Journal: Clinical and Translational Medicine

Article Title: Persistent high glucose induced EPB41L4A‐AS1 inhibits glucose uptake via GCN5 mediating crotonylation and acetylation of histones and non‐histones

doi: 10.1002/ctm2.699

Figure Lengend Snippet: EPB41L4A‐AS1 is up‐regulated in patients with type 2 diabetes mellitus (T2DM) and T2DM cell models. (A‐B) The expression of EPB41L4A‐AS1 in the liver (n = 10) and muscle (n = 16) tissues of patients with T2DM or insulin resistance compared with that in normal controls. Data are from the GEO databases GSE23343 and GSE6798 datasets, Mann–Whitney U test. (C) Glucose uptake in cells before and after treatment at high glucose concentrations (n = 3). (D) Glucose consumption in cells treated at normal glucose concentrations for 6 h after pre‐treatment with high‐ or normal‐glucose DMEM (n = 3). (E‐H) EPB41L4A‐AS1 was measured in cells treated at high glucose concentrations for 0, 0.25, 0.5, 2, 12 and 24 h (n = 3). One‐way analysis of variance, followed by Tukey's post‐hoc test. (I‐J) The expression of EPB41L4A‐AS1 in cells incubated with 5 mM glucosamine for 24 h (n = 3)

Article Snippet: An EPB41L4A‐AS1 RNA probe was labelled with Quasar 570 (Biosearch Technologies, SMF‐1063–5).

Techniques: Expressing, MANN-WHITNEY, Incubation

Journal: Clinical and Translational Medicine

Article Title: Persistent high glucose induced EPB41L4A‐AS1 inhibits glucose uptake via GCN5 mediating crotonylation and acetylation of histones and non‐histones

doi: 10.1002/ctm2.699

Figure Lengend Snippet: High glucose or glucosamine concentration up‐regulates the expression of EPB41L4A‐AS1 by enhancing TP53 expression. As shown, EAS1 represents EPB41L4A‐AS1; (A‐C) Pearson correlation analysis between EPB41L4A‐AS1 and TP53 expression in human liver and muscle tissues based on data from the GTEx database, and Pearson's correlation analysis of EPB41L4A‐AS1 and TP53 expression based on liver cancer data from the TCGA database. Images were obtained from the GEPIA website. (D‐G) TP53 expression was measured in cells treated at high glucose concentrations for 0, 0.25, 0.5, 2, 12 and 24 h (n = 3). (H) TP53 expression in cells incubated with 5 mM glucosamine; One‐way analysis of variance, followed by Tukey's post‐hoc test. (I‐J) TP53 expression was measured in cells treated at high glucose concentrations or with 5 mM glucosamine. (K) Diagram showing four mutation sites in the siRNA‐target sequence of P53 cDNA without a change in the amino‐acid sequence (mutP53), which could confer resistance to the P53 siRNA used in this study. (L‐O) EPB41L4A‐AS1 expression was analyzed in cells treated at high glucose concentrations for 24 h after transfection with NC, siP53 or siP53, and the mutP53 plasmid for 24 h (n = 3). (P‐S) The RNA expression of EPB41L4A‐AS1 was analyzed using cells treated with 5 mM glucosamine for 24 h after transfection with NC, siP53 or siP53, and the mutP53 plasmid for 24 h (n = 3)

Article Snippet: An EPB41L4A‐AS1 RNA probe was labelled with Quasar 570 (Biosearch Technologies, SMF‐1063–5).

Techniques: Concentration Assay, Expressing, Incubation, Mutagenesis, Sequencing, Transfection, Plasmid Preparation, RNA Expression

Journal: Clinical and Translational Medicine

Article Title: Persistent high glucose induced EPB41L4A‐AS1 inhibits glucose uptake via GCN5 mediating crotonylation and acetylation of histones and non‐histones

doi: 10.1002/ctm2.699

Figure Lengend Snippet: EPB41L4A‐AS1 regulates glucose uptake and mitochondrial respiration. As shown, EAS1 represents EPB41L4A‐AS1; (A‐D) Glucose uptake in cells treated at high glucose concentrations (n = 3). (E) Schematic representation of the animal experiments. (F‐G) Body weight of mice at days 0 and 21. (H) Measurement of blood glucose levels after AAV‐NC mice (n = 8) and AAV‐EAS1 mice (n = 8) were injected with 10% glucose injection (2 mg/g body weight) at 0, 15, 30, 45, 60, 90 and 120 min. (I) RT‐PCR detection of human lncRNA EPB41L4A‐AS1 expression in the liver, muscle, lung and adipose tissues of AAV‐NC and AAV‐EAS1 mice, using mouse β‐actin as the loading control. (J‐L) The oxygen consumption rate (OCR) measured using the Seahorse XFp assay in cells treated at high glucose concentrations for 24 h. (M‐O) The OCR was measured (using the Seahorse XFp assay) in cells treated at high glucose concentrations for 24 h after the stable knockdown of EPB41L4A‐AS1 (n = 3). (P‐Q) ATP production estimated using the Seahorse XFp mitochondrial respiration assay (n = 3)

