Journal: Cell Death Discovery

Article Title: Overexpression of circRNA SNRK targets miR-103-3p to reduce apoptosis and promote cardiac repair through GSK3β/β-catenin pathway in rats with myocardial infarction

doi: 10.1038/s41420-021-00467-3

Figure Lengend Snippet: A Graphical representation showing the predicted sites of miR-103-3p for binding to circSNRK and the corresponding mutation. B Luciferase activity of primary cardiomyocytes transfected with luciferase-circSNRK-WT and luciferase-circSNRK-Mut. ** P < 0.01 vs. miR-103-3p - NC. ( n = 3). C qRT-PCR analysis of miR-103-3p in vector or circSNRK overexpression plasmid treated primary cardiomyocytes. *** p < 0.001 vs. vector ( n = 6). D qRT-PCR analysis of circSNRK and SNRK mRNA in miR-NC or miR-103-3p treated primary cardiomyocytes. *** p < 0.001 vs. vector ( n = 6). E Luciferase reporter assay was used to test the luciferase activity of SNRK promoter region ( n = 3). F Luciferase activity of primary cardiomyocytes transfected with luciferase-SNRK 3′ UTR-WT and luciferase-SNRK 3′ UTR-Mut. ** P < 0.01 vs. miR-103-3p-NC + SNRK-WT, ### P < 0.001 versus miR-103-3P-NC + circSNRK+ SNRK-WT ( n = 3). G SNRK protein level after overexpression of circSNRK. *** p < 0.001 vs. vector ( n = 6). H SNRK protein level after overexpression of miR-103-3p. *** p < 0.001 vs. miR103-3p-NC ( n = 6). I SNRK protein level after downregulation of miR - 103-3p. *** p < 0.001 vs. miR-103-3p-NC ( n = 6). J SNRK protein level after circSNRK and miR-103-3p interference in cardiomyocytes. * P < 0.05 vs. Vector + miR-NC, ### P < 0.001 vs. circSNRK + miR-NC, && P < 0.01 vs. circSNRK + miR-103-3p mimics ( n = 3).

Article Snippet: HEK-293T cells were co-transfected with miR-103-3p mimics and miR-103-3p-NC (50 nM; Ribobio, Guangzhou, China), and a reporter plasmid containing the 3′ UTR of circSNRK (250 ng/well) (GeneChem, Shanghai, China) inserted downstream of the luciferase reporter gene (PGL3-circSNRK-UTR) were transfected by Lipofectamine 2000 in a 96-well plate.

Techniques: Binding Assay, Mutagenesis, Luciferase, Activity Assay, Transfection, Quantitative RT-PCR, Plasmid Preparation, Over Expression, Reporter Assay

Journal: Cell Death Discovery

Article Title: Overexpression of circRNA SNRK targets miR-103-3p to reduce apoptosis and promote cardiac repair through GSK3β/β-catenin pathway in rats with myocardial infarction

doi: 10.1038/s41420-021-00467-3

Figure Lengend Snippet: A – C Primary cardiomyocytes were transfected with Vector, circSNRK, circSNRK + miR-103-3p mimics and circSNRK + miT-103-3p mimics + SNRK in H/SD conditions. Flow cytometry analysis ( n = 3) ( A ) and TUNEL analysis ( B ) in primary cardiomyocytes after circSNRK, miR-103-3p, and SNRK interference. White arrows indicate apoptotic cells. bar = 50 μm. And Cell Counting Kit-8 (CCK-8) assay ( n = 6). C ** P < 0.01, *** p < 0.001 vs. Vector, ## P < 0.01, ### p < 0.001 vs. circSNRK, &&& p < 0.001 vs. circSNRK + miR-103-3p mimics ( n = 6). D BCL-2, BAX, cleaved-caspase-3/caspase-3 levels in hypoxic and ischemic primary cardiomyocytes after circSNRK, miR-103-3p, and SNRK interference. ** P < 0.01, *** p < 0.001 vs. Vector, ## P < 0.01, ### p < 0.001 vs. circSNRK, & P < 0.05, &&& p < 0.001 vs. circSNRK + mimics ( n = 3). E EdU staining in isolated NRCMs after circSNRK , miR-103-3p, and SNRK interference and quantification of EdU positive primary cardiomyocytes. White arrows indicate EdU positive primary cardiomyocytes. *** p < 0.001 vs. Vector, ### p < 0.001 vs. circSNRK, &&& p < 0.001 vs. circSNRK + miR-103-3p mimics; bar = 50 µm. ( n = 6). G Ki67 immunofluorescence staining in isolated primary cardiomyocytes after circSNRK, miR-103-3p, and SNRK interference and quantification of Ki67 positive primary cardiomyocytes. White arrows indicate Ki67-positive primary cardiomyocytes. ** P < 0.01 vs. Vector, ## P < 0.01 vs. circSNRK, &&& p < 0.001 vs. circSNRK + miR-103-3p mimics; bar = 50 µm. ( n = 6). H Flow cytometry analysis of primary cardiomyocytes transfected with miR-NC and miR-103-3p inhibitor. ** P < 0.01, *** p < 0.001 vs. vector group, ## p < 0.01 ### p < 0.001 vs. circSNRK, && p < 0.01 &&& p < 0.001 vs. circSNRK + miR-103-3p mimics ( n = 3).

