lncRNA XIST Research Articles

Exosomal lncRNA XIST promotes perineural invasion of pancreatic cancer cells via miR-211-5p/GDNF.

Perineural invasion (PNI) is an essential form of tumor metastasis in multiple malignant cancers, such as pancreatic cancer, prostate cancer, and head and neck cancer. Growing evidence has revealed that pancreatic cancer recurrence and neuropathic pain positively correlate with PNI. Therefore, targeting PNI is a proper strategy for pancreatic cancer treatment. Exosomal lncRNA derived from pancreatic cancer cells is an essential component of the tumor microenvironment. However, whether exosomal lncXIST derived from pancreatic cancer cells can promote PNI and its exact mechanism remains to be elucidated. We show that lncXIST mediates nerve-tumor crosstalk via exosomal delivery. Our data reveal that exosomal lncXIST derived from pancreatic cancer cells is delivered to neural cells and promotes their release of glial-cell-line-derived neurotrophic factor (GDNF), essential in facilitating the PNI of pancreatic cancer. Mechanistically, microRNA-211-5p negatively regulates GDNF, and lncXIST serves as a miR-211-5p sponge. The function of exosomes in the dynamic interplay between nerves and cancer is confirmed in both in vivo and in vitro PNI models. Therefore, targeting pancreatic cancer cell-derived exosomal lncXIST may provide clues for a promising approach for developing a new strategy to combat PNI of pancreatic cancer.

LncRNA XIST promotes bladder cancer progression by modulating miR-129-5p/TNFSF10 axis

BackgroundThe differential expression, biological function, and ceRNA regulatory mechanism of lncRNA XIST in bladder cancer (BC) were investigated, and its clinical values for the early diagnosis of bladder cancer patients were elucidated.MethodsqRT-PCR was employed to detect the expression patterns of lncRNA XIST, miR-129-5p and TNFSF10. The biological functions were measured by CCK8 assay, wound healing assay and transwell assay. Bioinformatics analysis and Dual-Luciferase reporter assay were employed to evaluate the interactions between the lncRNA XIST, miR-129-5p and TNFSF10.ResultsLncRNA XIST and TNFSF10 were highly expressed and miR-129-5p was low expressed (P < 0.05) in bladder cancer cell line. The depletion of lncRNA XIST inhibited BC proliferation, migration and invasion. Mechanistically, lncRNA XIST could sponge miR-129-5p to regulate TNFSF10 expression in bladder cancer. Furthermore, compared with adjacent tissues, lncRNA XIST and miR-129-5p were lowly expressed (P < 0.01) in bladder cancer tissues, and TNFSF10 was highly expressed (P < 0.001). miR-129-5p and TNFSF10 were associated with the risk of bladder cancer (P < 0.05); the difference in AUC values for the diagnosis of bladder cancer by lncRNA XIST (AUC = 0.739), miR-129-5p (AUC = 0.850) and TNFSF10 (AUC = 0.753) was statistically significant (P < 0.01), and the three genes combined AUC was 0.900, 95%CI was 0.842–0.958 with a sensitivity of 83.3% and specificity of 86.7%.ConclusionXIST, an elevated lncRNA in bladder cancer, inhibition of which could suppress the progression of BC. LncRNA XIST and miR-129-5p could form ceRNA to regulate the expression of TNFSF10.

High Expression of lncRNA XIST Assists in the Diagnosis of Parkinson’s Disease

High Expression of lncRNA XIST Assists in the Diagnosis of Parkinson’s Disease

Long noncoding RNA XIST inhibition promotes Leydig cell apoptosis by acting as a competing endogenous RNA for microRNA-145a-5p that targets SIRT1 in late-onset hypogonadism.