Article Snippet: An EPB41L4A‐AS1 RNA probe was labelled with Quasar 570 (Biosearch Technologies, SMF‐1063–5).

Techniques: Injection, Reverse Transcription Polymerase Chain Reaction, Expressing, Respiration Assay

Journal: Clinical and Translational Medicine

Article Title: Persistent high glucose induced EPB41L4A‐AS1 inhibits glucose uptake via GCN5 mediating crotonylation and acetylation of histones and non‐histones

doi: 10.1002/ctm2.699

Figure Lengend Snippet: EPB41L4A‐AS1 regulates glucose uptake through GLUT2 or GLUT4. As shown, EAS1 represents EPB41L4A‐AS1; (A‐D) GLUT2 protein level assayed by western blotting of proteins from HepG2 (n = 3) or L02 (n = 3) cells with transient EPB41L4A‐AS1/EPB41L4A‐AS1‐ATG‐mut over‐expression or stable EPB41L4A‐AS1 knockdown, Student's t ‐test. (E‐F) Cell membrane surface GLUT2 protein levels in HepG2 (n = 3) or L02 (n = 3) cells stimulated with 100 nM insulin for 30 min after the transient over‐expression of EPB41L4A‐AS1/EPB41L4A‐AS1‐ATG‐mut for 48 h or after the stable knockdown of EPB41L4A‐AS1, as measured by using western blotting. (G‐H) Total GLUT4 protein level (n = 3) and cell membrane surface GLUT4 protein level (n = 3) in A673 cells stimulated with 100 nM insulin for 30 min after the transient over‐expression of EPB41L4A‐AS1/EPB41L4A‐AS1‐ATG‐mut for 48 h or after the stable knockdown of EPB41L4A‐AS1, as measured using western blotting

Article Snippet: An EPB41L4A‐AS1 RNA probe was labelled with Quasar 570 (Biosearch Technologies, SMF‐1063–5).

Techniques: Western Blot, Over Expression

Journal: Clinical and Translational Medicine

Article Title: Persistent high glucose induced EPB41L4A‐AS1 inhibits glucose uptake via GCN5 mediating crotonylation and acetylation of histones and non‐histones

doi: 10.1002/ctm2.699

Figure Lengend Snippet: EPB41L4A‐AS1 negatively regulates GLUT4 transcription by increasing H3K27 crotonylation and PGC1‐β lysine acetylation. As shown, EAS1 represents EPB41L4A‐AS1; (A‐B) Pearson's correlation analysis of TP53 or EAS1 and GLUT4 (SLC2A4) expression in human muscle and adipose tissues. Data were obtained from the GTEx RNA‐seq database, and images were obtained from the GEPIA website. (C) GLUT4 mRNA levels measured using qPCR after the over‐expression of EAS1/EAS1‐ATG‐mut for 48 h or after the stable knockdown of EAS1 (n = 3), Student's t ‐test. (D) Primer locations in the GLUT4 promoter, as designed for chromatin immunoprecipitation‐qPCR. (E) H3K27ac enrichment in the GLUT4 promoter in A673 cells with EAS1/EAS1‐ATG‐mut over‐expression or EAS1 knockdown (n = 3). Data shown as log 2 (fold change); EAS1 and EAS1‐ATG‐mut expression was normalized to that of the vector (control, value = 0), whereas shEAS1 expression was normalized to that of shNC (control, value = 0). (F) H3K14ac enrichment in the GLUT4 promoter in A673 cells after the over‐expression of EAS1 or EAS1‐ATG‐mut plasmids for 48 h and in A673 cells with stable EAS1 knockdown (n = 3). (G) Total H3K27cr levels were measured by immunoblotting using stable EAS1 knock‐down A673 cells or A673 cells with EAS1 or EAS1‐ATG‐mut over‐expression (n = 3). (H) Interaction between EPB41L4A‐AS1 and H3K27cr (n = 3). (I) H3K27cr enrichment in the GLUT4 promoter was measured with the ChIP assay (n = 3). (J‐K) A673 cells were incubated with 10 mM crotonate or at high glucose concentrations or both for 24 h, and qRT‐PCR was conducted to measure the EPB41L4A‐AS1 (J) and GLUT4 levels (K) (n = 3). (L) shNC and shEAS1 A673 cells were incubated with or without glucose at high concentrations and 10 mM crotonate for 24 h, and ChIP‐qPCR was conducted to detect the occupation of H3K27cr in the GLUT4 promoter region (n = 3). (M‐N) Immunoprecipitation for measuring the level of lysine acetylation in PGC1‐β in A673 cells (n = 3). (O‐Q) GCN5 and PGC1‐β interactions were analyzed by the immunoprecipitation assay in stable EAS1 knock‐down A673 cells (n = 3) and EAS1‐ATG‐mut‐over‐expressing A673 cells (n = 3)

