Journal: Oncology reports

Article Title: Erb‑B2 Receptor Tyrosine Kinase 2 is negatively regulated by the p53‑responsive microRNA‑3184‑5p in cervical cancer cells.

doi: 10.3892/or.2020.7862

Figure Lengend Snippet: Figure 1. ERBB2 expression is upregulated in patient‑derived cervical cancer tissues and is associated with a poor prognosis. (A) RT‑qPCR and (B) WB analysis of ERBB2 transcript and protein expression, respectively, in patient‑derived cervical cancer tissues (n=65) vs. matched healthy cervical tissues (n=65). Data were analyzed via Wilcoxon signed‑rank test. (C) RT‑qPCR and (D) WB analysis of ERBB2 transcript and protein expression, respectively, in stage I/II vs. stage III/IV patient‑derived cervical cancer tissues (n=43 stage I/II; n=22 Stage III/IV). Data were analyzed via Mann‑Whitney U test. (E) RT‑qPCR and (F) WB analysis of ERBB2 transcript and protein expression, respectively, in lymph node metastatic and non‑metastatic patient‑derived cervical cancer biopsies [n=46 lymph node (‑); n=19 lymph node (+)]. Data were analyzed via Mann‑Whitney U test. (G) Survival analysis using the Kaplan‑Meier method according to high (above the median) or low (below the median) ERBB2 mRNA expression (n=32 in each cohort). The P‑value was calculated using the log‑rank test. For purposes of comparison across cohorts, the median ERBB2 mRNA and protein expression levels (normalized to the RT‑qPCR housekeeping control and WB loading control GAPDH) in the normal cohort have been set to 1.0. Data in box plots are expressed as the median ± IQRs (boxes) and absolute ranges (whiskers). n=3. **P<0.01. RT‑qPCR, reverse transcription‑quantitative PCR; WB, western blotting; ERBB2, Erb‑B2 Receptor Tyrosine Kinase 2; Pt, patient.

Article Snippet: A pMirTarget firefly luciferase reporter plasmid (cat. no. PS100062) containing the wild-type (WT) 3'-UTR of human ERBB2 (ERBB2-3'-UTRWT; cat. no. SC208188) was obtained from OriGene Technologies, Inc. Mutations were introduced using a QuikChangeTM Site-Directed Mutagenesis kit (Agilent Technologies, Inc.) into the putative miR-3184-5p binding site on ERBB2-3'-UTRWT to create the mutant (MU) ERBB2-3'-UTRMU.

Techniques: Expressing, Mann-Whitney U-Test, Comparison, Control, Western Blot

Journal: Oncology reports

Article Title: Erb‑B2 Receptor Tyrosine Kinase 2 is negatively regulated by the p53‑responsive microRNA‑3184‑5p in cervical cancer cells.

doi: 10.3892/or.2020.7862

Figure Lengend Snippet: Figure 2. ERBB2 overexpression in cervical cancer cell lines stimulates viability, invasion and sphere‑formation. (A) Confirmation of ERBB2 KD in siERBB2 cells and OE in ERBB2 vec cells via WB. GAPDH was used as the loading control. (B) Invasion of siERBB2 vs. siCtrl cells via Transwell assay. (C) Invasion of ERBB2 vec vs. Ctrl vec cells via Transwell assay. (D) Cellular viability of siERBB2, siCtrl, ERBB2 vec and Ctrl vec cells quantified using a Cell Counting Kit‑8. (E) Sphere‑formation of siERBB2 vs. siCtrl cells. (F) Sphere‑formation of ERBB2 vec vs. Ctrl vec cells. (G) Analysis of metastasis‑associated and cancer stem cell biomarkers mRNA and protein expression in siERBB2, siCtrl, ERBB2 vec and Ctrl vec cells via RT‑qPCR and WB, respectively. GAPDH was used as the RT‑qPCR housekeeping control and WB loading control. Data are expressed as the mean ± SEM (n=3). **P<0.01 vs. siCtrl or Ctrl vec analyzed via unpaired Student's t‑test. KD, knockdown; OE, overexpression; WB, western blotting; RT‑qPCR, reverse transcription‑quantitative PCR; si, small interfering; Ctrl, control; vec, vector; ERBB2, Erb‑B2 Receptor Tyrosine Kinase 2.

