Journal: Cell Proliferation

Article Title: Long non‐coding RNA HOTAIR/microRNA‐206 sponge regulates STC2 and further influences cell biological functions in head and neck squamous cell carcinoma

doi: 10.1111/cpr.12651

Figure Lengend Snippet: HOTAIR sponges miR‐206. A, the binding site between HOTAIR, miR‐206 and miR‐206‐Mut predicted by bioinformatics website; B, the luciferase activity in each group detected by dual luciferase reporter gene assay; C, the expression of miR‐206 in each group measured by RT‐qPCR; D, the binding of HOTAIR and AGO assessed by RIP; E, HOTAIR and miR‐206 localization by fluorescence double staining. The measurement data amongst multiple groups in panel B and C were analysed by one‐way ANOVA; the independent sample t test was used for statistical analysis between two groups; the data in FIGURE D were analysed by independent sample t test; the experiment was performed in triplicates. * P < 0.05 vs the miR‐206‐Mut, blank or IgG groups. RIP, RNA‐binding protein immunoprecipitation; IgG, immunoglobulin G

Article Snippet: Ribo TM lncRNA FISH probe Mix (Red) and Ribo TM miR‐206 FISH probe Mix (Green) (Guangzhou RIBOBIO Co., Ltd., Guangzhou, Guangdong, China) were performed.

Techniques: Binding Assay, Luciferase, Activity Assay, Reporter Gene Assay, Expressing, Quantitative RT-PCR, Fluorescence, Double Staining, RNA Binding Assay, Immunoprecipitation

Journal: Cell Proliferation

Article Title: Long non‐coding RNA HOTAIR/microRNA‐206 sponge regulates STC2 and further influences cell biological functions in head and neck squamous cell carcinoma

doi: 10.1111/cpr.12651

Figure Lengend Snippet: miR‐206 targets STC2. A, verification of the targeting relationship between miR‐206 and STC2; B, the luciferase activity of STC2‐Wt and STC2‐Mut detected by dual luciferase reporter assay; C, the expression of STC2 and miR‐206 assessed by RT‐qPCR; D, the grey value of STC2 protein band in response to the treatment of miR‐206 mimic or miR‐206 inhibitor; E, the protein level of STC2 determined by Western blot analysis; one‐way ANOVA was used for different factors analysis, and the independent sample t test was used for statistical analysis between two groups; the experiment was performed in triplicates; data were represented as mean ± standard deviation; *, P < 0.05 vs the NC group or the blank group. Wt; wide type; Mut, mutant

Article Snippet: Ribo TM lncRNA FISH probe Mix (Red) and Ribo TM miR‐206 FISH probe Mix (Green) (Guangzhou RIBOBIO Co., Ltd., Guangzhou, Guangdong, China) were performed.

Techniques: Luciferase, Activity Assay, Reporter Assay, Expressing, Quantitative RT-PCR, Western Blot, Standard Deviation, Mutagenesis

Journal: Cell Proliferation

Article Title: Long non‐coding RNA HOTAIR/microRNA‐206 sponge regulates STC2 and further influences cell biological functions in head and neck squamous cell carcinoma

doi: 10.1111/cpr.12651

Figure Lengend Snippet: miR‐206 inhibits the activation of the PI3K/AKT signalling pathway by targeting STC2. A, the expression of miR‐206 and mRNA levels of STC2, PI3K and AKT detected by RT‐qPCR; B, the grey value of STC2, PI3K, p‐PI3K, AKT and p‐AKT; C, the protein levels of STC2, p‐PI3K/PI3K and p‐AKT/AKT determined by Western blot analysis; one‐way ANOVA was used for analysis amongst groups, and the experiment was performed in triplicates; data were represented as mean ± standard deviation; * P < 0.05 vs the blank or the NC group

Article Snippet: Ribo TM lncRNA FISH probe Mix (Red) and Ribo TM miR‐206 FISH probe Mix (Green) (Guangzhou RIBOBIO Co., Ltd., Guangzhou, Guangdong, China) were performed.