Article Snippet: An EPB41L4A‐AS1 RNA probe was labelled with Quasar 570 (Biosearch Technologies, SMF‐1063–5).

Techniques: Expressing, RNA Sequencing Assay, Over Expression, Chromatin Immunoprecipitation, Plasmid Preparation, Western Blot, Incubation, Quantitative RT-PCR, Immunoprecipitation

Journal: Clinical and Translational Medicine

Article Title: Persistent high glucose induced EPB41L4A‐AS1 inhibits glucose uptake via GCN5 mediating crotonylation and acetylation of histones and non‐histones

doi: 10.1002/ctm2.699

Figure Lengend Snippet: EPB41L4A‐AS1 regulates H3K27cr via interaction with GCN5. As shown, EAS1 represents EPB41L4A‐AS1; (A) Immunoblot‐assayed pan crotonyl lysine of proteins and H3K27cr in HepG2 cells with or without GCN5 over‐expression. (B) The total H3K27cr levels were tested by western blotting after GCN5 knockdown or GCN5‐GFP over‐expression in A673 cells. (C) Immunoprecipitation analysis of the interaction between H3K27cr and GCN5 in A673 cells. (D) H3K27cr and GCN5 co‐localization in A673 cells and human primary skeletal muscle cells (HSkMC) was detected by immunofluorescence. (E) EPB41L4A‐AS1 interaction with GCN5 in A673 cells evaluated by RNA immunoprecipitation‐qPCR analysis (n = 3). Two different primers were designed to detect EPB41L4A‐AS1. (F) Interaction between EPB41L4A‐AS1 RNA and GCN5 in A673 cells evaluated using the RNA pull‐down assay, with each anti‐sense RNA as a control. (G) EPB41L4A‐AS1 RNA and GCN5 interaction in L02 cells evaluated using the RNA pull‐down assay, RNA without poly(A) as the negative control. (H) Co‐localization of EPB41L4A‐AS1 RNA (red) and GCN5 protein (green) in A673 cells. (I) The binding sites between lncRNA EPB41L4A‐AS1 and GCN5 predicted using catRAPID. (J) The diagram shows the deletion region of EPB41L4A‐AS1. (K) EPB41L4A‐AS1 and the ORF region with or without binding sites, predicted using catRAPID deletion, were used for the RNA pull‐down assay, and GCN5 expression was measured by western blotting. (L) Pearson's correlation analysis of GCN5 and GLUT4 expression in human muscle and adipose tissues. Data were obtained from the GTEx RNA‐seq database, and images were obtained from the GEPIA website. (M‐N) GLUT4 protein (n = 3) and mRNA (n = 3) levels were assayed after the transient knockdown of GCN5 in A673 cells (n = 3). (O) H3K27cr enrichment in the GLUT4 promoter in A673 cells with or without GCN5 knockdown or GCN5 over‐expression (n = 3), as measured by chromatin immunoprecipitation‐qPCR. Data shown as log 2 (fold change); siGNC5‐1 and siGCN5‐2 expression was normalized to that of the negative control (NC) (control, value = 0), whereas GCN5 expression was normalized to that of the vector (control, value = 0), Student's t ‐test. (P) GCN5 enrichment in the GLUT4 promoter region was measured after A673 cells were incubated with 10 mM crotonate at high glucose concentrations for 24 h (n = 3)

Article Snippet: An EPB41L4A‐AS1 RNA probe was labelled with Quasar 570 (Biosearch Technologies, SMF‐1063–5).

