LncRNA SNHG1 promotes sepsis‐induced myocardial injury by inhibiting Bcl‐2 expression via DNMT1

Myocardial injury is a frequently occurring complication of sepsis. This study aims to investigate the molecular mechanism of long noncoding RNA (lncRNA) small nucleolar RNA host gene 1 (SNHG1)‐mediated DNA methyltransferase 1/B‐cell lymphoma‐2 (DNMT1/Bcl‐2) axis in sepsis‐induced myocardial injury. Mice and HL‐1 cells were treated with lipopolysaccharide (LPS) to establish animal and cellular models simulating sepsis and inflammation. LncRNA SNHG1 was screened out as a differentially expressed lncRNA in sepsis samples through microarray profiling, and the upregulated expression of lncRNA SNHG1 was confirmed in myocardial tissues of LPS‐induced septic mice and HL‐1 cells. Further experiments suggested that silencing of lncRNA SNHG1 reduced the inflammation and apoptotic rate of LPS‐induced HL‐1 cells. LncRNA SNHG1 inhibited Bcl‐2 expression by recruiting DNMT1 to Bcl‐2 promoter region to cause methylation. Inhibition of Bcl‐2 promoter methylation reduced the inflammation and apoptotic rate of LPS‐induced HL‐1 cells. In vivo experiments substantiated that lncRNA SNHG1 silencing alleviated sepsis‐induced myocardial injury in mice. Taken together, lncRNA SNHG1 promotes LPS‐induced myocardial injury in septic mice by downregulating Bcl‐2 through DNMT1‐mediated Bcl‐2 methylation.     

DNA Methylation Modulates Nociceptive Sensitization after Incision

DNA methylation is a key epigenetic mechanism controlling DNA accessibility and gene expression. Blockade of DNA methylation can significantly affect pain behaviors implicated in neuropathic and inflammatory pain. However, the role of DNA methylation with regard to postoperative pain has not yet been explored. In this study we sought to investigate the role of DNA methylation in modulating incisional pain and identify possible targets under DNA methylation and contributing to incisional pain. DNA methyltranferase (DNMT) inhibitor 5-Aza-2′-deoxycytidine significantly reduced incision-induced mechanical allodynia and thermal sensitivity. Aza-2′-deoxycytidine also reduced hindpaw swelling after incision, suggesting an anti-inflammatory effect. Global DNA methylation and DNMT3b expression were increased in skin after incision, but none of DNMT1, DNMT3a or DNMT3b was altered in spinal cord or DRG. The expression of proopiomelanocortin Pomc encoding β-endorphin and Oprm1 encoding the mu-opioid receptor were upregulated peripherally after incision; moreover, Oprm1 expression was further increased under DNMT inhibitor treatment. Finally, local peripheral injection of the opioid receptor antagonist naloxone significantly exacerbated incision-induced mechanical hypersensitivity. These results suggest that DNA methylation is functionally relevant to incisional nociceptive sensitization, and that mu-opioid receptor signaling might be one methylation regulated pathway controlling sensitization after incision.     

Antinociceptive effect of VSL#3 on visceral hypersensitivity in a rat model of irritable bowel syndrome: a possible action through nitric oxide pathway and enhance barrier function

Irritable bowel syndrome (IBS) is a functional bowel disorder characterized by visceral hypersensitivity and altered bowel function. There are increasing evidences suggested that VSL#3 probiotics therapy has been recognized as an effective method to relieve IBS-induced symptoms. The aim of this study was to examine the effects of VSL#3 probiotics on visceral hypersensitivity (VH), nitric oxide (NO), fecal character, colonic epithelium permeability, and tight junction protein expression. IBS model was induced by intracolonic instillation of 4% acetic acid and restraint stress in rats. After subsidence of inflammation on the seventh experimental day, the rats were subjected to rectal distension, and then the abdominal withdrawal reflex and the number of fecal output were measured, respectively. Also, colonic permeability to Evans blue was measured in vivo, and tight junction protein expression was studied by immunohistochemistry and immunoblotting method. Rats had been pretreated with VSL#3 or aminoguanidine (NOS inhibitor) or VSL#3+ aminoguanidine before measurements. The rats at placebo group showed hypersensitive response to rectal distension (P < 0.05) and defecated more stools than control rats (P < 0.05), whereas VSL#3 treatment significantly attenuated VH and effectively reduced defecation. Aminoguanidine reduced the protective effects of VSL#3 on VH. A pronounced increase in epithelial permeability and decreased expression of tight junction proteins (occludin, ZO-1) in placebo group were prevented by VSL#3, but not aminoguanidine. VSL#3 treatment reduce the hypersensitivity, defecation, colonic permeability and increase the expression of tight junction proteins (occludin, ZO-1). As the part of this effect was lowered by NOS inhibitor, NO might play a role in the protective effect of VSL#3 to some extent.     