Techniques: Activation Assay, Expressing, Quantitative RT-PCR, Western Blot, Standard Deviation

Journal: Cell Proliferation

Article Title: Long non‐coding RNA HOTAIR/microRNA‐206 sponge regulates STC2 and further influences cell biological functions in head and neck squamous cell carcinoma

doi: 10.1111/cpr.12651

Figure Lengend Snippet: miR‐206 overexpression or STC2 silencing leads to the inhibition of HNSCC cell proliferation, migration and invasion, and elevation of apoptosis. A‐B, HNSCC cell migration detected by transwell assay (× 100); C‐D, HNSCC cell invasion by transwell assay; E‐F, HNSCC cell migration ability and healing rate by scratch test; G, CCK‐8 for detection of growth of HNSCC cells; H, EDU assay for proliferation of HNSCC cells; I, EDU fluorescence intensity statistical results in each group; J‐K, flow cytometry for cell cycle entry and cell cycle ratio of HNSCC cells; L‐M, flow cytometry for HNSCC cell apoptosis in each group. One‐way ANOVA was used for analysis amongst groups, and the experiment was performed in triplicates; data were represented as mean ± standard deviation; * P < 0.05 vs the blank or NC group

Article Snippet: Ribo TM lncRNA FISH probe Mix (Red) and Ribo TM miR‐206 FISH probe Mix (Green) (Guangzhou RIBOBIO Co., Ltd., Guangzhou, Guangdong, China) were performed.

Techniques: Over Expression, Inhibition, Migration, Transwell Assay, CCK-8 Assay, EdU Assay, Fluorescence, Flow Cytometry, Standard Deviation

Journal: Cell Proliferation

Article Title: Long non‐coding RNA HOTAIR/microRNA‐206 sponge regulates STC2 and further influences cell biological functions in head and neck squamous cell carcinoma

doi: 10.1111/cpr.12651

Figure Lengend Snippet: HOTAIR silencing or miR‐206 overexpression suppresses tumour growth in nude mice injected with transfected HNSCC cells. A, the tumour volume of nude mice measured by a vernier caliper; B, xenograft tumours in nude mice observed by naked eyes; C, tumour weight of nude mice; one‐way ANOVA was used for analysis amongst groups, and the experiment was performed in triplicates; data were represented as mean ± standard deviation; * P < 0.05 vs the blank or NC group

Article Snippet: Ribo TM lncRNA FISH probe Mix (Red) and Ribo TM miR‐206 FISH probe Mix (Green) (Guangzhou RIBOBIO Co., Ltd., Guangzhou, Guangdong, China) were performed.

Techniques: Over Expression, Injection, Transfection, Standard Deviation

Journal: Cell Proliferation

Article Title: Long non‐coding RNA HOTAIR/microRNA‐206 sponge regulates STC2 and further influences cell biological functions in head and neck squamous cell carcinoma

doi: 10.1111/cpr.12651

Figure Lengend Snippet: HOTAIR competitively binds to miR‐206, thereby promoting STC2 expression, activating PI3K/AKT signalling pathway

Article Snippet: Ribo TM lncRNA FISH probe Mix (Red) and Ribo TM miR‐206 FISH probe Mix (Green) (Guangzhou RIBOBIO Co., Ltd., Guangzhou, Guangdong, China) were performed.

Techniques: Expressing

Journal: Therapeutic Advances in Medical Oncology

Article Title: The effect of CELLFOOD TM on radiotherapy or combined chemoradiotherapy: preclinical evidence

doi: 10.1177/1758835919878347

Figure Lengend Snippet: CF modulated HIF1α, PGK1 and VEGF expression in cancer cell lines. Western blot analysis of HIF1α, PGK1 and VEGF in Cal27, MSTO and HCT-116 (HCT) cells. (a) HIF1α, PGK1 and VEGF expression basal and after treatment of Cal27, MSTO and HCT cells with 100 µmol/l CoCl 2 for 4, 6, 24 h; (b) HIF1α modulation under hypoxia (CoCl 2 ) after 4 h of treatment with CF (5 μl per ml of medium corresponding to a 1:200 dilution); (c) PGK1 and VEGF expressions under hypoxia (CoCl 2 ) after 24 h of treatment with CF (5 μl per ml of medium corresponding to a 1:200 dilution). Cal27, carcinoma of the tongue cell line; MSTO, MSTO-211H mesothelioma cell line; HCT-116, human colon carcinoma cell line; CoCl2, cobalt dichloride; HIF1α, hypoxia-inducible factor 1 alpha; PGK1, phosphoglycerate kinase 1; VEGF, vascular endothelial growth factor. CF, CELLFOOD™.