Leydig cell (LCs) apoptosis is responsible for decreased serum testosterone levels during late-onset hypogonadism (LOH). Our study was designed to illustrate the regulatory effect of lncRNA XIST on LCs and to clarify its molecular mechanism of action in LOH. The Leydig cells (TM3) was treated by 300μM H2O2 for 8h to establish Leydig cell oxidative stress model in vitro. The expression levels of lncRNA XIST in the testicular tissues of patients with LOH were measured using fluorescence in situ hybridization (FISH). The interaction between lncRNA XIST/SIRT1 and miR-145a-5p was assessed using starBase and dual-luciferase reporter gene assays. Apoptotic cells and Caspase3 activity were determined by flow cytometry (FCM) assay. Testosterone concentration was determined by ELISA. Moreover, histological assessment of testicles in mice was performed by using HE staining and the TUNEL assay was used to determine apoptosis. We found that the lncRNA XIST was downregulated in the testicular tissues of LOH patients and mice and in H2O2-induced TM3 cells. XIST siRNA significantly promoted apoptosis, enhanced Caspase3 activity and reduced testosterone levels in H2O2-stimulated TM3 cells. Further studies showed that the miR-145a-5p inhibitor reversed the effect of XIST-siRNA on H2O2-induced Leydig cell apoptosis. MiR-145a-5p negatively regulated SIRT1 expression, and SIRT1-siRNA reversed the effects of the miR-145a-5p inhibitor on H2O2 stimulated TM3 cells. The in vivo experiments indicated that silencing of the lncRNA XIST aggravated LOH symptoms in mice. Inhibition of lncRNA XIST induces Leydig cell apoptosis through the miR-145a-5p/SIRT1 axis in the progression of LOH.

Long noncoding RNA XIST promotes cell proliferation and migration in diabetic foot ulcers through the miR-126-3p/EGFR axis

BackgroundThe prevalence of diabetic foot ulcers (DFUs) has caused serious harm to human health. To date, a highly effective treatment is lacking. Long noncoding RNA X-inactive specific transcript (lncRNA XIST) has been the subject of mounting research studies, all of which have found that it serves as a protective factor against certain diseases; however, its function in DFUs is not entirely understood. This study was performed to determine the importance of the lncRNA XIST in the pathogenesis and biological function of DFUs.MethodsDiabetic ulcer skin from rats was analysed using haematoxylin-eosin (HE), Masson’s trichrome, and immunohistochemistry (IHC) staining. The differences in the expression of genes and proteins were examined with real-time quantitative polymerase chain reaction (RT–qPCR) and Western blotting. Next, the interaction was verified with a dual luciferase gene reporter assay. In addition, CCK-8, Transwell, and wound healing assays were used to assess the proliferation and migration of HaCaT cells.ResultsThe lncRNA XIST and epidermal growth factor receptor (EGFR) were downregulated, while microRNA-126-3p (miR-126-3p) was increased in diabetic ulcer rat skin tissues and high glucose-induced HaCaT cells. In addition, we found that the lncRNA XIST binds to miR-126-3p and that EGFR is directly targeted by miR‑126‑3p. Silencing XIST contributed to upregulated miR-126-3p expression, thus lowering EGFR levels and inhibiting the proliferative and migratory abilities of high glucose-treated HaCaT cells; however, the miR-126-3p inhibitor and overexpression of EGFR reversed this effect.ConclusionDecreased lncRNA XIST expression inhibits the proliferative and migratory abilities of high glucose-induced HaCaT cells by modulating the miR-126-3p/EGFR axis, causing delayed wound healing.

