were set up, the interference efficiency was detected by real-time PCR, the expression level of MGMT was detected by Western blotting, and the effect of miR-143 on the level of MGMT were analyzed. The NC inhibitor group, the miR-143 inhibitor group, the NC mimics group and the miR-143 mimics group were treated with different interventions, and the dual luciferase reporter assay was used to observe the changes of lncRNA SNHG14 and MGMT luciferase activities, and to verify the relationship among lncRNA SNHG14, miR-143 and MGMT. Finally, a NC group and a lncRNA SNHG14 overexpression group were set up, and the changes in the abundance of miR-143 and MGMT in each group were detected by RNA-binding protein immunoprecipitation experiments, and the competitive binding relationship among lncRNA SNHG14, miR-143 and MGMT was analyzed. RESULTS: Compared with the normoxia group, the hypoxia group could promote the expression of lncRNA SNHG14 in glioma cells. Compared with the control group, the expression of lncRNA SNHG14 could be significantly inhibited in the TMZ group (P<0.05). Compared with the si-NC group, the expression of lncRNA SNHG14 in the si-SNHG14 group could be effectively inhibited, and the expression level of MGMT was significantly decreased, and the apoptosis rate was significantly increased (all P<0.05). With the increase of TMZ concentrations, the glioma cell viability in the si-SNHG14 group was significantly lower than that in the si-NC group, and the cell viability in the hypoxia group was significantly higher than that in the normoxia group (both P<0.05). Online tool prediction found that miR-143 had binding sites with lncRNA SNHG14 and MGMT. The abundance of miR-143 in the hypoxia group was significantly lower than that in the normoxic group, and the abundance of miR-143 in the si-SNHG14 group was significantly higher than that in the si-NC group (both P<0.05). The miR-143 mimics group or the miR-143 inhibitor group could significantly over-express or under-express miR-143 (both P<0.05). But there was no significant difference between the NC mimics group (or the NC inhibitor group) and the control group (both P>0.05). The level of MGMT protein could significantly up-regulate in the miR-143 inhibitor group, and on the contrary which could significantly down-regulate in the miR-143 mimics group (both P<0.01). The dual luciferase reporter assay showed that there was no significant difference between the NC mimics group (or the NC inhibitor group) and the control group (both P>0.05). The wild-type SNHG14 and MGMT luciferase activities were significantly down-regulated in the miR-143 mimics group, which were significantly up-regulated in the miR-143 inhibitor group (P<0.01 and P<0.05, respectively), but there was no significant change in the luciferase activities of mutant SNHG14 and MGMT (both P>0.05). The results of the RNA-binding protein immunoprecipitation experiment showed that: compared with the NC group, more lncRNA SNHG14 was bound to the precipitated argonaute 2 protein in the cells in the lncRNA SNHG14 overexpression group, but the abundance of MGMT mRNA was decreased significantly, and there were significant differences (both P<0.01). There was a targeting regulatory relationship among lncRNA SNHG14, miR-143 and MGMT. CONCLUSIONS: The up-regulated lncRNA SNHG14 can target miR-143, relieve the inhibition of miR-143 on MGMT, and promote the TMZ resistance in the hypoxia-induced glioma cells.

Long noncoding RNA SNHG14 knockdown exerts a neuroprotective role in MPP+-induced Parkinson's disease cell model through mediating miR-135b-5p/KPNA4 axis.

BACKGROUND: Parkinson's disease (PD) is a neurodegenerative disease resulted from the loss of dopaminergic neurons. Here, we analyzed the role of long noncoding RNA (lncRNA) small nucleolar RNA host gene 14 (SNHG14) in PD using 1-methyl-4-phenyl pyridine (MPP+)-induced PD cell model. METHODS: Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blot assay were performed to determine RNA and protein expression, respectively. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry (FCM) analysis were conducted to analyze cell viability and apoptosis. Enzyme-Linked Immunosorbent Assay (ELISA) was conducted to analyze the release of inflammatory cytokines. Cytotoxicity was assessed using reactive oxygen species (ROS) assay kit, superoxide dismutase (SOD) activity assay kit and lactate dehydrogenase (LDH) activity assay kit. Dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay were performed to confirm the interaction between microRNA-135b-5p (miR-135b-5p) and SNHG14 or karyopherin subunit alpha 4 (KPNA4). RESULTS: MPP+ treatment elevated the expression of SNHG14 in SK-N-SH cells in a dose and time-dependent manner. SNHG14 knockdown alleviated MPP+-induced apoptosis, inflammation, and cytotoxicity in SK-N-SH cells. SNHG14 interacted with miR-135b-5p, and SNHG14 silencing-mediated effects were partly overturned by miR-135b-5p knockdown in PD cell model. Besides, miR-135b-5p interacted with the 3' untranslated region (3'UTR) of KPNA4, and KPNA4 overexpression partly reversed miR-135b-5p overexpression-induced effects in PD cell model. SNHG14 knockdown reduced the protein level of KPNA4 partly by up-regulating miR-135b-5p in SK-N-SH cells. CONCLUSION: SNHG14 promoted MPP+-induced neuro injury in PD cell model through mediating miR-135b-5p/KPNA4 axis.