Article Snippet: After electrophoresis, proteins were transferred to polyvinylidene difluoride membranes and probed with antibodies against HIF1α [HIF1α (H-206); Santa Cruz Biotechnology, Santa Cruz, California, US], PGK1 [PGK1 (Y-12); Santa Cruz Biotechnology], VEGF [VEGF (A-20); Santa Cruz Biotechnology] and α-tubulin (Calbiochem, Darmstadt, Germany) Goat antimouse immunoglobulin G horseradish peroxidase-conjugated secondary antibodies (Bio-Rad Laboratories) were used.

Techniques: Expressing, Western Blot

Journal: Therapeutic Advances in Medical Oncology

Article Title: The effect of CELLFOOD TM on radiotherapy or combined chemoradiotherapy: preclinical evidence

doi: 10.1177/1758835919878347

Figure Lengend Snippet: Effect of ROS on HIF1α, PGK1 and VEGF expression. (a) The histogram shows the results of three independent experiments: the percentage of levels of ROS with respect to untreated MSTO cells (CNTR) after the addition of 50–200 μmol/l H 2 O 2 (−NAC) and after cotreatment with 5 mmol/l NAC (+NAC). Data are expressed as mean ± SD of at least three independent experiments. ROS effect on the expression of the HIF1α protein, PGK1 and VEGF was detected by western blot in all experiments, (b) represents a single experiment. The relative bands intensities of the proteins of interest after 50–200 μmol/l H 2 O 2 (c) and 50–200 μmol/l H 2 O 2 with NAC (d) were quantified by the Scion Image software in all experiment. * p < 0.05 versus CNTR. CNTR, with vehicle; HIF1α, hypoxia-inducible factor 1 alpha; NAC, N-acetyl-L-cysteine; PGK1, phosphoglycerate kinase 1; ROS, reactive oxygen species; SD, standard deviation; VEGF, vascular endothelial growth factor.

Article Snippet: After electrophoresis, proteins were transferred to polyvinylidene difluoride membranes and probed with antibodies against HIF1α [HIF1α (H-206); Santa Cruz Biotechnology, Santa Cruz, California, US], PGK1 [PGK1 (Y-12); Santa Cruz Biotechnology], VEGF [VEGF (A-20); Santa Cruz Biotechnology] and α-tubulin (Calbiochem, Darmstadt, Germany) Goat antimouse immunoglobulin G horseradish peroxidase-conjugated secondary antibodies (Bio-Rad Laboratories) were used.

Techniques: Expressing, Western Blot, Software, Standard Deviation

Journal: Therapeutic Advances in Medical Oncology

Article Title: The effect of CELLFOOD TM on radiotherapy or combined chemoradiotherapy: preclinical evidence

doi: 10.1177/1758835919878347

Figure Lengend Snippet: Effect of CF in association with therapeutic treatments on tumour growth and HIF1α, PGK1 and VEGF expression in mesothelioma mice xenograft. (a) Action of the CF alone and with RT(4) on the tumour growth. * significant versus CNTR, ¤ significant versus CNTR + RT(4); (b) action of the ST alone and with CF on the tumour growth; * significant versus CNTR; (c) action of the RT(4) alone, with ST and with ST + CF on the tumour growth; * significant versus CNTR + RT(4); ¤ significant versus ST + RT(4); (d) a representative western blot of HIF1α, PGK1 and VEGF of masses removed by mice untreated and treated with ST, RT(4) and CF alone and in combination, and quantification of protein band intensities by Scion Image software and tubulin-normalization (e); (f) immunohistochemistry of tumour explants; the scale bar is 30 µmol/l. Data are expressed as mean ± SD (standard deviation) of all determinations of explants for each group. * p < 0.05 versus RT(4). # p < 0.05 versus CF + RT(4). CF, CELLFOOD™; CNTR, with vehicle; RT(4), irradiation treatment (RT) with 4 Gy; RT(8), RT with 8 Gy; ST, standard therapy; HIF1α, hypoxia-inducible factor 1 alpha; PGK1, phosphoglycerate kinase 1; VEGF, vascular endothelial growth factor; H/E, haematoxylin and eosin staining.

