Journal: American Journal of Cancer Research

Article Title: KLK11 suppresses cellular proliferation via inhibition of Wnt/β-catenin signaling pathway in esophageal squamous cell carcinoma

doi:

Figure Lengend Snippet: The expression of KLK11 is significantly lower in ESCC tissues. A and B. Indicated detection of KLK11 expression in dataset TCGA. C and D. Indicated KLK11 protein expression in 8 pairs of ESCC tissues and their para-carcinoma tissues by western blotting. KLK11 expression in ESCC tissues were significantly lower than those in the matched adjacent non-tumor tissues. E. KLK11 protein level was measured by immunohistochemical analysis in normal tissues. F. KLK11 protein level was measured by immunohistochemical analysis in ESCC tissues. Representative images are showed at 100 × and 200 × magnifications, respectively. G. KLK11 protein level in 100 ESCC tissues and matched normal tissues. Data were representative of three independent experiments (mean and SEM). *P < 0.05.

Article Snippet: Lentivirus vectors and transfection Three pairs of short hairpin RNA (shRNA) targeting KLK11 lentivirus vectors (shKLK11#1) and negative controls (scramble, NC) were specifically synthesized by Synbio Technologies (Suzhou, China).

Techniques: Expressing, Western Blot, Immunohistochemical staining

Journal: American Journal of Cancer Research

Article Title: KLK11 suppresses cellular proliferation via inhibition of Wnt/β-catenin signaling pathway in esophageal squamous cell carcinoma

doi:

Figure Lengend Snippet: KLK11 expression was significantly associated with clinical characteristics

Article Snippet: Lentivirus vectors and transfection Three pairs of short hairpin RNA (shRNA) targeting KLK11 lentivirus vectors (shKLK11#1) and negative controls (scramble, NC) were specifically synthesized by Synbio Technologies (Suzhou, China).

Techniques: Expressing

Journal: American Journal of Cancer Research

Article Title: KLK11 suppresses cellular proliferation via inhibition of Wnt/β-catenin signaling pathway in esophageal squamous cell carcinoma

doi:

Figure Lengend Snippet: Summary of univariate and multivariate Cox regression analyses of overall survival

Article Snippet: Lentivirus vectors and transfection Three pairs of short hairpin RNA (shRNA) targeting KLK11 lentivirus vectors (shKLK11#1) and negative controls (scramble, NC) were specifically synthesized by Synbio Technologies (Suzhou, China).

Techniques: Expressing

Journal: American Journal of Cancer Research

Article Title: KLK11 suppresses cellular proliferation via inhibition of Wnt/β-catenin signaling pathway in esophageal squamous cell carcinoma

doi:

Figure Lengend Snippet: Low KLK11 expression was associated with poor prognosis in ESCC patients. ESCC samples from the tissue microarray were divided as two different groups, high KLK11 expression and low KLK11 expression group. Patients’ survival information was provided by the manufacture. Kaplan-Meier analysis of overall survival was done for the two group.

Article Snippet: Lentivirus vectors and transfection Three pairs of short hairpin RNA (shRNA) targeting KLK11 lentivirus vectors (shKLK11#1) and negative controls (scramble, NC) were specifically synthesized by Synbio Technologies (Suzhou, China).

Techniques: Expressing, Microarray

Journal: American Journal of Cancer Research

Article Title: KLK11 suppresses cellular proliferation via inhibition of Wnt/β-catenin signaling pathway in esophageal squamous cell carcinoma

doi:

Figure Lengend Snippet: The expression of KLK11 is significantly lower in ESCC cell lines. QPCR (A) and western blotting (B) showed the expression of KLK11 in seven ESCC cell lines and HEEC. KLK11 knockdown efficiency was confirmed by qPCR (C) and western blotting (D) in ESCC cells. qPCR (E) and western blotting (F) confirmed the restoration of KLK11 expression in four groups of TE-1 cells. qPCR (G) and western blotting (H) confirmed the restoration of KLK11 expression in four groups of EC18 cells. Results were showed as mean ± SD (*P < 0.05) of triplicate determination from three independent experiments.

Article Snippet: Lentivirus vectors and transfection Three pairs of short hairpin RNA (shRNA) targeting KLK11 lentivirus vectors (shKLK11#1) and negative controls (scramble, NC) were specifically synthesized by Synbio Technologies (Suzhou, China).