Article Snippet: A pMirTarget firefly luciferase reporter plasmid (cat. no. PS100062) containing the wild-type (WT) 3'-UTR of human ERBB2 (ERBB2-3'-UTRWT; cat. no. SC208188) was obtained from OriGene Technologies, Inc. Mutations were introduced using a QuikChangeTM Site-Directed Mutagenesis kit (Agilent Technologies, Inc.) into the putative miR-3184-5p binding site on ERBB2-3'-UTRWT to create the mutant (MU) ERBB2-3'-UTRMU.

Techniques: Over Expression, Control, Transwell Assay, CCK-8 Assay, Expressing, Knockdown, Western Blot, Plasmid Preparation

Journal: Oncology reports

Article Title: Erb‑B2 Receptor Tyrosine Kinase 2 is negatively regulated by the p53‑responsive microRNA‑3184‑5p in cervical cancer cells.

doi: 10.3892/or.2020.7862

Figure Lengend Snippet: Figure 3. ERBB2 controls cervical cancer cell viability and invasion by regulating PIK3CA protein expression. (A) Schematic diagram of the ERBB2‑ERRB3 complex interacting with PI3K(p85), thereby promoting the downstream phosphorylation of AKT and mTOR. (B) IP in cervical cancer cell lysates with antibodies against ERBB3 or IgG control. Expression levels of ERBB3, ERBB2 and PI3K(p85) in the IP fraction were assessed via WB. PIK3CA mRNA expression in transfected (C) HeLa and (D) SiHa cells assessed via RT‑qPCR. GAPDH was used as the housekeeping control. PIK3CA, p‑AKT/AKT and p‑mTOR/mTOR protein expression in transfected (E) HeLa and (F) SiHa cells assessed via WB. GAPDH was used as the loading control. (G) Invasion of transfected HeLa cells assessed via Transwell assay. (H) Cellular viability of transfected HeLa cells quantified using Cell Counting Kit‑8. (I) Sphere‑formation of transfected HeLa cells. Data are expressed as the mean ± SEM (n=3). *P<0.05 and **P<0.01 vs. siCtrl or Ctrl vec; †P<0.05 and ††P<0.01 vs. siERBB2 or ERBB2 vec. Data were analyzed via one‑way ANOVA. IP, immunoprecipitation; WB, western blotting; RT‑qPCR, reverse transcription‑quantitative PCR; si, small interfering; vec, vector; ERBB2, Erb‑B2 Receptor Tyrosine Kinase 2; p‑, phosphorylated; PIK3CA, phosphatidylinositol‑4,5‑bisphosphate 3‑kinase catalytic subunit α.

Article Snippet: A pMirTarget firefly luciferase reporter plasmid (cat. no. PS100062) containing the wild-type (WT) 3'-UTR of human ERBB2 (ERBB2-3'-UTRWT; cat. no. SC208188) was obtained from OriGene Technologies, Inc. Mutations were introduced using a QuikChangeTM Site-Directed Mutagenesis kit (Agilent Technologies, Inc.) into the putative miR-3184-5p binding site on ERBB2-3'-UTRWT to create the mutant (MU) ERBB2-3'-UTRMU.