Effect of lncRNA XIST on acute myeloid leukemia cells via miR-142-5p-PFKP axis

ABSTRACT Acute myeloid leukemia (AML) is the common blood cancer in hematopoietic system-related diseases and has a poor prognosis. Studies have shown that long non-coding RNAs (lncRNAs) are closely related to the pathogenesis of a variety of diseases, including AML. However, the specific molecular mechanism remains unclear. Hence, the objective of this study was to investigate the effect and mechanism of lncRNA X inactive specific transcript (lncRNA XIST) on AML. To achieve our objective, some tests were performed. Quantitative real-time polymerase chain reaction (qRT-PCR) was utilized to detect the expression of lncRNA XIST, miR-142-5p and the platelet isoform of phosphofructokinase (PFKP). The targeting relationship between miR-142-5p and lncRNA XIST and PFKP was verified by Pearson correlation analysis, dual-luciferase reporter assay, and pull-down assay. Functional experiments were used to analyze the effect and mechanism of action of knocking down lncRNA XIST on THP-1 and U937 cells. Compared with bone marrow cells, lncRNA XIST and PFKP expression levels were up-regulated and miR-142-5p expression levels were down-regulated in AML. Further analysis revealed that lncRNA XIST targeted and bound to miR-142-5p, and PFKP was a target gene of miR-142-5p. Knockdown of lncRNA XIST significantly promoted miR-142-5p expression to down-regulate PFKP in THP-1 and U937 cells, while the cell proliferation, cell viability, and cell cycle arrest were inhibited and apoptosis was increased. Knockdown of miR-142-5p reversed the functional impact of lncRNA XIST knockdown on AML cells. In conclusion, down-regulation of lncRNA XIST can affect the progression of AML by regulating miR-142-5p.

LncRNA XIST facilitates the odontogenic differentiation of dental pulp stem cells via the FUS/ZBTB16

LncRNA XIST facilitates the odontogenic differentiation of dental pulp stem cells via the FUS/ZBTB16

Advancing Wound Healing: Innovative Pathway Strategies for Enhanced Recovery

As the largest organ of the body, it is crucial that the skin, and all it protects, remain healthy and functioning. However, burn injuries may result as a serious threat to the skin, possibly forming greater health issues depending on the severity of the issue. Burn wound injury is characterized as damage to the skin or other organic tissue originating from sources such as heat, friction, radiation, radioactivity, electricity, and/or chemical contact. Different degrees of burn wounds make it increasingly more difficult to treat the more severe the wound is. For this reason, it is crucial to study how skin can not only heal itself via its own equipped healing related pathways, but also what treatments exist outside of the ongoing biochemical mechanisms in order to accelerate healing and repair the body’s greatest defense. More standard forms of treatment that currently exist adopt a more holistic approach, treating at the surface level, while treatments targeting the molecular level are less well studied. This review paper assesses several novel therapeutic alternatives including, but not limiting to, targeting lncRNA XIST, IL-33, and miR-19b through analyzing the molecular pathways in which wound healing is performed.

LncRNA xist regulates sepsis associated neuroinflammation in the periventricular white matter of CLP rats by miR-122-5p/PKCη Axis.

Neuroinflammation is a common feature of many neurological diseases, and remains crucial for disease progression and prognosis. Activation of microglia and astrocytes can lead to neuroinflammation. However, little is known about the role of lncRNA xist and miR-122-5p in the pathogenesis of sepsis-associated neuroinflammation (SAN). This study aims to investigate the role of lncRNA xist and miR-122-5p in the pathogenesis of SAN. Levels of miR-122-5p and proinflammatory mediators were detected in the cerebrospinal fluid (CSF) of patients with intracranial infection (ICI) by ELISA and qRT-PCR. miRNA expression in the periventricular white matter (PWM) in rats was analyzed by high-throughput sequencing. Levels of lncRNA xist, miR-122-5p and proinflammatory mediators in the PWM were measured using qRT-PCR and western blot. Bioinformatics analysis was used to predict the upstream and downstream of miR-122-5p. The interaction between miR-122-5p and its target protein was validated using luciferase reporter assay. BV2 and astrocytes were used to detect the expression of lncRNA xist, miR-122-5p. The level of miR-122-5p was significantly decreased in the CSF of ICI patients, while the expression of IL-1β and TNF-α were significantly upregulated. Furthermore, it was found that the expression of IL-1β and TNF-α were negatively correlated with the level of miR-122-5p. A high-throughput sequencing analysis showed that miR-122-5p expression was downregulated with 1.5-fold changes in the PWM of CLP rats compared with sham group. Bioinformatics analysis found that lncRNA xist and PKCη were the upstream and downstream target genes of miR-122-5p, respectively. The identified lncRNA xist and PKCη were significantly increased in the PWM of CLP rats. Overexpression of miR-122-5p or knockdown of lncRNA xist could significantly downregulate the level of PKCη and proinflammatory mediators from activated microglia and astrocytes. Meanwhile, in vitro investigation showed that silencing lncRNA xist or PKCη or enhancing the expression of miR-122-5p could obviously inhibit the release of proinflammatory mediators in activated BV2 cells and astrocytes. LncRNA xist could regulate microglia and astrocytes activation in the PWM of CLP rats via miR-122-5p/PKCη axis, further mediating sepsis associated neuroinflammation.