Article Snippet: After electrophoresis, proteins were transferred to polyvinylidene difluoride membranes and probed with antibodies against HIF1α [HIF1α (H-206); Santa Cruz Biotechnology, Santa Cruz, California, US], PGK1 [PGK1 (Y-12); Santa Cruz Biotechnology], VEGF [VEGF (A-20); Santa Cruz Biotechnology] and α-tubulin (Calbiochem, Darmstadt, Germany) Goat antimouse immunoglobulin G horseradish peroxidase-conjugated secondary antibodies (Bio-Rad Laboratories) were used.

Techniques: Expressing, Western Blot, Software, Immunohistochemistry, Standard Deviation, Irradiation, Staining

Journal: Therapeutic Advances in Medical Oncology

Article Title: The effect of CELLFOOD TM on radiotherapy or combined chemoradiotherapy: preclinical evidence

doi: 10.1177/1758835919878347

Figure Lengend Snippet: Schematic representation of a model explaining the association between CF and radio- and chemotherapy. (a) Before irradiation (baseline), the tumour mass consists of well-oxygenated (green) and nonoxygenated (red) cells. RT kills a higher number of well-oxygenated than hypoxic cells with ROS production and increased HIF1α expression. In the case of CF + RT treatment, CF is expected to increase the fraction of oxygenated cells reducing the hypoxic fraction, making RT more effective (b). This implies a greater production of ROS and reduction of HIF1α (b) compared with RT alone (a). After the CF administration, when ST is added to RT (c), we expected a further increase in ROS, attributable to the action of CISP and an overall reduction in HIF1α expression. ROS, reactive oxygen species; HIF1α, hypoxia-inducible factor 1 alpha; RT, irradiation therapy; CF, CELLFOOD™; ST, standard therapy.

Article Snippet: After electrophoresis, proteins were transferred to polyvinylidene difluoride membranes and probed with antibodies against HIF1α [HIF1α (H-206); Santa Cruz Biotechnology, Santa Cruz, California, US], PGK1 [PGK1 (Y-12); Santa Cruz Biotechnology], VEGF [VEGF (A-20); Santa Cruz Biotechnology] and α-tubulin (Calbiochem, Darmstadt, Germany) Goat antimouse immunoglobulin G horseradish peroxidase-conjugated secondary antibodies (Bio-Rad Laboratories) were used.

Techniques: Irradiation, Expressing

Journal: Theranostics

Article Title: Hyperglycemia facilitates EV71 replication: Insights into miR-206-mediated regulation of G3BP2 promoting EV71 IRES activity