Techniques: Expressing, Phospho-proteomics, Control, Transfection, Transwell Assay, CCK-8 Assay, Immunoprecipitation, Western Blot, Plasmid Preparation

Journal: Oncology reports

Article Title: Erb‑B2 Receptor Tyrosine Kinase 2 is negatively regulated by the p53‑responsive microRNA‑3184‑5p in cervical cancer cells.

doi: 10.3892/or.2020.7862

Figure Lengend Snippet: Figure 4. miR‑3184‑5p attenuates cervical cancer cell viability and invasion by targeting ERBB2. (A) Putative binding location for miR‑3184‑5p on ERBB2 3'‑UTR via TargetScan analysis. (B) miR‑3184‑5p expression in HeLa and SiHa cervical cancer cell lines compared with in the non‑cancerous human H8 cervical epithelial cell line assessed via RT‑qPCR. U6 was used as the housekeeping control. **P<0.01 vs. H8; ††P<0.01 vs. SiHa. Luciferase reporter assay of ERBB2‑3'‑UTRWT or ERBB2‑3'‑UTRMU in (C) HeLa or (D) SiHa cells transfected with miR‑3184‑5p mimic or inhibitor, respectively. **P<0.01 vs. Ctrl mimic or Ctrl inhib. WB of (E) HeLa and (F) SiHa cells transfected with miR‑3184‑5p mimic or inhibitor, respectively. (G) Invasion of transfected HeLa cells assessed via Transwell assay. (H) Cellular viability of transfected HeLa cells quantified using Cell Counting Kit‑8. (I) Sphere‑formation of transfected HeLa cells. Data are expressed as the mean ± SEM (n=3). **P<0.01 vs. Ctrl vec; ††P<0.01 vs. miR‑3184‑5p mimic. Data were analyzed via one‑way ANOVA. UTR, untranslated region; WT, wild‑type; MU, mutant; Ctrl, control; inhib, inhibitor; WB, western blotting; RT‑qPCR, reverse transcription‑quantitative PCR; miR, microRNA; vec, vector; ERBB2, Erb‑B2 Receptor Tyrosine Kinase 2; PIK3CA, phosphatidylinositol‑4,5‑bisphosphate 3‑kinase catalytic subunit α.

Article Snippet: A pMirTarget firefly luciferase reporter plasmid (cat. no. PS100062) containing the wild-type (WT) 3'-UTR of human ERBB2 (ERBB2-3'-UTRWT; cat. no. SC208188) was obtained from OriGene Technologies, Inc. Mutations were introduced using a QuikChangeTM Site-Directed Mutagenesis kit (Agilent Technologies, Inc.) into the putative miR-3184-5p binding site on ERBB2-3'-UTRWT to create the mutant (MU) ERBB2-3'-UTRMU.

Techniques: Binding Assay, Expressing, Control, Luciferase, Reporter Assay, Transfection, Inhibition, Transwell Assay, CCK-8 Assay, Mutagenesis, Western Blot, Plasmid Preparation

Journal: Oncology reports

Article Title: Erb‑B2 Receptor Tyrosine Kinase 2 is negatively regulated by the p53‑responsive microRNA‑3184‑5p in cervical cancer cells.

doi: 10.3892/or.2020.7862

Figure Lengend Snippet: Figure 5. p53‑activating Mithramycin A boosts miR‑3184‑5p expression, which lowers ERBB2 expression and attenuates viability and invasion of cervical cancer cell lines. (A) p53, p21 and ERBB2 protein expression in cervical cancer cultures incubated with MM or vehicle (DMSO) assessed via WB. GAPDH was used as the loading control. (B) miR‑3184‑5p expression in cervical cancer cultures incubated with MM or vehicle assessed via RT‑qPCR. U6 was used as the housekeeping control. (C) p53 and ERBB2 protein expression in cervical cancer cultures transfected with a p53 overexpression plasmid or empty plasmid control assessed via WB. GAPDH was used as the loading control. (D) miR‑3184‑5p expression in cervical cancer cultures transfected with a p53 overexpression plasmid or empty plasmid control assessed via RT‑qPCR. U6 was used as the housekeeping control. (E) Representative images of Transwell and sphere‑formation assays in (E) HeLa and (F) SiHa cells, and quantitative analysis of viability, invasion and sphere‑formation of cells treated with MM or vehicle. (G) Schematic diagram of the p53 activator MM rescuing miR‑3184‑5p expression, thereby suppressing ERBB2 transcription. This attenuates PIK3CA activity, which stimulates cervical cancer cell viability, invasion and sphere‑formation. Data are expressed as the mean ± SEM (n=3). **P<0.01 analyzed via unpaired Student's t‑test. MM, Mithramycin A; Ctrl, control; WB, western blotting; RT‑qPCR, reverse transcription‑quantitative PCR; miR, microRNA; vec, vector; ERBB2, Erb‑B2 Receptor Tyrosine Kinase 2; p‑, phosphorylated; PIK3CA, phosphatidylinositol‑4,5‑bisphosphate 3‑kinase catalytic subunit α.