Long non-coding RNA-small nucleolar RNA host gene 7 regulates inflammatory responses following spinal cord injury by regulating the microRNA-449a/TNF-α-induced protein 3-interacting protein 2 axis.

The current study aimed to explore the anti-inflammatory effects of long non-coding RNA-small nucleolar RNA host gene 7 (lncRNA-SNHG7) and its mechanism in spinal cord injury (SCI) models. SCI models were established both in vivo and in vitro. Reverse transcription-quantitative PCR was performed to determine the expression levels of lncRNA-SNHG7 in SCI models. Bioinformatics analysis and dual-luciferase reporter assays were carried out to confirm the interaction between lncRNA-SNHG7 with microRNA (miR)-499a and TNF-α-induced protein 3-interacting protein 2 (TNIP2). In addition, cell viability, apoptosis, and the secretion of inflammatory cytokines were assessed by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay, flow cytometric analysis, and enzyme linked immunosorbent assay (ELISA), respectively. The results showed that lncRNA-SNHG7 was markedly downregulated in the SCI model group. LncRNA-SNHG7 directly bound to miR-499a, which in turn directly targeted TNIP2. In addition, TNIP2 was significantly decreased in SCI rats and lipopolysaccharide (LPS)-treated PC-12 cells. The in vitro results in PC-12 cells revealed that lncRNA-SNHG7 overexpression attenuated neuronal cell death and SCI-mediated inflammatory responses by regulating miR-449a expression. Furthermore, miR-499a knockdown inhibited LPS-induced PC-12 cell injury by targeting TNIP2. In conclusion, lncRNA-SNHG7 modulates the apoptosis and inflammation of PC-12 cells by regulating the miR-449a/TNIP2/NF-κB signaling pathway.

Transcriptomic Analysis of Cardiomyocyte Extracellular Vesicles in Hypertrophic Cardiomyopathy Reveals Differential snoRNA Cargo.

Hypertrophic cardiomyopathy (HCM) is characterized by increased left ventricular wall thickness that can lead to devastating conditions such as heart failure and sudden cardiac death. Despite extensive study, the mechanisms mediating many of the associated clinical manifestations remain unknown and human models are required. To address this, human-induced pluripotent stem cell (hiPSC) lines were generated from patients with a HCM-associated mutation (c.ACTC1G301A) and isogenic controls created by correcting the mutation using CRISPR/Cas9 gene editing technology. Cardiomyocytes (hiPSC-CMs) were differentiated from these hiPSCs and analyzed at baseline, and at increased contractile workload (2 Hz electrical stimulation). Released extracellular vesicles (EVs) were isolated and characterized after a 24-h culture period and transcriptomic analysis performed on both hiPSC-CMs and released EVs. Transcriptomic analysis of cellular mRNA showed the HCM mutation caused differential splicing within known HCM pathways, and disrupted metabolic pathways. Analysis at increasing contraction frequency showed further disruption of metabolic gene expression, with an additive effect in the HCM background. Intriguingly, we observed differences in snoRNA cargo within HCM released EVs that specifically altered when HCM hiPSC-CMs were subjected to increased workload. These snoRNAs were predicted to have roles in post-translational modifications and alternative splicing, processes differentially regulated in HCM. As such, the snoRNAs identified in this study may unveil mechanistic insight into unexplained HCM phenotypes and offer potential future use as HCM biomarkers or as targets in future RNA-targeting therapies.

Long Non-Coding RNA Small Nucleolar RNA Host Gene 1 Alleviates Sepsis-Associated Myocardial Injury by Modulating the miR-181a-5p/XIAP Axis in vitro.

