Journal: bioRxiv

Article Title: Human-specific lncRNA TMEM9B-AS1 is downregulated in skeletal muscle of individuals with type 2 diabetes and regulates ribosomal biogenesis

doi: 10.1101/2024.07.05.602204

Figure Lengend Snippet: a. Unique protein interactors of TMEM9B-AS1, determined by using the overlapping proteins in three individual TMEM9B-AS1 RNA pull-downs and TMEM9B-AS1 binding proteins according to the CLIP-SEQ data found in publicly available ENCORI database b. Functional enrichment analysis for the biological processes on STRING database using unique protein interactors of TMEM9B-AS1 achieved by the comparison of specific interactors of TMEM9B-AS1 from RNA pulldown data and CLIP-SEQ data from ENCORI database. CRD-mediated mRNA stabilization is the top enriched term. Strength= 2,99, FDR=0,0023 . c. Schematic representation of interaction between MYC mRNA and RNA-binding proteins, IGF2BPs via the CRD region on MYC mRNA, which facilitates the stabilization of MYC mRNA. d. mRNA levels of MYC upon TMEM9B-AS1 silencing. n=6, * p<0.05. e. Schematic representation of RNA pull-down experiments to determine the binding efficiency of RNA-binding proteins IGF2BP1 and IGF2BP2 to CRD region on MYC mRNA in the presence or absence of TMEM9B-AS1. f. Western blot for IGF2BP1 and IGF2BP2 upon RNA-pulldowns. Negative control RNA was used to determine the RNA pull-down specificity over MYC CRD. RNA pull-down specificity of g. IGF2BP1 and h. IGF2BP2. RNA binding efficiency of i. IGF2BP1 and j. IGF2BP2 upon two RNA pull-downs in control and TMEM9B-AS1 silencing conditions. Data were normalized to the individual input levels of IGF2BP1 and IGF2BP2.

Article Snippet: siRNA transfection was performed using either control siRNA, Stealth Control #1 medium GC (12935300, Invitrogen) or TMEM9B-AS1 Stealth siRNA (HSS162366, Thermo Fisher Scientific), IGF2BP1 Stealth siRNA (HSS173800, Thermo Fisher Scientific), IGF2BP2 Stealth siRNA (HSS173802, Thermo Fisher Scientific) (10nM final concentration) 6 days after induction of differentiation.

Techniques: Binding Assay, Functional Assay, Comparison, RNA Binding Assay, Western Blot, Negative Control, Control

Journal: bioRxiv

Article Title: Human-specific lncRNA TMEM9B-AS1 is downregulated in skeletal muscle of individuals with type 2 diabetes and regulates ribosomal biogenesis

doi: 10.1101/2024.07.05.602204

Figure Lengend Snippet: Western blot results showing the protein levels of a. IGF2BP1(n=8) b. IGF2BP2 (n=8) c. MYC (n=6) upon TMEM9B-AS1 silencing in human myotubes. d. Western blot results for MYC protein levels upon IGF2BP1 and IGF2BP2 siRNA silencing (n=6). Band area ratios were calculated by the normalization of the band intensity of the protein of interest to a selected ponceau band. *p<0.05.

Article Snippet: siRNA transfection was performed using either control siRNA, Stealth Control #1 medium GC (12935300, Invitrogen) or TMEM9B-AS1 Stealth siRNA (HSS162366, Thermo Fisher Scientific), IGF2BP1 Stealth siRNA (HSS173800, Thermo Fisher Scientific), IGF2BP2 Stealth siRNA (HSS173802, Thermo Fisher Scientific) (10nM final concentration) 6 days after induction of differentiation.

Techniques: Western Blot

Journal: bioRxiv

Article Title: Human-specific lncRNA TMEM9B-AS1 is downregulated in skeletal muscle of individuals with type 2 diabetes and regulates ribosomal biogenesis

doi: 10.1101/2024.07.05.602204

Figure Lengend Snippet: a. Expression of IGF2BP1 in different human tissues from FANTOM 5 data. b. siRNA silencing efficiencies of IGF2BP1 and IGF2BP2. QPCR results showing the expression levels of c. POLI, d. POLII and e. POLIII dependent ribosomal RNA levels upon IGF2BP1 and IGF2BP2 silencing. a. f. mRNA levels of MYC upon IGF2BP1 silencing. g. Results of the time course mRNA stability experiment of human myotubes treated with Actinomycin D for blocking transcription upon siRNA silencing of TMEM9B-AS1 and IGF2BP1 . QPCR data normalized to the housekeeping gene(s) n=6, * p<0.05, ** p<0.01, *** p<0.001. h. Immunocytochemistry images showing Desmin protein levels (shown as red) in differentiated human myotubes in two different donors: Donor 1 and Donor 2. DAPI was used for the staining of the nuclei (shown as blue). Scale bar is 100 µm.