doi: 10.7150/thno.93883

Figure Lengend Snippet: Identification of miR-206 upregulation in the brainstem during EV71 infection and its modulation by glucose level. (A) miRNA microarray analysis of two neuroblastoma cell lines infected with EV71. SH-SY5Y and IMR-32 cells were infected with EV71 (MOI = 3) and assessed at 6 h post-infection. Differentially expressed miRNAs were identified through comparative analysis of miRNA expression levels between infected and uninfected cells. (B) qRT-PCR analysis of miR-206 expression levels in the brainstem of EV71-infected hSCARB2-Tg mice. Mice (n = 5) were infected with EV71 or left uninfected (Ctrl), and qPCR was performed on isolated total RNA at the indicated dpi. Statistical significance was assessed using a two-sided T-test, with a significance threshold set at p < 0.05. (C) miR-206 in situ hybridization (ISH) in the brainstem of EV71-infected hSCARB2-Tg mice. The miR-206 probe was utilized to visualize miR-206 expression, while VP-1 antibody labeled EV71-infected cells within brainstem tissue. Scale bar represents 5 μm. (D) qRT-PCR assessment of miR-206 expression in the brainstem of EV71-infected hSCARB2-Tg mice under insulin or STZ treatment. qRT-PCR analysis employed primers specific to miR-206 and normalized to GAPDH. Values are presented as mean ± SEM. (E) qRT-PCR evaluation of miR-206 expression in EV71-infected IMR32 and SH-SY5Y cells and assayed at the indicated hours post-infection (hpi). (F) qRT-PCR measurement of miR-206 expression in EV71-infected cells treated with increasing levels of glucose. SH-SY5Y cells were infected with or without EV71 (MOI = 3) and miR-206 levels were measured at 6 hpi in culture media containing 6, 12, 18, or 24 mM of glucose, with mannitol used for adjustments. (G) Luciferase reporter activity of the miR-206 promoter in EV71-infected cells treated with increasing glucose levels. Luciferase reporter activity of the miR-206 promoter (-995 to +113) in EV71-infected cells treated with increasing levels of glucose. SH-SY5Y cells were transfected with a luciferase reporter carrying the miR-206 promoter region. The cells were then infected with or without EV71 (MOI = 3) and exposed to media containing escalating glucose concentrations (5, 12, or 24 mM), with mannitol adjustments. The Promega Luciferase Reporter Assay Kit was employed to conduct the luciferase reporter assay, evaluating miR-206 promoter activity at 6 hpi.

Article Snippet: LNA-modified microRNA miR-206 antisense oligonucleotide probes were labeled with Biotin (MDBio Inc.) and the signal was amplified using Tyramide amplification solution (Thermo Fisher Scientific).

Techniques: Infection, Microarray, Expressing, Quantitative RT-PCR, Isolation, In Situ Hybridization, Labeling, Luciferase, Activity Assay, Transfection, Reporter Assay

Journal: Theranostics

Article Title: Hyperglycemia facilitates EV71 replication: Insights into miR-206-mediated regulation of G3BP2 promoting EV71 IRES activity

doi: 10.7150/thno.93883

Figure Lengend Snippet: miR-206 exhibits significant impacts on EV71 neurovirulence and insulin secretion in hSCARB2-Tg mice. (A) qRT-PCR analysis of viral load in the brainstem of EV71-infected hSCARB2-Tg mice, with or without antagomiR-206 (AM206) treatment (n = 5). Statistical significance was determined using a two-sided T-test, with a significance threshold set at p < 0.05. (B) Survival assessment of EV71-infected hSCARB2-Tg mice, with or without AM206 treatment. Seven-day-old hSCARB2-Tg mice infected with EV71 were administered LNA miR-206 antagomir (AM206, 1.2 mg/kg, n = 15) or (Ctrl, n = 14). The log-rank test was utilized to determine the statistical significance in survival rates between the treatment groups. (C) Representative micrographs depicting brainstem gliosis in EV71-infected hSCARB2-Tg mice, with or without AM206 treatment. Scale bar represents 20 μm. (D) Brainstem expression of proinflammatory cytokines in EV71-infected hSCARB2-Tg mice, with or without AM206 treatment. RNA was extracted from the brainstem tissue of mice at 5 dpi and subjected to qRT-PCR analysis using TNFα-specific, and IL-6-specific primers, normalized to GAPDH (n = 5). (E) Proposed role of miR-206 in targeting GCK-mediated insulin-glucose homeostasis. (F) Representative immunohistochemistry (IF) images displaying GCK levels in the pancreas of EV71-infected hSCARB2-Tg mice, with or without AM206 treatment. Paraffin-embedded sections were stained using the VP1/2 antibody to detect EV71, while islet staining was performed using an insulin antibody. Scale bar denotes 20 μm. (G) qRT-PCR quantification of GCK levels in the pancreas of EV71-infected hSCARB2-Tg mice, with or without AM206 treatment (n = 5). (H) Plasma insulin levels in EV71-infected hSCARB2-Tg mice, with or without AM206 treatment. Insulin levels were measured using the mouse Insulin ELISA assay (n = 5). (I) Plasma glucose levels in EV71-infected mice, with or without AM206 treatment. Glucose levels were measured using a blood glucose assay (n = 5).