Association of XIST/miRNA155/Gab2/TAK1 cascade with the pathogenesis of anti-phospholipid syndrome and its effect on cell adhesion molecules and inflammatory mediators.

Anti-phospholipid syndrome (APS) is an autoimmune disease characterized by thrombosis and miscarriage events. Still, the molecular mechanisms underlying APS, which predisposes to a wide spectrum of complications, are being explored. Seventy patients with primary and secondary APS were recruited, in addition to 35 healthy subjects. Among APS groups, the gene expression levels of XIST, Gab2, and TAK1 were higher along with declined miRNA155 level compared with controls. Moreover, the sera levels of ICAM-1, VCAM-1, IL-1ꞵ, and TNF-α were highly elevated among APS groups either primary or secondary compared with controls. The lncRNA XIST was directly correlated with Gab2, TAK1, VCAM-1, ICAM-1, IL-1ꞵ, and TNF-α. The miRNA155 was inversely correlated with XIST, Gab2, and TAK1. Moreover, ROC curve analyses subscribed the predictive power of the lncRNA XIST and miRNA155, to differentiate between primary and secondary APS from control subjects. The lncRNA XIST and miRNA155 are the upstream regulators of the Gab2/TAK1 axis among APS patients via influencing the levels of VCAM-1, ICAM-1, IL1ꞵ, and TNF-α which propagates further inflammatory and immunological streams. Interestingly, the study addressed that XIST and miRNA155 may be responsible for the thrombotic and miscarriage events associated with APS and provides new noninvasive molecular biomarkers for diagnosing the disease and tracking its progression.

Hypoxic bone marrow mesenchymal stem cell-derived exosomal lncRNA XIST attenuates lipopolysaccharide-induced acute lung injury via the miR-455-3p/Claudin-4 axis

Mesenchymal stem cell-derived exosomes and long non-coding RNAs (lncRNAs) have been identified to play a role in acute lung injury (ALI). In this study, we investigated whether exosomal lncRNAs could regulate ALI and the underlying mechanisms. Bone marrow mesenchymal stem cells (BM-MSCs) were pretreated with hypoxia or normoxia, and exosomes were subsequently extracted from normoxic BM-MSCs (Nor-exos) and hypoxic BM-MSCs (Hypo-exos). A rat model of ALI was established via an airway perfusion of lipopolysaccharide (LPS). Exosomes were administered via the tail vein to evaluate the in vivo effect of exosomes in ALI. LPS-exposed RLE-6TN cells were incubated with exosomes to explore their in vitro effect in ALI. A luciferase reporter assay was used to evaluate the interaction between lncRNA XIST and miR-455-3p, as well as miR-455-3p and Claudin-4. We found that the exosomes attenuated LPS-induced ALI and Hypo-Exos exerted a greater therapeutic effect compared with Nor-exos both in vitro and in vivo. Moreover, an abundance of lncRNA XIST was observed in Hypo-exos compared with Nor-exos. Mechanistically, LncRNA XIST functioned as a miR-455-3p sponge and targeted Claudin-4 in ALI. Our results provide novel insight into the role of exosomal lncRNA XIST for the treatment of ALI. Thus, hypoxic pretreatment may represent an effective method for improving the therapeutic effects of exosomes.