Techniques: Expressing, Western Blot, Knockdown

Journal: American Journal of Cancer Research

Article Title: KLK11 suppresses cellular proliferation via inhibition of Wnt/β-catenin signaling pathway in esophageal squamous cell carcinoma

doi:

Figure Lengend Snippet: KLK11 inhibited the proliferation of ESCC cells. A. KLK11 inhibited the proliferation of TE-1 cells by MTT assays. B. KLK11 inhibited the proliferation of EC18 cells by MTT assays. C. Colony formation assays showed that KLK11 overexpression inhibited TE-1 and EC18 cells proliferation. These effects were abolished by treatment with shKLK11. D. Flow cytometry analysis of cell cycle phase revealed a G0/G1 arrest induced by KLK11. Data were representative of three independent experiments (mean and SEM). *P < 0.05.

Article Snippet: Lentivirus vectors and transfection Three pairs of short hairpin RNA (shRNA) targeting KLK11 lentivirus vectors (shKLK11#1) and negative controls (scramble, NC) were specifically synthesized by Synbio Technologies (Suzhou, China).

Techniques: Over Expression, Flow Cytometry

Journal: American Journal of Cancer Research

Article Title: KLK11 suppresses cellular proliferation via inhibition of Wnt/β-catenin signaling pathway in esophageal squamous cell carcinoma

doi:

Figure Lengend Snippet: KLK11 affected the expression of the related factors in Wnt/β-catenin pathway and cell cycle-mediated factors. A. The expression of p-GSK-3β/GSK-3β in EC18 and TE-1 cells after lentivirus vectors delivery in each group as measured by western blot. B. The TCF/LEF luciferase ratio reported the activity of Wnt/β-catenin pathway in the indicated cells. C. Expression of CDK4, cyclin D1, CDK6, p-Rb, Rb, β-catenin (cytosol), and β-catenin (nucleus) were determined in TE-1 and EC18 cells transfected with KLK11. KLK11 inhibited the proliferation of ESCC by regulating the expression of the above proteins. D. Analysis of Ki67, cyclin D1, CDK4, CDK6, and c-myc expression in KLK11-overexpression or KLK11-knockdown ESCC cells by qPCR. Data were representative of three independent experiments (mean and SEM). *P < 0.05.

Article Snippet: Lentivirus vectors and transfection Three pairs of short hairpin RNA (shRNA) targeting KLK11 lentivirus vectors (shKLK11#1) and negative controls (scramble, NC) were specifically synthesized by Synbio Technologies (Suzhou, China).

Techniques: Expressing, Western Blot, Luciferase, Activity Assay, Transfection, Over Expression, Knockdown

Journal: American Journal of Cancer Research

Article Title: KLK11 suppresses cellular proliferation via inhibition of Wnt/β-catenin signaling pathway in esophageal squamous cell carcinoma

doi:

Figure Lengend Snippet: KLK11 down regulated the expression of Ki67 and cyclin D1 through deactivation of the Wnt/β-catenin signaling pathway. A, B. ESCC cells were pre-treated with XAV-939 for 24 h before harvesting, and the detection of Ki67, cyclin D1, c-myc, and expression levels were performed by qPCR. C, D. Treatment of EC18 and TE-1 cells with XAV-939, the expression of Ki67, cyclin D1, β-catenin (cytosol), and β-catenin (nucleus) were significantly inhibited. Data were representative of three independent experiments (mean and SEM). *P < 0.05.

Article Snippet: Lentivirus vectors and transfection Three pairs of short hairpin RNA (shRNA) targeting KLK11 lentivirus vectors (shKLK11#1) and negative controls (scramble, NC) were specifically synthesized by Synbio Technologies (Suzhou, China).

Techniques: Expressing

Journal: American Journal of Cancer Research

Article Title: KLK11 suppresses cellular proliferation via inhibition of Wnt/β-catenin signaling pathway in esophageal squamous cell carcinoma

doi:

Figure Lengend Snippet: KLK11 suppressed ESCC cells proliferation through inhibiting the activity of the Wnt/β-catenin pathway. A, B. XAV-939 was added to TE-1 and EC18 cells transfected with shRNA, and the proliferation of TE-1 and EC18 cells was detected by MTT assays. C. XAV-939 was added to TE-1 and EC18 cells transfected with shRNA, and the proliferation of TE-1 and EC18 cells was detected by colony formation assays. D. The relationship between KLK11 and the Wnt/β-catenin pathway. Data were representative of three independent experiments (mean and SEM). *P < 0.05.

Article Snippet: Lentivirus vectors and transfection Three pairs of short hairpin RNA (shRNA) targeting KLK11 lentivirus vectors (shKLK11#1) and negative controls (scramble, NC) were specifically synthesized by Synbio Technologies (Suzhou, China).