Article Snippet: LNA-modified microRNA miR-206 antisense oligonucleotide probes were labeled with Biotin (MDBio Inc.) and the signal was amplified using Tyramide amplification solution (Thermo Fisher Scientific).

Techniques: Quantitative RT-PCR, Infection, Expressing, Immunohistochemistry, Staining, Enzyme-linked Immunosorbent Assay, Glucose Assay

Journal: Theranostics

Article Title: Hyperglycemia facilitates EV71 replication: Insights into miR-206-mediated regulation of G3BP2 promoting EV71 IRES activity

doi: 10.7150/thno.93883

Figure Lengend Snippet: miR-206 boosting viral replication through G3BP2-mediated activation of virus IRES activity. (A) Work flow of a threefold approach for identifying potential miR-206 target genes. In silico prediction identified 833 potential candidates using TargetScanVert. GO enrichment analysis identified 20 RNA binding genes among these candidates. Ultimately, microarray analysis of differentially expressed genes in EV71-infected SH-SY5Y cells highlighted G3BP2 as the sole significantly regulated target gene within this RNA-binding gene set. Lower panel depicting sequence alignment between the mature miR-206's seed region and its potential target gene G3BP2, conserved in mouse and human. (B) qRT-PCR quantification of G3BP2 expression levels in the brainstem of hSCARB2-Tg with or without EV71 infection. RNA was extracted from the brainstem tissue of mice at 3 or 5 dpi and subjected to qRT-PCR analysis using G3BP2-specific primers, normalized to GAPDH (n = 6). (C) qRT-PCR measurement of G3BP2 levels in the brainstem of EV71-infected hSCARB2-Tg mice, with or without AM206 treatment (n = 5). (D) Western blot analysis of G3BP2 in the brainstem of EV71-infected hSCARB2-Tg mice, with or without AM206 treatment. Brainstem lysates were subjected to western blotting using G3BP2 and GAPDH antibodies. (E) Hypothetical model illustrating miR-206 targeting virus IRES activity through G3BP2. (F) Assessment of miR-206's impact on EV71-IRES activity using dual luciferase assay. SH-SY5Y cells were transfected with a luciferase vector containing EV71 IRES, with or without AM206 treatment or miR-206 overexpression. (G) Influence of G3BP2 on EV71-IRES Activity. SH-SY5Y cells were transfected with luciferase vector containing EV71 IRES, along with G3BP2 overexpression or the G3BP2 inhibitor C108. (H) Dual effects of miR-206 and G3BP3 on virus load. SH-SY5Y cells were treated with miR-206 antagomer AM206, or AM206 in combination with the G3BP2 inhibitor C108, followed by EV71 infection. Virus titers were quantified using qRT-PCR. (I) Brainstem viral loads in EV71-infected hSCARB2-Tg mice, with or without C108 treatment. Seven-day-old hSCARB2-Tg mice were subcutaneously injected with the EV71 5746 (C2) strain, followed by C108 (30 mg/kg) injection on day 2, and were subsequent sacrificed on 5 dpi (n = 4). (J) qRT-PCR measurement of TNFα and IL6 levels in the brainstem of hSCARB2-Tg mice, with or without C108 treatment (n = 5). (K) Survival analysis of EV71-infected hSCARB2-Tg mice, with or without C108 treatment. Seven-day-old hSCARB2-Tg mice infected with EV71 on day 0 were administered C108 (n = 9) or control treatment (n = 9) on day 1. Statistical significance was assessed using the two-sided log-rank test, with p < 0.05 indicating statistical significance.

Article Snippet: LNA-modified microRNA miR-206 antisense oligonucleotide probes were labeled with Biotin (MDBio Inc.) and the signal was amplified using Tyramide amplification solution (Thermo Fisher Scientific).

Techniques: Activation Assay, Virus, Activity Assay, In Silico, RNA Binding Assay, Microarray, Infection, Sequencing, Quantitative RT-PCR, Expressing, Western Blot, Luciferase, Transfection, Plasmid Preparation, Over Expression, Injection