Journal: Cancers

Article Title: miR766-3p and miR124-3p Dictate Drug Resistance and Clinical Outcome in HNSCC

doi: 10.3390/cancers14215273

Figure Lengend Snippet: The role of miR124-3p and miR766-3p target genes in HNSCC drug resistance. ( A ) Expression analysis of miR124-3p and miR766-3p direct target genes and downstream target genes by Western blot in HNSCC cell lines (CAL27 and FaDu), with or without transfection with miRNA inhibitors or miRNA mimics. Left: Western blotting showed the expression of miR124-3p target gene (CREBRF) and CREBRF target genes (ATG5 and CREB3). Right: Western blotting showed the expression of miR766-3p target gene (NR3C2) and NR3C2 target genes (β-catenin and c-Myc). Quantitative data (relative expression levels after β-actin-corrected) from three independent experiments are disclosed below each protein band. Data represent the mean ± SD (n = 3). ( B ) Target gene analysis in sensitive (CAL27) vs. resistant (CAL27/FP-R) HNSCC cell lines. Quantitative data (relative expression levels after β-actin-corrected) is shown below each protein band. ( C ) The effect of NR3C2 and/or CREBRF knockdown on drug-induced cytotoxicity in CAL27 and FaDu. Cells were transfected by 10 nM siRNA (single or combined) for 24 h followed by 72 h exposure to the indicated drug. Cytotoxicity was determined by MTT assay. IC 50 values are listed in . ( D ) Measurement of apoptosis in CAL27 or FaDu cells in response to cisplatin or 5-FU ± 24 h prior transfection with 10 nM siRNA. After 24 h of drug treatments, cells were labeled with anti-annexin V-FITC antibody and PI and then analyzed with flow cytometry. A two-way ANOVA with Bonferroni’s correction for multiple comparisons was used to analyze group comparisons, and data are presented as means ± SD (n = 3). * p < 0.05, *** p < 0.001 (vs. control siRNA), and ### p < 0.001 (vs. untreated).

Article Snippet: The polyvinylidene difluoride membranes were incubated with specific antibodies, including CREBRF rabbit monoclonal antibody (mAb) (ThermoFisher, PA5-68552; 1:1000), CREB3 rabbit mAb (Proteintech, 11275-1-AP; 1:1000), NR3C2 mouse mAb (Novus, NB300-562; 1:1000), c-Myc rabbit mAb (Novus, NB600-336; 1:1000), ATG5 rabbit mAb (Novus, NB110-53818SS; 1:1000), β-catenin mouse mAb (Novus, NBP1-54467SS; 1:1000), and β-actin mouse mAb (Sigma-Aldrich, A3854; 1:1000).

Techniques: Expressing, Western Blot, Transfection, Knockdown, MTT Assay, Labeling, Flow Cytometry, Control

Journal: Cancers

Article Title: miR766-3p and miR124-3p Dictate Drug Resistance and Clinical Outcome in HNSCC

doi: 10.3390/cancers14215273

Figure Lengend Snippet: Downregulation of CREBRF and NR3C2 increase poor prognosis in HNSCC. ( A ) Histological analysis of miR124-3p and miR766-3p target gene expression (CREBRF-ATG5/CREB3, NR3C2-β-catenin/c-Myc) in Responder vs. Non-Responder HNSCC clinical samples. Magnification, x100. Scale bar, 210 µm. The quantitative data from all specimens are shown in the bar chart. Each dot in the graph represents an individual clinical sample. Two-sided unpaired Student t test was used to analyze comparisons, and data are presented as means ± SEM. * p < 0.05 and *** p < 0.001. ( B ) Histological analysis of tumor morphology in relation to miR124-3p and miR766-3p target gene expression. Representative images of CREBRF, ATG5, CREB3, NR3C2, β-catenin, and c-Myc expression in the serial section of responder and non-responder HNSCC specimens. BV: Blood Vessel. T: Tumor. N: Normal tissue. The invasive cancer cells are indicated by red arrowhead. Magnification, ×200. Scale bar, 100 µm. ( C ) Summary of resistance mechanisms regulated by miR124-3p and miR766-3p. Our data indicated that upon acquired resistance in HNSCC cells or in non-responder HNSCC tumors, the levels of miR124-3p and miR766-3p go up, which in turn down-regulate its direct target genes: CREBRF and NR3C2, and consequently the expression of downstream targets of CREBRF (ATG5/CREB3) and NR3C2 (β-catenin/c-Myc) increased in resistant tumors, which are positively correlated with poor prognosis. Thus, by enhancing the CREBRF-ATG5/CREB3 and NR3C2-β-catenin/c-Myc axis, miR124-3p and miR766-3p support aggressive HNSCC progression.

Article Snippet: The polyvinylidene difluoride membranes were incubated with specific antibodies, including CREBRF rabbit monoclonal antibody (mAb) (ThermoFisher, PA5-68552; 1:1000), CREB3 rabbit mAb (Proteintech, 11275-1-AP; 1:1000), NR3C2 mouse mAb (Novus, NB300-562; 1:1000), c-Myc rabbit mAb (Novus, NB600-336; 1:1000), ATG5 rabbit mAb (Novus, NB110-53818SS; 1:1000), β-catenin mouse mAb (Novus, NBP1-54467SS; 1:1000), and β-actin mouse mAb (Sigma-Aldrich, A3854; 1:1000).

Techniques: Targeted Gene Expression, Expressing

Journal: iScience

Article Title: CREB3L4 promotes hepatocellular carcinoma progression and decreases sorafenib chemosensitivity by promoting RHEB-mTORC1 signaling pathway

doi: 10.1016/j.isci.2024.108843

Figure Lengend Snippet:

Article Snippet: CREB3L4 , Proteintech , Cat No. 13630-1-AP; RRID: AB_2276550.

Techniques: Virus, Recombinant, CCK-8 Assay, Chromatin Immunoprecipitation, Reporter Assay, Expressing, Sequencing, Plasmid Preparation, Software