Article Snippet: HEK-293T cells were co-transfected with miR-103-3p mimics and miR-103-3p-NC (50 nM; Ribobio, Guangzhou, China), and a reporter plasmid containing the 3′ UTR of circSNRK (250 ng/well) (GeneChem, Shanghai, China) inserted downstream of the luciferase reporter gene (PGL3-circSNRK-UTR) were transfected by Lipofectamine 2000 in a 96-well plate.

Techniques: Transfection, Plasmid Preparation, Flow Cytometry, TUNEL Assay, Cell Counting, CCK-8 Assay, Staining, Isolation, Immunofluorescence

Journal: Cell Communication and Signaling : CCS

Article Title: Concomitant induction of SLIT3 and microRNA-218–2 in macrophages by toll-like receptor 4 activation limits osteoclast commitment

doi: 10.1186/s12964-023-01226-w

Figure Lengend Snippet: TLR4 activation enhances miRNA-218 in OCPs, which suppresses osteoclast differentiation by directly inhibiting RANK expression. A The mouse OCPs were incubated with LPS for 4 h and miR-218–2 expression was measured by qPCR. B WT or Tlr4 −/− OCPs were incubated with 10 ng/mL LPS for 4 h and miR-218–2 expression was determined using qPCR. C WT or Slit3 knockdown OCPs were incubated with 0, 1, or 10 ng/mL LPS for 4 h and miR-218–2 expression was then assayed. D Schematic representation of miR-218–2-3p targeting sites in the Tnfrsf11a 3’-UTR. E, F OCPs were transfected with or without the miR-218–2-3p mimic and miR-218–2-3p inhibitor (100 nM), and then incubated with or without 10 ng/mL LPS for 4 h and subjected to qPCR analysis of the relative Tnfrsf11a mRNA expression levels ( E ). Moreover, these transfected cells were incubated with or without LPS for 24 h and then the RANK protein expression level was analyzed by immunoblotting and quantified with the ImageJ software ( F ). G Schematic representation of firefly luciferase constructs containing the CMV promoter, luciferase coding region, and a fragment of the Tnfrsf11a 3’-UTR ( G, left ). NIH3T3 cells were co-transfected with miR-218–2-3p , firefly luciferase constructs ( Tnfrsf11a 3’-UTR WT, Mut1, Mut2, or Mut1 + 2), and Renilla luciferase control for the dual-luciferase assay. The relative luciferase activity represents the dual luciferase activity ratio (i.e., firefly/Renilla luciferase). WT, wild type; Mut 1, mutation at site 1; Mut 2, mutation at site 2; Mut1 + 2, mutation at both sites 1 and 2 ( G, right ). H The transfected OCPs with or without miR-218–2-3p mimic or miR-218–2-3p inhibitor were cultured with M-CSF and RANKL in the presence or absence of LPS (10 ng/mL), and in the presence or absence of anti-miR (Qiagen). These cells were then fixed, stained for TRAP ( H, left ), and the number of TRAP + MNCs containing three or more nuclei (MNCs; ≥ 3 nuclei) were counted under a light microscope ( H, right ). Scale bar, 100 μm. Data are shown as mean with s.d., * P < 0.05, ** P < 0.005, *** P < 0.0001. All representative data from three independent experiments are shown

Article Snippet: An miRNA mimic ( miR-218–2-3p ) and its inhibitor anti-miR ( anti-miR-218–2-3p ) were purchased from Qiagen for miRNA functional assays.