Article Snippet: siRNA transfection was performed using either control siRNA, Stealth Control #1 medium GC (12935300, Invitrogen) or TMEM9B-AS1 Stealth siRNA (HSS162366, Thermo Fisher Scientific), IGF2BP1 Stealth siRNA (HSS173800, Thermo Fisher Scientific), IGF2BP2 Stealth siRNA (HSS173802, Thermo Fisher Scientific) (10nM final concentration) 6 days after induction of differentiation.

Techniques: Expressing, Blocking Assay, Immunocytochemistry, Staining

Journal: bioRxiv

Article Title: Human-specific lncRNA TMEM9B-AS1 is downregulated in skeletal muscle of individuals with type 2 diabetes and regulates ribosomal biogenesis

doi: 10.1101/2024.07.05.602204

Figure Lengend Snippet: Single tissue expression pattern of a. MYC, b. TMEM9B-AS1, and c. IGF2BP1 on GTEX database Arrows are indicating the expression level of TMEM9B-AS1, IGF2BP1 and IGF2BP2 in myocytes and satellite cells in skeletal muscle tissue. d. Expression level of MYC, TMEM9B-AS1 and IGF2BP1 in skeletal muscle fibers and satellite stem cells on the single-cell data platform, CZ CELLxGENE Discover . e. Comparison of the expression level of IGF2BP1 mRNA in human and rodent skeletal muscle cells and tissues.

Article Snippet: siRNA transfection was performed using either control siRNA, Stealth Control #1 medium GC (12935300, Invitrogen) or TMEM9B-AS1 Stealth siRNA (HSS162366, Thermo Fisher Scientific), IGF2BP1 Stealth siRNA (HSS173800, Thermo Fisher Scientific), IGF2BP2 Stealth siRNA (HSS173802, Thermo Fisher Scientific) (10nM final concentration) 6 days after induction of differentiation.

Techniques: Expressing, Comparison

Journal: Journal of Translational Medicine

Article Title: METTL3-mediated NDUFB5 m6A modification promotes cell migration and mitochondrial respiration to promote the wound healing of diabetic foot ulcer

doi: 10.1186/s12967-024-05463-6

Figure Lengend Snippet: m6A modificated by METTL3 and IGF2BP2 may enhance HUVECs NDUFB5 expression. ( A ) The predicted m6A sites in NDUFB5 with use of SRAMP. AGEs (200 µg/mL) was used to stimulate HUVECs for 48 h. ( B ) MeRIP was used to detect the m6A level of NDUFB5 3’UTR in HUVECs. ( C ) Dual luciferase reporter gene assay of NDUFB5 3’UTR activity in HUVECs. Ulcer tissue samples from DFU patients of DUSS grade 1–2 or 3–4 and skin tissues from normal trauma patients were collected. ( D ) qRT-PCR analysis and ( E ) WB analysis of METTL3 expression in tissues. Different concentrations of AGEs were applied to stimulate HUVECs for 48 h. ( F ) qRT-PCR analysis and ( G ) WB analysis of METTL3 expression in HUVECs. HUVECs were transfected with METTL3 expression vector or blank vector and stimulated with 200 µg/ml AGEs or vehicle for 48 h, and HUVECs in control group were transfected with blank vector and stimulated with vehicle. ( H ) MeRIP was applied to assess the m6A level of NDUFB5 3’UTR in HUVECs. ( I ) Dual luciferase reporter gene assay of NDUFB5 3’UTR activity in HUVECs. ( J ) qRT-PCR analysis and ( K ) WB analysis of NDUFB5 expression in HUVECs. ( L ) NDUFB5 mRNA stability analysis in actinomycin D-treated HUVECs. HUVECs were transfected with siIGF2BP2 or siNC and stimulated with 200 µg/ml AGEs or vehicle for 48 h, and HUVECs in control group were transfected with siNC and stimulated with vehicle. ( M ) qRT-PCR analysis and ( N ) WB analysis of NDUFB5 expression in HUVECs. ( O ) AGEs (200 µg/mL) was used to stimulate HUVECs for 48 h. RIP assay of enrichment of NDUFB5 3’UTR by anti-IGF2BP2. * P < 0.05, ** P < 0.01, *** P < 0.001 vs. control or 0 µg/mL group. # P < 0.05, ### P < 0.001 vs. AGEs + vector or AGEs + siNC group

Article Snippet: To knockdown NDUFB5 or IGF2BP2, siRNAs targeting NDUFB5 or IGF2BP2 was synthesized (Addgene, Watertown, USA).