Increased Expression of Colonic Mucosal Melatonin in Patients with Irritable Bowel Syndrome Correlated with Gut Dysbiosis

Dysregulation of the gut microbiota/gut hormone axis contributes to the pathogenesis of irritable bowel syndrome (IBS). Melatonin plays a beneficial role in gut motility and immunity. However, altered expression of local mucosal melatonin in IBS and its relationship with the gut microbiota remain unclear. Therefore, we aimed to detect the colonic melatonin levels and microbiota profiles in patients with diarrhea-predominant IBS (IBS-D) and explore their relationship in germ-free (GF) rats and BON-1 cells. Thirty-two IBS-D patients and twenty-eight healthy controls (HCs) were recruited. Fecal specimens from IBS-D patients and HCs were separately transplanted into GF rats by gavage. The levels of colon mucosal melatonin were assessed by immunohistochemical methods, and fecal microbiota communities were analyzed using 16S rDNA sequencing. The effect of butyrate on melatonin synthesis in BON-1 cells was evaluated by ELISA. Melatonin levels were significantly increased and negatively correlated with visceral hypersensitivity in IBS-D patients. GF rats inoculated with fecal microbiota from IBS-D patients had high colonic melatonin levels. Butyrate-producing Clostridium cluster XIVa species, such as Roseburia species and Lachnospira species, were positively related to colonic mucosal melatonin expression. Butyrate significantly increased melatonin secretion in BON-1 cells. Increased melatonin expression may be an adaptive protective mechanism in the development of IBS-D. Moreover, some Clostridium cluster XIVa species could increase melatonin expression via butyrate production. Modulation of the gut hormone/gut microbiota axis offers a promising target of interest for IBS in the future.     

肠道菌群与腹泻型肠易激综合征 相关性的研究进展

肠易激综合征(IBS)是一种常见的功能性胃肠道疾病,其特征是反复发作的腹痛,伴随排便频率与大便性状的改变。腹泻为主的肠易激综合征(IBS-D)是其主要亚型,主要表现是腹痛和腹泻。目前IBS-D的发病机制尚不完全明确,但大量的研究提示可能与胃肠道动力紊乱、黏膜通透性和肠上皮屏障功能改变、内脏高敏感性增加、"脑-肠-菌"轴失调、肠道感染与炎症反应激活、精神心理因素异常等有关。随着研究的不断深入,发现肠道菌群与IBS-D的关系密切,调节肠道菌群的益生菌干预成为缓解IBS-D相关症状的手段之一。本研究就近十余年来肠道菌群情况与IBS-D关系的研究现状作一综述。     

Schisandra chinensis reverses visceral hypersensitivity in a neonatal-maternal separated rat model

Visceral hypersensitivity is an important characteristic feature of functional gastrointestinal disorders, such as irritable bowel syndrome (IBS). This study evaluated the effect of Schisandra chinensis on visceral hyperalgesia induced by neonatal maternal separation (NMS) in an IBS rat model. The visceromotor responses to colorectal balloon distension (CRD) were measured by abdominal withdrawal reflex (AWR) and electromyographic (EMG) activities. NMS control rats (receiving vehicle) underwent aggravated visceral pain in response to CRD as compared to normal rats, evidenced by the reduced pain threshold, enhanced AWR scores and EMG responses. Treatment with a 70% ethanol extract of S. chinensis (0.3g/kg and 1.5g/kg/day) for 7 days resulted in an increase in the pain threshold (NMS control: 19.1±1.0mmHg vs low-dose: 24.8±1.3mmHg and high-dose: 25.2±1.8mmHg, p<0.01), and abolished the elevated AWR and EMG responses to CRD in NMS rats (AUC values of EMG response curve were: 1952±202 in NMS control group vs 1074±90 in low-dose group and 1145±92 in high-dose group, p<0.001), indicating that S. chinensis could reverse the visceral hypersensitivity induced by early-life stress event. The result of ELSA measurement shows that the elevated serotonin (5-HT) level in the distal colon of NMS rats returned to normal level after treatment with S. chinensis. Moreover, the increase in pain threshold in rats treated with S. chinensis was associated with a decline of the mRNA level of 5-HT(3) receptor in the distal colon. All available results demonstrate that S. chinensis can reverse visceral hypersensitivity induced by neonatal-maternal separation, and the effect may be mediated through colonic 5-HT pathway in the rat.     