Techniques: Activation Assay, Expressing, Incubation, Knockdown, Transfection, Western Blot, Software, Luciferase, Construct, Control, Activity Assay, Mutagenesis, Cell Culture, Staining, Light Microscopy

Journal: Cancer Management and Research

Article Title: circRNA MYLK Accelerates Cervical Cancer via Up-Regulation of RHEB and Activation of mTOR Signaling

doi: 10.2147/CMAR.S238172

Figure Lengend Snippet: CircMYLK sponges miR-1301-3p in CC cells. ( A, B ) The cytoplasmic or nuclear abundance of circMYLK in HCC94 and C-33A was elucidated in cellular fractionation and FISH assay. ( C ) Top 50 miRNAs from starBase underwent qRT-PCR analysis in HCC94 and C-33A compared with Ect1/E6E7 to pinpoint the downregulated miRNAs; 8 overexpressed eight miRNAs were obtained. ( D ) Luciferase reporter assay of circMYLK activity responding to indicated miRNA mimics. ( E ) CircMTLK and miR-1301-3p were enriched in Ago2 group in RIP assay. ( F ) Bioinformatics predications of the binding sites between circMYLK and miR-1301-3p. ( G ) Luciferase activity of circMYLK-WT, circMYLK-MUT upon the transfection of miR-1301-3p mimics or NC mimics. ( H ) RNA pull-down monitored the enrichment of circMYLK treated with bio-NC, bio-miR-1301-3p-wt, or bio-miR-1301-3p-wt probe. ( I – K ) Colony formation potential, cell viability and proliferation of circMYLK-silenced cells could be reversed by miR-1301-3p inhibitor. ( L ) CircMYLK-silenced cell apoptosis could be suppressed by miR-1301-3p inhibitor in TUNEL. *P < 0.05; **P < 0.01.

Article Snippet: Additionally, miR-1301-3p mimics and NC (negative control) mimics, miR-1301-3p inhibitor and NC inhibitor, were also produced by Genepharm.

Techniques: Cell Fractionation, Quantitative RT-PCR, Luciferase, Reporter Assay, Activity Assay, Binding Assay, Transfection, TUNEL Assay

Journal: Cancer Management and Research

Article Title: circRNA MYLK Accelerates Cervical Cancer via Up-Regulation of RHEB and Activation of mTOR Signaling

doi: 10.2147/CMAR.S238172

Figure Lengend Snippet: CircMYLK sponges miR-1301-3p to activate RHEB-dependent mTOR pathway. ( A, B ) Log2 fold change of the candidate mRNAs following the transfection of miR-1301-3p mimics and NC mimics into HCC94 and C-33A. ( C ) The abundance of circMYLK, miR-1301-3p and RHEB in Ago2 protein. ( D ) The binding sites between miR-1301-3p and RHEB disclosed by bioinformatics analysis. ( E ) Overexpression efficacy of circMYLK. ( F ) Luciferase reporter confirmed the interacting sequence of miR-1301-3p and RHEB; circMYLK could reverse the suppressed activity of RHEB-wt. ( G ) The impact of circMYLK/miR-1301-3p on RHEB expression. ( H ) The suppressive effect of sh/circMYLK#1 on RHEB, EIF5, and mTORC1 expression. **P < 0.01.

Article Snippet: Additionally, miR-1301-3p mimics and NC (negative control) mimics, miR-1301-3p inhibitor and NC inhibitor, were also produced by Genepharm.