Techniques: Expressing, Luciferase, Reporter Gene Assay, Activity Assay, Quantitative RT-PCR, Transfection, Plasmid Preparation, Control

Journal: iScience

Article Title: CircEZH2 promotes gallbladder cancer progression and lipid metabolism reprogramming through the miR-556-5p/SCD1 axis

doi: 10.1016/j.isci.2024.110428

Figure Lengend Snippet: CircEZH2 is upregulated in GBC tissues and cell lines (A) The RT-qPCR method was applied to detect the expression levels of circEZH2 in 29 GBC tumor tissues and adjacent normal tissues. (B) The relative expression of circEZH2 in human GBC cell lines and a human normal biliary epithelial cells (H69) by RT-qPCR. (C) The junction site of circEZH2 was confirmed by amplification with divergent primers and subsequent detection through Sanger sequencing. (D) RT-qPCR analysis for RNA stabilization of circEZH2 or EZH2 treated with Actinomycin D (2 μg/mL) at the indicated time point. (E and F) RT-qPCR analysis of circEZH2 by divergent primers and convergent primers in cDNA from total RNA or RNase R-treated RNA. (G) Fluorescence in situ hybridization (FISH) was performed on circEZH2. Data are shown as mean ± SD. ∗ p < 0.05. ns., not significant.

Article Snippet: The siRNA against circEZH2, IGF2BP2, si-negative control (si-NC), miR-556 mimic, miR-556 inhibitor, miR-mimic NC and miR-inhibitor NC were synthesized by Ribobio (Guangzhou, China).

Techniques: Quantitative RT-PCR, Expressing, Amplification, Sequencing, Fluorescence, In Situ Hybridization

Journal: iScience

Article Title: CircEZH2 promotes gallbladder cancer progression and lipid metabolism reprogramming through the miR-556-5p/SCD1 axis

doi: 10.1016/j.isci.2024.110428

Figure Lengend Snippet: Correlation between clinicopathological characteristics and circEZH2 expression in 29 cases of GBC patients

Article Snippet: The siRNA against circEZH2, IGF2BP2, si-negative control (si-NC), miR-556 mimic, miR-556 inhibitor, miR-mimic NC and miR-inhibitor NC were synthesized by Ribobio (Guangzhou, China).

Techniques: Expressing

Journal: iScience

Article Title: CircEZH2 promotes gallbladder cancer progression and lipid metabolism reprogramming through the miR-556-5p/SCD1 axis

doi: 10.1016/j.isci.2024.110428

Figure Lengend Snippet: circEZH2 promotes GBC progression in vitro and in vivo (A) The relative expression of circEZH2 was detected in NOZ and SGC-996 cells by RT-qPCR analysis after cells were transfected with siNC, sicircEZH2-1, or sicircEZH2-2. (B and C) CCK-8 assay for cell proliferation capacity after cells were transfected with siRNA at 0–3 days. (D) 5-Ethynyl-2′-deoxyuridine (EdU) proliferation assay. Knockdown of circEZH2 inhibits DNA synthesis in NOZ and SGC-996 cells. The original magnification was 200×. (E and F) Colony formation assay was conducted to verify the impact of knockdown circEZH2. (G) The percentage cell-cycle phase distribution was analyzed by flow cytometry after NOZ and SGC-996 cells were transfected with siNC, sicircEZH2-1, or sicircEZH2-2. (H–K) The volume and weight of subcutaneous xenograft tumors ( n = 5 mice per group). (L) The expression of Ki67 and cyclinD1 by IHC staining. Data are shown as mean ± SD. ∗ p < 0.05.

Article Snippet: The siRNA against circEZH2, IGF2BP2, si-negative control (si-NC), miR-556 mimic, miR-556 inhibitor, miR-mimic NC and miR-inhibitor NC were synthesized by Ribobio (Guangzhou, China).