Techniques: Activity Assay, Transfection, shRNA

Journal: American Journal of Cancer Research

Article Title: KLK11 suppresses cellular proliferation via inhibition of Wnt/β-catenin signaling pathway in esophageal squamous cell carcinoma

doi:

Figure Lengend Snippet: KLK11 inhibited the proliferation of ESCC in vivo. (A, B) Tumor xenograft volume (A) and tumor size (B) in shRNA-KLK11-treated nude mice were bigger than those in the scramble group. Tumor sizes in KLK11 overexpression nude mice were smaller than those in the vector group. (C) Analysis of Ki67, cyclin D1, and KLK11 expression in mice tumor tissues with KLK11 overexpression or KLK11 knockdown by qPCR. (D) Analysis of KLK11 and Cyclin D1 in xenografts by Immunohistochemistry. Data were representative of three independent experiments (mean and SEM). *P < 0.05.

Article Snippet: Lentivirus vectors and transfection Three pairs of short hairpin RNA (shRNA) targeting KLK11 lentivirus vectors (shKLK11#1) and negative controls (scramble, NC) were specifically synthesized by Synbio Technologies (Suzhou, China).

Techniques: In Vivo, shRNA, Over Expression, Plasmid Preparation, Expressing, Knockdown, Immunohistochemistry

Journal: Cell Death & Disease

Article Title: Hsa_circ_0021727 (circ-CD44) promotes ESCC progression by targeting miR-23b-5p to activate the TAB1/NFκB pathway

doi: 10.1038/s41419-022-05541-x

Figure Lengend Snippet: A Heat maps of differentially expressed circRNAs in three ESCC tissues (T) and adjacent normal tissues (P). B Volcano plots showing changes in circRNA expression between cancerous and paracancerous tissues. C Schematic and Sanger sequencing of hsa_circ_0021727. D Agarose gel electrophoresis detection of hsa_circ_0021727 in KYSE510 cells. E The relative RNA levels were examined by qRT-PCR after treatment with RNase R or mock among total RNAs derived from KYSE 510 cells. F Random hexamer or oligo (dT)18 primers were utilized for reverse transcription assays. The relative RNA levels were examined using qRT-qPCR and normalized to those generated using random hexamer primers. G The subcellular localization of hsa_circ_0021727 in KYSE510 and TE-1 cells performed using FISH. H and I RNA-ISH detection of the expression of hsa_circ_0021727 in ESCC tissues and adjacent tissues. J Kaplan–Meier analysis of the OS in 162 patients with ESCC with high or low expression of hsa_circ_0021727. Values are expressed as the means ± SD; * P < 0.05.

Article Snippet: CircRNA overexpression and short hairpin RNA (shRNA) lentiviral vectors, miRNA lentiviral vectors, and matching negative control vectors were designed and synthesized by Synbio Technologies (Suzhou, China).

Techniques: Expressing, Sequencing, Agarose Gel Electrophoresis, Quantitative RT-PCR, Derivative Assay, Random Hexamer, Reverse Transcription, Generated

Journal: Cell Death & Disease

Article Title: Hsa_circ_0021727 (circ-CD44) promotes ESCC progression by targeting miR-23b-5p to activate the TAB1/NFκB pathway

doi: 10.1038/s41419-022-05541-x

Figure Lengend Snippet: A and C qRT-PCR detection of the expression efficiency of hsa_circ_0021727 after cell transfection. B and D Wound healing assays for the migration ability of KYSE510 and TE-1 cells after cell transfection. E Transwell assays examining the invasive ability of KYSE510 and TE-1 cells after transfection of the hsa_circ_0021727 overexpression or knockdown vectors. F Three-dimensional (3D) spheroid invasion assays showing the effects of hsa_circ_0021727 on the growing antennae of KYSE510 and TE-1 cells. G and H MTT assays detecting the proliferation of KYSE510 and TE-1 cells after transfection of hsa_circ_0021727 overexpression or knockdown vectors. I Colony formation assays examining the proliferative capacity of KYSE510 and TE-1 cells transfected with hsa_circ_0021727 overexpression or knockdown lentiviral vectors. J EdU immunofluorescence assays detecting the proliferative capacity of KYSE510 and TE-1 cells. Values are expressed as the means ± SD; * P < 0.05.

Article Snippet: CircRNA overexpression and short hairpin RNA (shRNA) lentiviral vectors, miRNA lentiviral vectors, and matching negative control vectors were designed and synthesized by Synbio Technologies (Suzhou, China).