Techniques: Transfection, Binding Assay, Over Expression, Luciferase, Sequencing, Activity Assay, Expressing

Journal: Journal of Translational Medicine

Article Title: Exosomal LncRNA LINC00659 transferred from cancer-associated fibroblasts promotes colorectal cancer cell progression via miR-342-3p/ANXA2 axis

doi: 10.1186/s12967-020-02648-7

Figure Lengend Snippet: CAFs-derived exosomal LINC00659 acts as a sponge for miR-342-3p to regulate ANXA2 expression. Note: After NFs-exo and CAFs-exo were co-incubated with SW48 cells, qRT-PCR was applied to measure miR-342-3p expression in SW48 cells ( a ). The levels of ANXA2 in SW48 cells were assessed by qRT-PCR and Western blot, respectively ( b , c ). Following transfection of miR-342-3p mimics or inhibitors in SW48 cells, the expression of miR-342-3p in SW48 cells were measured by qRT-PCR ( d ). The levels of ANXA2 in SW48 cells were detected by qRT-PCR and Western blot, respectively ( e , f ). Prediction of binding sites and designed mutation sites of LINC00659 and ANXA2, LINC00659 and miR-342-3p was performed by the online software Starbase ( g ). Dual luciferase reporter assay was used to test the interactions of LINC00659 and miR-342-3p ( h ) as well as ANXA2 and miR-342-3p (i). N = 3, * P < 0.05, ** P < 0.01, *** P < 0.001; CAFs-exo CAFs-derived exosomes, NFs-exo NFs-derived exosomes

Article Snippet: pcDNA3.1-LINC00659, pcDNA3.1-ANXA2, pcDNA3.1, sh-LINC00659, miR-342-3p mimic, miR-342-3p inhibitor and their negative controls were obtained from Shanghai GenePharma Co. Ltd (Shanghai, China).

Techniques: Derivative Assay, Expressing, Incubation, Quantitative RT-PCR, Western Blot, Transfection, Binding Assay, Mutagenesis, Software, Luciferase, Reporter Assay

Journal: Journal of Translational Medicine

Article Title: Exosomal LncRNA LINC00659 transferred from cancer-associated fibroblasts promotes colorectal cancer cell progression via miR-342-3p/ANXA2 axis

doi: 10.1186/s12967-020-02648-7

Figure Lengend Snippet: CAFs-derived exosomal LINC00659 promotes CRC cell progression via blocking miR-342-3p. Note: Cell proliferation, cell migration, cell invasion and EMT related markers were analyzed by CCK-8 ( a , b ) and clone formation assay ( c , d ), cell scratch ( e , f ), Transwell ( g , h ), qRT-PCR ( i , j ) and Western blot ( k , l ), respectively. N = 3, * P < 0.05, ** P < 0.01; CAFs cancer-associated fibroblasts, CRC colorectal cancer, EMT epithelial mesenchymal transformation

Article Snippet: pcDNA3.1-LINC00659, pcDNA3.1-ANXA2, pcDNA3.1, sh-LINC00659, miR-342-3p mimic, miR-342-3p inhibitor and their negative controls were obtained from Shanghai GenePharma Co. Ltd (Shanghai, China).

Techniques: Derivative Assay, Blocking Assay, Migration, CCK-8 Assay, Tube Formation Assay, Quantitative RT-PCR, Western Blot, Transformation Assay

Journal: Cancer & Metabolism

Article Title: Blocking lncRNA-SNHG16 sensitizes gastric cancer cells to 5-Fu through targeting the miR-506-3p-PTBP1-mediated glucose metabolism

doi: 10.1186/s40170-022-00293-w

Figure Lengend Snippet: SNHG16 downregulates miR-506-3p in GC by sponging it. a The potential binding between SNHG16 and miR-506-3p was predicted from starBase. b miR-506-3p expressions were detected in gastric tumors ( n =55), and their matched normal gastric tissues by qRT-PCR. c Kaplan-Meier Plotter analyzes the survival rates of GC patients with high or low miR-506-3p expressions. d Pearson coefficient analysis shows a significantly negative correlation between SNHG16 and miR-506-3p in gastric tumors. e Expressions of miR-506-3p were detected in AGS parental and 5-Fu-resistant cells. f , g AGS and SGC-7901 cells were transfected with control miRNA or miR-506-3p. Cells were treated with 5-Fu, and cell viability were determined by MTT assay. h GC cells were transfected with control or SNHG16 siRNA. Expressions of miR-506-3p were detected by qRT-PCR. i Luciferase assay was performed in AGS and SGC-7901 cells transfected with control or miR-506-3p plus WT-SNHG16 or mut-SNHG16. Data were presented as mean±S.D. * p <0.05; ** p <0.01; *** p <0.001

Article Snippet: Control miRNA and miR-506-3p precursor were purchased from GenePharma Co. (Shanghai, China).