OBJECTIVE: Sepsis is a systemic inflammatory response syndrome that results in severe myocardial injury. This study aimed to explore the role and mechanism of long non-coding RNA (lncRNA) small nucleolar RNA host gene 1 (SNHG1) in sepsis-induced myocardial injury in vitro. METHODS: Embryonic rat ventricular myocardial cell line (H9c2) was treated with lipopolysaccharide (LPS) to simulate sepsis-induced myocardial injury in vitro. A quantitative real-time polymerase chain reaction was executed to determine the expression of SNHG1 and microRNA (miR)-181a-5p. 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-h-tetrazolium bromide assay was employed to measure cell viability. The levels of inflammatory factors (tumor necrosis factor alpha [TNF-α], interleukin 6 [IL-6], and IL-1ß) were measured by enzyme-linked immunosorbent assay. Oxidative stress was assessed by measuring malondialdehyde, superoxide dismutase, and lactate dehydrogenase. The targeted interrelations among SNHG1, miR-181a-5p, and X-linked inhibitor of apoptosis protein (XIAP) were verified by dual-luciferase reporter assay. Relative protein expression of XIAP was detected by western blot. RESULTS: SNHG1 and XIAP were down-regulated, and miR-181a-5p was up-regulated in LPS-induced H9c2 cells. Overexpression of SNHG1 or inhibition of miR-181a-5p facilitated cell viability and repressed inflammation and oxidative stress in LPS-treated H9c2 cells. MiR-181a-5p was a target of and negatively regulated by SNHG1. At the same time, XIAP was a target gene of and inversely modulated by miR-181a-5p. In addition, XIAP was positively regulated by SNHG1. Up-regulation of miR-181a-5p or silencing of XIAP reversed the inhibitory effects of SNHG1 on inflammation and oxidative stress, as well as the promoting effects on cell viability in LPS-induced H9c2 cells. CONCLUSION: SNHG1 protected H9c2 cells against LPS-induced injury through modulating the miR-181a-5p/XIAP axis.

Long non-coding RNA small nucleolar RNA host gene 1 alleviates the progression of epilepsy by regulating the miR-181a/BCL-2 axis in vitro.

PURPOSE: Long non-coding RNAs (lncRNAs) have been reported to be involved in regulating epilepsy. The purpose of this study is to investigate the possibly regulatory mechanism of small nucleolar RNA host gene 1 (SNHG1) on epilepsy. METHODS: Quantitative real-time PCR was utilized to detect the expression of SNHG1, microRNA (miR)-181a, and B-cell lymphoma-2 (BCL-2). Through an enzyme-linked immunosorbent assay, the levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, IL-6, and cyclooxygenase-2 (COX-2) were determined. The viability and apoptosis of CTX-TNA2 cells were measured using MTT assay and flow cytometry analysis, respectively. Western blot assay was performed to analyze the protein levels of Bcl-2, BCL2-associated X, and Caspase-3. The relationships between miR-181a and SNHG1/BCL-2 were confirmed by the dual-luciferase reporter assay. RESULTS: SNHG1 expression was down-regulated in EP tissues and kainic acid (KA)-induced CTX-TNA2 cells. The apoptosis and release of inflammatory factors (TNF-α, IL-1ß, IL-6, and COX-2) in KA-induced CTX-TNA2 cells were suppressed by SNHG1 overexpression and promoted by miR-181a up-regulation. In addition, we confirmed that SNHG1 targeted miR-181a, whereas BCL-2 was a target gene of miR-181a. Negative correlations between SNHG1 and miR-181a, as well as miR-181a and BCL-2 were exhibited. Both the up-regulation of miR-181a and down-regulation of BCL-2 reversed the inhibiting effects of SNHG1 on apoptosis and inflammatory response of KA-induced CTX-TNA2 cells, and the promoting effect upon cell viability. CONCLUSIONS: SNHG1 alleviated the progression of EP by modulating the miR-181a/BCL-2 axis in vitro, thus SNHG1 could act as a possible therapeutic target for treating EP.

hNUDT16: a universal decapping enzyme for small nucleolar RNA and cytoplasmic mRNA.

Human NUDT16 (hNUDT16) is a decapping enzyme initially identified as the human homolog to the Xenopus laevis X29. As a metalloenzyme, hNUDT16 relies on divalent cations for its cap-hydrolysis activity to remove m7GDP and m²²7GDP from RNAs. Metal also determines substrate specificity of the enzyme. So far, only U8 small nucleolar RNA (snoRNA) has been identified as the substrate of hNUDT16 in the presence of Mg²(+). Here we demonstrate that besides U8, hNUDT16 can also actively cleave the m7GDP cap from mRNAs in the presence of Mg²(+) or Mn²(+). We further show that hNUDT16 does not preferentially recognize U8 or mRNA substrates by our cross-inhibition and quantitative decapping assays. In addition, our mutagenesis analysis identifies several key residues involved in hydrolysis and confirms the key role of the REXXEE motif in catalysis. Finally an investigation into the subcellular localization of hNUDT16 revealed its abundance in both cytoplasm and nucleus. These findings extend the substrate spectrum of hNUDT16 beyond snoRNAs to also include mRNA, demonstrating the pleiotropic decapping activity of hNUDT16.