Techniques: Western Blot, Over Expression, Immunoprecipitation, Immunofluorescence, Pull Down Assay, Negative Control, Binding Assay, Expressing, RNA Sequencing Assay, Chromatin Immunoprecipitation, Plasmid Preparation, Incubation

Journal: Clinical and Translational Medicine

Article Title: Persistent high glucose induced EPB41L4A‐AS1 inhibits glucose uptake via GCN5 mediating crotonylation and acetylation of histones and non‐histones

doi: 10.1002/ctm2.699

Figure Lengend Snippet: EPB 41L4A‐AS1 regulates TXNIP transcription via enhancement of H3K14 and H3K27 acetylation. As shown, EAS1 represents EPB41L4A‐AS1; (A) TXNIP and EPB41L4A‐AS1 expression correlation in muscle and liver tissues; data obtained from GTEx database and the GEPIA website are plotted in the diagram. (B‐D) The mRNA expression of TXNIP (n = 3) measured using qRT‐PCR. (E‐F) TXNIP protein levels in HepG2 (n = 3), A673 (n = 3),and L02 (n = 3) cells after the stable knockdown of EPB41L4A‐AS1 or the transient over‐expression of EPB41L4A‐AS1 or EPB41L4A‐AS1‐ATG‐mut, as measured using western blotting. (G) Schematic representation of the primer location in the TXNIP promoter region. (H) H3K27cr enrichment in the TXNIP promoter measured by chromatin immunoprecipitation (ChIP)‐qPCR in HepG2 cells over‐expressing EPB41L4A‐AS1 or EPB41L4A‐AS1‐ATG‐mut or with EPB41L4A‐AS1 knockdown (n = 3), Student's t ‐test. (I‐J) H3K27ac and H3K14ac enrichment in the TXNIP promoter of A673 cells after the over‐expression of EPB41L4A‐AS1 or EPB41L4A‐AS1‐ATG‐mut or the stable knockdown of EPB41L4A‐AS1 (n = 3), as evaluated using ChIP‐qPCR analysis, Student's t ‐test. (K‐L) GCN5 was transiently knocked down in A673 cells for 48 h using GCN5‐specific siRNAs, and the GCN5 and TXNIP protein and mRNA levels were measured by immunoblotting (n = 3) and qPCR (n = 3), respectively. (M) GCN5 occupation in the TXNIP promoter in A673 cells (n = 3) was assayed by ChIP‐qPCR. (N) The interaction between MLXIP and GCN5 in A673 cells. (O) RNA immunoprecipitation‐qPCR analysis of the interaction between EPB41L4A‐AS1 and MLXIP in A673 cells (n = 3). (P) MLXIP and TXNIP RNA expression correlation determined by Pearson's correlation analysis in muscle and liver tissues. Data were obtained from the GTEx database, and the diagram was obtained from the GEPIA website. (Q) MLXIP enrichment at the TXNIP promoter in A673 cells after the stable knockdown of EPB41L4A‐AS1 or over‐expression of EPB41L4A‐AS1 or EPB41L4A‐AS1‐ATG‐mut (n = 3), as detected using ChIP‐qPCR

Article Snippet: An EPB41L4A‐AS1 RNA probe was labelled with Quasar 570 (Biosearch Technologies, SMF‐1063–5).

Techniques: Expressing, Quantitative RT-PCR, Over Expression, Western Blot, Chromatin Immunoprecipitation, Immunoprecipitation, RNA Expression

Journal: Clinical and Translational Medicine

Article Title: Persistent high glucose induced EPB41L4A‐AS1 inhibits glucose uptake via GCN5 mediating crotonylation and acetylation of histones and non‐histones

doi: 10.1002/ctm2.699

Figure Lengend Snippet: Summary of the molecular mechanism by which EPB41L4A‐AS1 affects glucose uptake. Persistently high glucose concentrations increase the expression of the lncRNA EPB41L4A‐AS1 via the enhancement of TP53 expression. Through interaction with GCN5 and up‐regulation of its own expression, the lncRNA EPB41L4A‐AS1 increases histone H3K27 crotonylation in the GLUT4 promoter region and non‐histone PGC1‐β acetylation, which inhibits GLUT4 transcription and suppresses glucose uptake in muscle cells. Conversely, EPB41L4A‐AS1 binding to GCN5 enhances H3K27 and H3K14 acetylation in the TXNIP promoter region to activate transcription by recruiting the transcriptional activator MLXIP, which enhances GLUT4/2 endocytosis and further suppresses glucose uptake

Article Snippet: An EPB41L4A‐AS1 RNA probe was labelled with Quasar 570 (Biosearch Technologies, SMF‐1063–5).

Techniques: Expressing, Binding Assay