Techniques: In Vitro, In Vivo, Expressing, Quantitative RT-PCR, Transfection, CCK-8 Assay, Proliferation Assay, Knockdown, DNA Synthesis, Colony Assay, Flow Cytometry, Immunohistochemistry

Journal: iScience

Article Title: CircEZH2 promotes gallbladder cancer progression and lipid metabolism reprogramming through the miR-556-5p/SCD1 axis

doi: 10.1016/j.isci.2024.110428

Figure Lengend Snippet: circEZH2 regulates lipid metabolism and ferroptosis in GBC (A and B) NOZ cells were transfected with siNC or sicircEZH2-2 for volcano plot of the differential metabolite analysis and metabolite principal-component analysis (PCA). (C) Metabolite pathway analysis after NOZ were transfected with siNC or sicircEZH2-2. (D and E) Glycerophospholipids contain SFA upregulated while Glycerophospholipids contain MUFA downregulated in NOZ cells after circEZH2 knockdown. (F) Flow cytometry was applied to detect lipid peroxidation level in NOZ and SGC-996 cells after incubated with a BODIPY C11 probe for 30 min. (G) The RT-qPCR method was applied to detect the mRNA levels of GPX4, SLC7A11, ACSL1, ACSL4, LPCAT3, PTGS2, CHAC1, HMOX1, and NRF2, which were normalized to β-actin. (H and I) GPX4 and NRF2 protein levels after circEZH2 knockdown in NOZ cells were detected by western blot. (J and K) GPX4 protein level in xenograft tumors tissue and GBC patients’ tumor or normal tissues were detected by immunohistochemistry. Data are shown as mean ± SD. ∗ p < 0.05. ns., not significant. MUFA, monounsaturated fatty acids; SFA, saturated fatty acids; LPC, lysophosphatidylcholines; LPE, lysophosphatidylethanolamines; PC, phosphatidylcholines; PE, phosphatidylethanolamines; PI, phosphatidylinositols; PS, phosphatidylserines.

Article Snippet: The siRNA against circEZH2, IGF2BP2, si-negative control (si-NC), miR-556 mimic, miR-556 inhibitor, miR-mimic NC and miR-inhibitor NC were synthesized by Ribobio (Guangzhou, China).

Techniques: Transfection, Knockdown, Flow Cytometry, Incubation, Quantitative RT-PCR, Western Blot, Immunohistochemistry

Journal: iScience

Article Title: CircEZH2 promotes gallbladder cancer progression and lipid metabolism reprogramming through the miR-556-5p/SCD1 axis

doi: 10.1016/j.isci.2024.110428

Figure Lengend Snippet: CircEZH2 acts as a sponge for miR-556-5p in GBC (A) Intersections among three circRNA target prediction algorithms (starBase, circBank, CircInteractome). (B) The relative expression of miR-556-5p and miR-495-3p was detected after NOZ cells were transfected with siNC, sicircEZH2-1, or sicircEZH2-2 by RT-qPCR analysis. (C) The bio-circEZH2 or bio-NC group complex was pulled down from NOZ cell lysate with streptavidin-coated magnetic beads, and the expression of miR-556-5p or miR-495-3p was detected by RT-qPCR. (D) The bio-miR-556-5p or bio-NC group complex was pulled down from NOZ cell lysate with streptavidin-coated magnetic beads, and the expression of circEZH2 was detected by RT-qPCR. (E) Schematic of the wild-type (WT) or mutant-type (MUT) circEZH2 luciferase reporter vectors was constructed according to the prediction from CircInteractome. (F and G) The luciferase activities of the circEZH2-WT or circEZH2-MUT luciferase reporter vector in NOZ or SGC-996 cells transfected with miR-556-5p mimics or miR-NC. (H) Flow cytometry was applied to detect lipid peroxidation level in NOZ and SGC-996 cells after transfected with siNC, sicircEZH2-2, or co-transfected with sicircEZH2-2+ miR-556-5p inhibitor. (I–K) CCK-8 and colony formation assay was performed to detect the ability of proliferation after transfected with siNC, sicircEZH2-2, or co-transfected with sicircEZH2-2+ miR-556-5p inhibitor. Data are shown as mean ± SD. ∗ p < 0.05. ns., not significant.

Article Snippet: The siRNA against circEZH2, IGF2BP2, si-negative control (si-NC), miR-556 mimic, miR-556 inhibitor, miR-mimic NC and miR-inhibitor NC were synthesized by Ribobio (Guangzhou, China).

Techniques: Expressing, Transfection, Quantitative RT-PCR, Magnetic Beads, Mutagenesis, Luciferase, Construct, Plasmid Preparation, Flow Cytometry, CCK-8 Assay, Colony Assay