Depleting long noncoding RNA HOTAIR attenuates chronic myelocytic leukemia progression by binding to DNA methyltransferase 1 and inhibiting PTEN gene promoter methylation

Long noncoding RNAs (lncRNAs) are known to play a key role in chronic myelocytic leukemia (CML) development, and we aimed to identify the involvement of the lncRNA HOX antisense intergenic RNA (HOTAIR) in CML via binding to DNA methyltransferase 1 (DNMT1) to accelerate methylation of the phosphatase and tensin homolog (PTEN) gene promoter. Bone marrow samples from CML patients and normal bone marrow samples from healthy controls were collected. HOTAIR, DNMT1, DNMT3A, DNMT3B, and PTEN expression was detected. The biological characteristics of CML cells were detected. The relationship among HOTAIR, DNMT1, and PTEN was verified. Tumor volume and weight in mice injected with CML cells were tested. We found that HOTAIR and DNMT1 expression was increased and PTEN expression was decreased in CML. We also investigated whether downregulated HOTAIR or DNMT1 reduced proliferation, colony formation, invasion, and migration and increased the apoptosis rate of CML cells. Moreover, we tested whether low expression of HOTAIR or DNMT1 reduced the volume and weight of tumors in mice with CML. Collectively, the results of this studied showed that depleted HOTAIR demonstrated reduced binding to DNMT1 to suppress CML progression, which may be related to methylation of the PTEN promoter.Subject terms: Cell biology, Diseases     

Impact of DNA methyltransferases on the epigenetic regulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor expression in malignant melanoma

Aberrant promoter methylation and resultant silencing of TRAIL decoy receptors were reported in a variety of cancers, but to date little is known about the relevance of this epigenetic modification in melanoma. In this study, we examined the methylation and the expression status of TRAIL receptor genes in cutaneous and uveal melanoma cell lines and specimens and their interaction with DNA methyltransferases (DNMTs) DNMT1, DNMT3a, and DNMT3b. DR4 and DR5 methylation was not frequent in cutaneous melanoma but on the contrary it was very frequent in uveal melanoma. No correlation between methylation status of DR4 and DR5 and gene expression was found. DcR1 and DcR2 were hypermethylated with very high frequency in both cutaneous and uveal melanoma. The concordance between methylation and loss of gene expression ranged from 91% to 97%. Here we showed that DNMT1 was crucial for DcR2 hypermethylation and that DNMT1 and DNMT3a coregulate the methylation status of DcR1. Our work also revealed the critical relevance of DcR1 and DcR2 expression in cell growth and apoptosis either in cutaneous or uveal melanoma. In conclusion, the results presented here claim for a relevant impact of aberrant methylation of decoy receptors in melanoma and allow to understand how the silencing of DcR1 and DcR2 is related to melanomagenesis.     

Downregulated lncRNA RCPCD promotes differentiation of embryonic stem cells into cardiac pacemaker-like cells by suppressing HCN4 promoter methylation

Long non-coding RNA (lncRNA) is receiving increasing attention in embryonic stem cells (ESCs) research. However, the roles of lncRNA in the differentiation of ESCs into pacemaker-like cells are still unclear. Therefore, the present study aims to explore the roles and mechanisms of lncRNA in the differentiation of ESCs into pacemaker-like cells. ESCs were cultured and induced differentiation to pacemaker-like cells. RNA sequencing was used to identify the differential expression lncRNAs during the differentiation of ESCs into pacemaker-like cells. Cell morphology observation, flow cytometry, quantitative real-time polymerase chain reaction, western blot, and immunofluorescence were used to detect the differentiation of ESCs into pacemaker-like cells. LncRNA and genes overexpression or knockdown through transfected adenovirus in the differentiation process. The fluorescence in situ hybridization (FISH) detected the lncRNA location in the differentiated ESCs. Luciferase reporter gene assay, methylation-specific PCR, chromatin immunoprecipitation assay, and RNA immunoprecipitation assay were performed to reveal the mechanism of lncRNA-regulating HCN4 expression. Rescue experiments were used to confirm that lncRNA regulates the differentiation of ESCs into pacemaker-like cells through HCN4. We cultured the ESCs and induced the differentiation of ESCs into pacemaker-like cells successfully. The expression of lncRNA RCPCD was significantly decreased in the differentiation of ESCs into pacemaker-like cells. Overexpression of RCPCD inhibited the differentiation of ESCs into pacemaker-like cells. RCPCD inhibited the expression of HCN4 by increasing HCN4 methylation at the promoter region through DNMT1, DNMT2, and DNMT3. RCPCD inhibited the differentiation of ESCs into pacemaker-like cells by inhibiting the expression of HCN4. Our results confirm the roles and mechanism of lncRNA RCPCD in the differentiation of ESCs into pacemaker-like cells, which could pave the path for the development of a cell-based biological pacemaker.Subject terms: Arrhythmias, Stem-cell research     