Article Snippet: A pMirTarget firefly luciferase reporter plasmid (cat. no. PS100062) containing the wild-type (WT) 3'-UTR of human ERBB2 (ERBB2-3'-UTRWT; cat. no. SC208188) was obtained from OriGene Technologies, Inc. Mutations were introduced using a QuikChangeTM Site-Directed Mutagenesis kit (Agilent Technologies, Inc.) into the putative miR-3184-5p binding site on ERBB2-3'-UTRWT to create the mutant (MU) ERBB2-3'-UTRMU.

Techniques: Expressing, Incubation, Control, Transfection, Over Expression, Plasmid Preparation, Activity Assay, Western Blot

Journal: Biosensors

Article Title: Dual Biomarker Strategies for Liquid Biopsy: Integrating Circulating Tumor Cells and Circulating Tumor DNA for Enhanced Tumor Monitoring

doi: 10.3390/bios15020074

Figure Lengend Snippet: Combined analysis of circulating tumor cells and circulating tumor DNA for the diagnosis and molecular profiling of gastrointestinal cancer.

Article Snippet: UC , 16 UC patients (6 blood samples from UTUC and 10 urine samples from bladder cancer patients) , Cynvenio LiquidBiopsy Blood Collection Kit (Targeting EpCAM, HER2, EGFR, and Trop2) , IF staining (CK+, CD45−, DAPI+) NGS using Ion AmpliSeq Cancer Hotspot Panel v2 (50 cancer-related genes) , MagMAX Cell-Free DNA Isolation Kit , NGS using Ion AmpliSeq Cancer Hotspot Panel v2 (50 cancer-related genes) , Combined NGS analysis of CTCs and cfDNA identified actionable mutations, with cfDNA revealing additional mutations not found in CTCs , [ ] .

Techniques: Biomarker Discovery, Isolation, Extraction, Selection, Single-cell Isolation, Staining, Mutagenesis, Synchronous Coefficient of Drag Alteration, Sequencing, Filtration, RNA In Situ Hybridization, Clinical Proteomics, Fluorescence

Journal: Biosensors

Article Title: Dual Biomarker Strategies for Liquid Biopsy: Integrating Circulating Tumor Cells and Circulating Tumor DNA for Enhanced Tumor Monitoring

doi: 10.3390/bios15020074

Figure Lengend Snippet: Combined analysis of circulating tumor cells and circulating tumor DNA for the diagnosis and molecular profiling of melanoma, urothelial cancer, and pancreatic cancer.

Article Snippet: UC , 16 UC patients (6 blood samples from UTUC and 10 urine samples from bladder cancer patients) , Cynvenio LiquidBiopsy Blood Collection Kit (Targeting EpCAM, HER2, EGFR, and Trop2) , IF staining (CK+, CD45−, DAPI+) NGS using Ion AmpliSeq Cancer Hotspot Panel v2 (50 cancer-related genes) , MagMAX Cell-Free DNA Isolation Kit , NGS using Ion AmpliSeq Cancer Hotspot Panel v2 (50 cancer-related genes) , Combined NGS analysis of CTCs and cfDNA identified actionable mutations, with cfDNA revealing additional mutations not found in CTCs , [ ] .

Techniques: Biomarker Discovery, Isolation, Extraction, Selection, Staining, DNA Extraction