Journal: iScience

Article Title: CircEZH2 promotes gallbladder cancer progression and lipid metabolism reprogramming through the miR-556-5p/SCD1 axis

doi: 10.1016/j.isci.2024.110428

Figure Lengend Snippet: CircEZH2 regulates SCD1 expression by interacting with miR-556-5p in GBC cells (A) Intersections among three miRNA target prediction algorithms (starBase, TargetScan, miRWalk) and a ferroptosis suppressor catalog in the FerrDb database. (B) Immunohistochemical staining was shown for SCD1 expression in GBC tumor tissues compared with adjacent normal tissues. (C) The protein expression of SCD1 was shown by western blot in NOZ cells after cells were transfected with siNC, sicircEZH2-1, and sicircEZH2-2. (D) SCD1 protein level in xenograft tumors tissue was detected by immunohistochemistry. (E and F) The mRNA or protein expression of SCD1 was shown by RT-qPCR or western blot in NOZ and SGC-996 cells after cells were transfected with siNC, sicircEZH2-2, or co-transfected with sicircEZH2-2+ miR-556-5p inhibitor. (G) The WT and corresponding MUT of the SCD1 mRNA 3′ UTR were constructed targeting miR-556-5p. (H and I) Luciferase reporter assays were performed in NOZ and SGC-996 cells transfected with miR-556-5p mimics, miR-NC and SCD1-WT or SCD1-MUT reporter vector. Data are shown as mean ± SD. ∗ p < 0.05. ns., not significant.

Article Snippet: The siRNA against circEZH2, IGF2BP2, si-negative control (si-NC), miR-556 mimic, miR-556 inhibitor, miR-mimic NC and miR-inhibitor NC were synthesized by Ribobio (Guangzhou, China).

Techniques: Expressing, Immunohistochemical staining, Staining, Western Blot, Transfection, Immunohistochemistry, Quantitative RT-PCR, Construct, Luciferase, Plasmid Preparation

Journal: iScience

Article Title: CircEZH2 promotes gallbladder cancer progression and lipid metabolism reprogramming through the miR-556-5p/SCD1 axis

doi: 10.1016/j.isci.2024.110428

Figure Lengend Snippet: The IGF2BP2-circEZH2 positive feedback loop upregulates the expression of circEZH2 (A) RNA pull-down assay combined with silver staining was used to detect circEZH2-related proteins. (B) Western blot analysis was performed to verify the interaction of circEZH2 and IGF2BP2 or AGO2. GAPDH was used as a negative control. (C) RNA immunoprecipitation (RIP) assay was performed to further verify the interaction between circEZH2 and IGF2BP2 in NOZ cells. IgG was used as a negative control. (D) Immunohistochemical staining was shown for IGF2BP2 expression in GBC tissues compared with adjacent normal tissues. (E) RT-qPCR was applied to detect the expression of circEZH2 in NOZ cells after transfected with siNC or siIGF2BP2. (F) RT-qPCR analysis for RNA stabilization of circEZH2 in NOZ cells after transfected with siNC or siIGF2BP2 at the indicated time point. (G) The m6A modification sites of circEZH2 was predicted from the SRAMP database. (H) RIP was applied to evaluate the level of m6a modification in circEZH2. (I and J) RT-qPCR and western blot were used to detect the expression of IGF2BP2 in NOZ or GBC-SD cells after transfected with siNC, sicircEZH2-2, pLCDH-vector, or pLCDH-circEZH2. (K) CircEZH2 knockdown and control NOZ cells were incubated with MG132 (10 μM) for 8 h. Protein levels of IGF2BP2 were measured by western blot. (L) Immunoprecipitation detected ubiquitination modification of IGF2BP2 with MG132 treatment in NOZ cells. IgG was used as a negative control. Data are shown as mean ± SD. ∗ p < 0.05. ns., not significant, Ub, ubiquitin.

Article Snippet: The siRNA against circEZH2, IGF2BP2, si-negative control (si-NC), miR-556 mimic, miR-556 inhibitor, miR-mimic NC and miR-inhibitor NC were synthesized by Ribobio (Guangzhou, China).

Techniques: Expressing, Pull Down Assay, Silver Staining, Western Blot, Negative Control, RNA Immunoprecipitation, Immunohistochemical staining, Staining, Quantitative RT-PCR, Transfection, Modification, Plasmid Preparation, Knockdown, Control, Incubation, Immunoprecipitation, Ubiquitin Proteomics

Journal: iScience

Article Title: CircEZH2 promotes gallbladder cancer progression and lipid metabolism reprogramming through the miR-556-5p/SCD1 axis

doi: 10.1016/j.isci.2024.110428

Figure Lengend Snippet:

Article Snippet: The siRNA against circEZH2, IGF2BP2, si-negative control (si-NC), miR-556 mimic, miR-556 inhibitor, miR-mimic NC and miR-inhibitor NC were synthesized by Ribobio (Guangzhou, China).

Techniques: Virus, Recombinant, SYBR Green Assay, Protease Inhibitor, Membrane, RNA Immunoprecipitation, Proliferation Assay, Silver Staining, Reporter Assay, Mass Spectrometry, Plasmid Preparation, Software