Investigating the role of DNMT1 gene expression on myocardial ischemia reperfusion injury in rat and associated changes in mitochondria

Altered DNA methylation and mitochondrial dysfunction are the two key features of myocardial ischemia reperfusion injury (I/R), but their association with I/R remains unknown. In the present study, the relationship between DNA methyl transferase1 (DNMT1), the key methylation gene, and the mitochondrial quality control genes in rat heart during I/R was explored. We used the Langendorff rat heart model with 30 min of ischemia followed by 60 min of reperfusion and subsequent inhibition of DNMT1 with 5-azacytidine to evaluate the role of DNA methylation in I/R. Reperfusion significantly increased the expression of the DNMT1 gene, enzyme activity, and global DNA methylation levels, along with decreased mitochondrial copy, electron transport chain (ETC) activities, and ATP level. This was in agreement with the significant downregulation of 11 mitochondrial genes PGC-1α, TFAM, POLG, MFN1 and MFN2, FIS1, PARKIN, OPTN, ND1, ND4L, Cyt B and COX1 in I/R induced rat hearts. The expression pattern of the mitochondrial genes PGC-1α, TFAM, ND1 and Cyt B showed a significant negative correlation with DNMT1 expression. Rate pressure product, index of cardiac performance negatively correlated with DNMT1 expression (r = -0.8231, p = 0.0456). However, DNMT1 inhibited rat hearts via 5-azacytidine significantly improved the heart from I/R injury and reversed the I/R associated changes in the gene expression of TFAM, POLG, PGC-1α, ND1, COX1 and Cyt B, and improved the overall mtDNA copies, with a subsequent improvement in the ETC enzyme activity and ATP levels. To conclude, I/R augmented the DNMT1 activity with a subsequent increase in cardiac injury via downregulating the mitochondrial functional genes.     

Dynamics of DNA Methylation during Early Development of the Preimplantation Bovine Embryo

There is species divergence in control of DNA methylation during preimplantation development. The exact pattern of methylation in the bovine embryo has not been established nor has its regulation by gender or maternal signals that regulate development such as colony stimulating factor 2 (CSF2). Using immunofluorescent labeling with anti-5-methylcytosine and embryos produced with X-chromosome sorted sperm, it was demonstrated that methylation decreased from the 2-cell stage to the 6–8 cell stage and then increased thereafter up to the blastocyst stage. In a second experiment, embryos of specific genders were produced by fertilization with X- or Y-sorted sperm. The developmental pattern was similar to the first experiment, but there was stage × gender interaction. Methylation was greater for females at the 8-cell stage but greater for males at the blastocyst stage. Treatment with CSF2 had no effect on labeling for DNA methylation in blastocysts. Methylation was lower for inner cell mass cells (i.e., cells that did not label with anti-CDX2) than for trophectoderm (CDX2-positive). The possible role for DNMT3B in developmental changes in methylation was evaluated by determining gene expression and degree of methylation. Steady-state mRNA for DNMT3B decreased from the 2-cell stage to a nadir for D 5 embryos >16 cells and then increased at the blastocyst stage. High resolution melting analysis was used to assess methylation of a CpG rich region in an intronic region of DNMT3B. Methylation percent decreased between the 6–8 cell and the blastocyst stage but there was no difference in methylation between ICM and TE. Results indicate that DNA methylation undergoes dynamic changes during the preimplantation period in a manner that is dependent upon gender and cell lineage. Developmental changes in expression of DNMT3B are indicative of a possible role in changes in methylation. Moreover, DNMT3B itself appears to be under epigenetic control by methylation.