High Expression of LncRNA XIST as an Index Helping to Diagnose Parkinson’s Disease

High Expression of LncRNA XIST as an Index Helping to Diagnose Parkinson’s Disease

System biology analysis to develop diagnostic biomarkers, monitoring pathological indexes, and novel therapeutic approaches for immune targeting based on maggot bioactive compounds and polyphenolic cocktails in mice with gastric cancer

Early diagnosis and prognosis are prerequisites for mitigating mortality in gastric cancer (GaCa). Identifying some causative or sensitive elements (coding RNA (cRNA)-non-cRNAs (ncRNAs)) can be very helpful in the early diagnosis of GaCa. Notably, despite significant development in the GaCa treatment, the outcome of patients does not remain satisfactory due to limitations such as multi-drug resistance and tumor relapse. Therefore, more attention has been drawn to complementary therapies and the use of supplements. In this regard, Polyphenol natural compounds (PNC) and maggot larvae (MaLa) alone or in combination were administered along with chemotherapy (paclitaxel) to N-methyl-N-nitrosourea (MNU)- induced murine tumor model. In addition, in order to identify potential diagnostic or prognostic biomarkers, transcriptomics analysis was performed through a bioinformatics approach. Then transcription profile of ncRNAs with their target hub genes was assessed through qPCR Real-Time, Western blot, and ELISA. According to the bioinformatics results, 17 hub genes (e.g., IL-6, CXCL8, MKI67, IL-2, IL-4, IL-10, IL-1β, SPP1, LOX, COL1A1, and IFN-γ) were explored that contribute towards inflammation and oxidative stress and ultimately GaCa development. Upstream of the mentioned hub genes, regulatory factors (lncRNA XIST and NEAT1) were also identified and introduced as prognosis and diagnosis biomarkers for GaCa. Our results showed that PNC alone and in combination with MaLa was able to reduce the size and number of tumors, which is related to the reduction of genes expression levels (including IL-6, CXCL8, MKI67, IL-2, IL-4, IL-10, IL-1β, SPP1, LOX, COL1A1, IFN-γ, NEAT1, and XIST). In conclusion, PNC and MaLa have the potential to be considered as complementary and improving chemotherapy due to their effective compounds. Also, the introduced hub gene and lncRNA in addition to diagnostic and prognostic biomarkers can be used as druggable proteins for novel therapeutic targeting of GaCa.

The Role of LncRNA XIST in Gynecologic Cancers

Abstract: Long non-coding RNAs (lncRNAs) are non-coding RNAs longer than 200 nucleotides that act by targeting translational and post-translational modifications, epigenetic regulators, and chromatin remodeling complexes. There has been increasing evidence that the lncRNA X-inactive specific transcript (lncRNA Xist) via targeting miRNAs and transcription factors plays a critical role in cell growth, proliferation, and differentiation. This lncRNA also has an important role in the progression of tumors and other human diseases by acting as a competing endogenous RNA (ceRNA). Accumulating evidence revealed that lncRNA Xist by targeting several signaling pathways is involved in the pathogenesis of gynecologic cancers. In this review, we focused on the recent functions of lncRNA Xist in breast, cervical, and ovarian cancers.

Prevalence of Diabetic Kidney Disease with Different Subtypes in Hospitalized Patients with Diabetes and Correlation Between eGFR and LncRNA XIST Expression in PBMCs.

Diabetic kidney disease (DKD) has become the leading cause of end-stage kidney disease (ESKD) in most countries. Recently, long noncoding RNA XIST has been found involved in the development of DKD. A total of 1184 hospitalized patients with diabetes were included and divided into four groups based on their estimated glomerular filtration rate (eGFR) and urinary albumin to creatinine ratio (UACR): normal control group (nDKD), DKD with normoalbuminuric and reduced eGFR (NA-DKD), DKD with albuminuria but without reduced eGFR (A-DKD), and DKD with albuminuria and reduced eGFR (Mixed), and then their clinical characteristics were analyzed. Peripheral blood mononuclear cells (PBMCs) of patients with DKD were isolated, and lncRNA XIST expression was detected by real-time quantitative PCR. The prevalence of DKD in hospitalized patients with diabetes mellutus (DM) was 39.9%, and the prevalence of albuminuria and decreased eGFR was 36.6% and 16.2%, respectively. NA-DKD, A-DKD, and Mixed groups accounted for 23.7%, 3.3%, and 12.9%, respectively. Women with DKD had considerably lower levels of lncRNA XIST expression in their PBMCs compared to nDKD. There was a significant correlation between eGFR level and lncRNA XIST expression (R = 0.390, P = 0.036) as well as a negative correlation between HbA1c and lncRNA XIST expression (R = -0.425, P = 0.027) in female patients with DKD. Our study revealed that 39.9% of DM inpatients who were admitted to the hospital had DKD. Importantly, lncRNA XIST expression in PBMCs of female patients with DKD was significantly correlated with eGFR and HbA1c.