Techniques: Binding Assay, Quantitative RT-PCR, Transfection, MTT Assay, Luciferase

Journal: Cancer & Metabolism

Article Title: Blocking lncRNA-SNHG16 sensitizes gastric cancer cells to 5-Fu through targeting the miR-506-3p-PTBP1-mediated glucose metabolism

doi: 10.1186/s40170-022-00293-w

Figure Lengend Snippet: miR-506-3p sensitizes GC cells to 5-Fu by direct targeting PTBP1. a The potential binding of miR-506-3p on PTBP1 3′UTR was predicted from starBase. b AGS and SGC-7901 cells were transfected with control miRNA or miR-506-3p. Protein expressions of PTBP1 were determined. c Luciferase assay was performed in AGS and d SGC-7901 cells transfected with control or miR-506-3p plus WT- or mut-3′UTR of PTBP1. e Pearson coefficient analysis shows a significantly negative correlation between miR-506-3p and PTBP1 in gastric tumors. f AGS and SGC-7901 cells were transfected with control, miR-506-3p alone, or plus PTBP1. Protein expressions of PTBP1 were examined. g , h The glucose uptake and lactate product from the above transfected cells were determined. i , j The above transfected cells were treated with 5-Fu at the indicated concentrations, and cell viability was determined by MTT assay. Data were presented as mean±S.D. * p <0.05; ** p <0.01; *** p <0.001

Article Snippet: Control miRNA and miR-506-3p precursor were purchased from GenePharma Co. (Shanghai, China).

Techniques: Binding Assay, Transfection, Luciferase, MTT Assay

Journal: Cancer & Metabolism

Article Title: Blocking lncRNA-SNHG16 sensitizes gastric cancer cells to 5-Fu through targeting the miR-506-3p-PTBP1-mediated glucose metabolism

doi: 10.1186/s40170-022-00293-w

Figure Lengend Snippet: SNHG16 promotes 5-Fu resistance through modulating the miR-506-3p-PTBP1-glycolysis axis. a , b AGS 5-Fu-resistant cells were transfected with control, SNHG16 alone, or plus miR-506-3p. Expressions of miR-506-3p and PTBP1 were examined by qRT-PCR and Western blot. c ECAR, d glucose uptake, and e glycolysis enzymes expressions from the above transfected cells were determined. f The above transfected cells were treated with 5-Fu at the indicated concentrations, and cell viabilities were determined by MTT and g Annexin V apoptosis assay. Data were presented as mean±S.D. * p <0.05; ** p <0.01; *** p <0.001

Article Snippet: Control miRNA and miR-506-3p precursor were purchased from GenePharma Co. (Shanghai, China).

Techniques: Transfection, Quantitative RT-PCR, Western Blot, Apoptosis Assay

Journal: Molecular Neurobiology

Article Title: Short-Term Memory Deficit Associates with miR-153-3p Upregulation in the Hippocampus of Middle-Aged Mice

doi: 10.1007/s12035-023-03770-5

Figure Lengend Snippet: Upregulation of the miR-153-3p in the HP impairs performance in the 6-DOT in adult mice. a After surgery and handling, 3-month-old mice were injected with mimic-miR-153-3p 3 h prior to performing the 6-DOT. b Bar charts show objects exploration for controls (scramble) and mimic-miR-153-3p injected mice. Only control mice ( N = 13) were able to discriminate the new object compared to all the other familiar ones (F1-F5), while mimic-miR-153-3p injected mice ( N = 12) showed no difference in exploration between the objects at test and a significantly lower exploration of the new object compared to controls. * p < 0.001 (Dunnett post-hoc test within group), # p < 0.001(Tukey HSD post-hoc test between groups). c Illustration of the placement injections in the dHP of control (cyan) and mimic-miR-153-3p injected (orange) mice

Article Snippet: Then, 200 µM of saline-formulated (0.15 M NaCl) LNA preparations mimic miR-153-3p (Exiqon, mimic miR-153-3p, product no: 470930–001) or mimic negative control (scramble; Exiqon, product no: 479903–001) were delivered into the dorsal hippocampus at a rate of 0.4 μl per 2 min, using a micropump (Harvard Apparatus, Holliston, MA, USA).

Techniques: Injection