LncRNA XIST Exacerbates Oxygen-Glucose Deprivation/Reoxygenation-Induced Cerebral Injury Through the miR-25-3p/TRAF3 Axis.

Ischemic stroke causes lethal damage to the brain. Identifying key regulators of OGD/R-induced cerebral injury is important for developing novel therapies for ischemic stroke. HMC3 and SH-SY5Y cells were treated with OGD/R as an in vitro ischemic stroke model. Cell viability and apoptosis were determined via CCK-8 assay and flow cytometry. Inflammatory cytokines were examined by ELISA. Luciferase activity was measured for evaluating the interaction of XIST, miR-25-3p, and TRAF3. Bcl-2, Bax, Bad, cleaved-caspase 3, total caspase 3, and TRAF3 were detected via western blotting. HMC3 and SH-SY5Y cells showed increased XIST expression and decreased miR-25-3p expression following OGD/R. Importantly, silencing of XIST and overexpression of miR-25-3p reduced apoptosis and inflammatory response following OGD/R. Furthermore, XIST worked as a miR-25-3p sponge, and miR-25-3p targeted TRAF3 to suppress its expression. Moreover, the knockdown of TRAF3 ameliorated OGD/R-induced injury. Loss of XIST-mediated protective effects was reversed by overexpression of TRAF3. LncRNA XIST exacerbates OGD/R-induced cerebral damage via sponging miR-25-3p and enhancing TRAF3 expression.

LncRNA XIST inhibition promotes M2 polarization of microglial and aggravates the spinal cord injury via regulating miR-124–3p / IRF1 axis

BackgroundSpinal cord injury (SCI) has a high disability rate and mortality rate. Recently, LncRNA XIST has been found to be involved in the regulation of inflammatory responses. Therefore, we aimed to investigate the role of XIST in the occurrence and development of SCI and the specific regulation mechanism. Methods: 100 ng/mL lipopolysaccharide (LPS) was used to treat mouse microglia BV2 cells. Hitting spinal cord was performed to C57BL/6 mice for establishing SCI model. Real-time reverse transcriptase-polymerase chain reaction (RT-qPCR), Western blot, Immunofluorescence (IF) and Enzyme linked immunosorbent assay (ELISA) experiments were used to explore the function of XIST, miR-124–3p and IRF1 in LPS-induced BV2 cells. RT-qPCR, Nissl staining, IF, Western blot and ELISA experiment were performed to study the function of XIST in SCI mice. Dual-luciferase reporter assay, RNA immunoprecipitation (RIP), RT-qPCR and Western blot assays were utilized to identify the interaction among XIST, miR-124–3p and IRF1. Results: XIST was upregulated in LPS-induced BV2 cells and spinal cord tissues of SCI mice. Overexpression of XIST promoted the M1 microphages polarization and cytokines concentration in LPS-stimulated BV2 cells, aggravated SCI of mice. Downregulated XIST promoted M1-to-M2 conversion of microglial and relieved the injury of SCI mice. Mechanism verification indicated that XIST acted as a molecular sponge of miR-124–3p and regulated IRF1 expression. Increased miR-124–3p or reduced IRF1 inhibited M1 polarization of microglial and decreased the production of inflammatory cytokines in LPS-induced BV2 cells. Increased XIST or decreased miR-124–3p had an opposite of on LPS-induced BV2 cells. Conclusion: Overexpression of XIST enhanced M1 polarization of microglia and promoted the level of inflammatory cytokines through sponging miR-124–3p and regulating IRF1 expression.

LncRNA XIST Depletion Prevents Cancer Progression in Invasive Pituitary Neuroendocrine Tumor by Inhibiting bFGF via Upregulation of microRNA-424-5p [Retraction

[This retracts the article DOI: 10.2147/OTT.S208329.].

A monoclonal antibody raised against human EZH2 cross-reacts with the RNA-binding protein SAFB.

The Polycomb Repressive Complex 2 (PRC2) is a conserved enzyme that tri-methylates Lysine 27 on Histone 3 (H3K27me3) to promote gene silencing. PRC2 is remarkably responsive to the expression of certain long noncoding RNAs (lncRNAs). In the most notable example, PRC2 is recruited to the X-chromosome shortly after expression of the lncRNA Xist begins during X-chromosome inactivation. However, the mechanisms by which lncRNAs recruit PRC2 to chromatin are not yet clear. We report that a broadly used rabbit monoclonal antibody raised against human EZH2, a catalytic subunit of PRC2, cross-reacts with an RNA-binding protein called Scaffold Attachment Factor B (SAFB) in mouse embryonic stem cells (ESCs) under buffer conditions that are commonly used for chromatin immunoprecipitation (ChIP). Knockout of EZH2 in ESCs demonstrated that the antibody is specific for EZH2 by western blot (no cross-reactivity). Likewise, comparison to previously published datasets confirmed that the antibody recovers PRC2-bound sites by ChIP-Seq. However, RNA-IP from formaldehyde-crosslinked ESCs using ChIP wash conditions recovers distinct peaks of RNA association that co-localize with peaks of SAFB and whose enrichment disappears upon knockout of SAFB but not EZH2. IP and mass spectrometry-based proteomics in wild-type and EZH2 knockout ESCs confirm that the EZH2 antibody recovers SAFB in an EZH2-independent manner. Our data highlight the importance of orthogonal assays when studying interactions between chromatin-modifying enzymes and RNA.

Combined inhibition of histone deacetylase and cytidine deaminase improves epigenetic potency of decitabine in colorectal adenocarcinomas

BackgroundTargeting the epigenome of cancerous diseases represents an innovative approach, and the DNA methylation inhibitor decitabine is recommended for the treatment of hematological malignancies. Although epigenetic alterations are also common to solid tumors, the therapeutic efficacy of decitabine in colorectal adenocarcinomas (COAD) is unfavorable. Current research focuses on an identification of combination therapies either with chemotherapeutics or checkpoint inhibitors in modulating the tumor microenvironment. Here we report a series of molecular investigations to evaluate potency of decitabine, the histone deacetylase inhibitor PBA and the cytidine deaminase (CDA) inhibitor tetrahydrouridine (THU) in patient derived functional and p53 null colon cancer cell lines (CCCL). We focused on the inhibition of cell proliferation, the recovery of tumor suppressors and programmed cell death, and established clinical relevance by evaluating drug responsive genes among 270 COAD patients. Furthermore, we evaluated treatment responses based on CpG island density.ResultsDecitabine caused marked repression of the DNMT1 protein. Conversely, PBA treatment of CCCL recovered acetylation of histone 3 lysine residues, and this enabled an open chromatin state. Unlike single decitabine treatment, the combined decitabine/PBA treatment caused > 95% inhibition of cell proliferation, prevented cell cycle progression especially in the S and G2-phase and induced programmed cell death. Decitabine and PBA differed in their ability to facilitate re-expression of genes localized on different chromosomes, and the combined decitabine/PBA treatment was most effective in the re-expression of 40 tumor suppressors and 13 genes typically silenced in cancer-associated genomic regions of COAD patients. Furthermore, this treatment repressed expression of 11 survival (anti-apoptotic) genes and augmented expression of X-chromosome inactivated genes, especially the lncRNA Xist to facilitate p53-mediated apoptosis. Pharmacological inhibition of CDA by THU or its gene knockdown prevented decitabine inactivation. Strikingly, PBA treatment recovered the expression of the decitabine drug-uptake transporter SLC15A1, thus enabling high tumor drug-loads. Finally, for 26 drug responsive genes we demonstrated improved survival in COAD patients.ConclusionThe combined decitabine/PBA/THU drug treatment improved drug potency considerably, and given their existing regulatory approval, our findings merit prospective clinical trials for the triple combination in COAD patients.