Techniques: Quantitative RT-PCR, Expressing, Transfection, Migration, Over Expression, Knockdown, Immunofluorescence

Journal: Cell Death & Disease

Article Title: Hsa_circ_0021727 (circ-CD44) promotes ESCC progression by targeting miR-23b-5p to activate the TAB1/NFκB pathway

doi: 10.1038/s41419-022-05541-x

Figure Lengend Snippet: A Image suggesting that hsa_circ_0021727 might target bound miRNAs. B Probe efficiency for qRT-PCR detection. C and D Relative levels of five miRNAs in KYSE510 and TE-1 cells lysates pulled down by the hsa_circ_0021727 probe or the oligo probe. E and F qRT-PCR detection of the levels of five miRNAs in KYSE510 and TE-1 cells transfected with hsa_circ_0021727 overexpression or knockdown lentiviral vectors. G A luciferase reporter gene assay performed in KYSE510 and TE-1 cells co-transfected with hsa_circ_0021727 overexpression vector, pGL3 plasmid, pGL3-miR-23b-5p-wt, and pGL3-miR-23b-5p-mut. H FISH analysis showing the colocalization between hsa_circ_0021727 and miR-23b-5p in KYSE510 and TE-1 cells. Values are expressed as the means ± SD; * P < 0.05.

Article Snippet: CircRNA overexpression and short hairpin RNA (shRNA) lentiviral vectors, miRNA lentiviral vectors, and matching negative control vectors were designed and synthesized by Synbio Technologies (Suzhou, China).

Techniques: Quantitative RT-PCR, Transfection, Over Expression, Knockdown, Luciferase, Reporter Gene Assay, Plasmid Preparation

Journal: Cell Death & Disease

Article Title: Hsa_circ_0021727 (circ-CD44) promotes ESCC progression by targeting miR-23b-5p to activate the TAB1/NFκB pathway

doi: 10.1038/s41419-022-05541-x

Figure Lengend Snippet: A and B Wound healing assays evaluating the cell migration ability of KYSE510 cells and TE-1 cells co-transfected with miR-23b-5p and hsa_circ_0021727 overexpression vectors. C Transwell assays showing that miR-23b-5p and hsa_circ_0021727 jointly affected the invasion ability of KYSE510 and TE-1 cells. D Three-dimensional (3D) spheroid invasion assays showing the effects of hsa_circ_0021727 and miR-23b-5p on the growing antennae of KYSE510 and TE-1 cells. E and F MTT assays demonstrating the proliferation capacity of KYSE510 and TE-1 cells co-transfected with miR-23b-5p and hsa_circ_0021727 overexpression vectors. G Colony formation assays indicating that the cell proliferation capability of KYSE510 and TE-1 cells transfected with miR-23b-5p was reversed when the cells were co-transfected with hsa_circ_0021727 (values are expressed as the means ± SD; * P < 0.05).

Article Snippet: CircRNA overexpression and short hairpin RNA (shRNA) lentiviral vectors, miRNA lentiviral vectors, and matching negative control vectors were designed and synthesized by Synbio Technologies (Suzhou, China).

Techniques: Migration, Transfection, Over Expression

Journal: Cell Death & Disease

Article Title: Hsa_circ_0021727 (circ-CD44) promotes ESCC progression by targeting miR-23b-5p to activate the TAB1/NFκB pathway

doi: 10.1038/s41419-022-05541-x

Figure Lengend Snippet: A Image showing the subcutaneous xenograft tumor tissues injected with KYSE510 cells co-transfected with miR-23b-5p and hsa_circ_0021727 overexpression vectors. B Graphs of tumor volumes in subcutaneous xenograft tumor models. C Image of lung metastatic nodules. Nude mice were injected with KYSE510 cells co-transfected with miR-23b-5p and hsa_circ_0021727 overexpression vectors. D Statistical analysis of lung metastatic nodules. E Lung tissue was stained using HE. Values are expressed as the means ± SD; * P < 0.05.

Article Snippet: CircRNA overexpression and short hairpin RNA (shRNA) lentiviral vectors, miRNA lentiviral vectors, and matching negative control vectors were designed and synthesized by Synbio Technologies (Suzhou, China).

Techniques: Injection, Transfection, Over Expression, Staining

Journal: Cell Death & Disease

Article Title: Hsa_circ_0021727 (circ-CD44) promotes ESCC progression by targeting miR-23b-5p to activate the TAB1/NFκB pathway

doi: 10.1038/s41419-022-05541-x

Figure Lengend Snippet: A Heatmap created from mRNA-seq (OE: overexpression, V: vector). B The top 20 enriched pathways in the mRNA data. C GSEA results showing that hsa_circ_0021727 correlated positively with NFκB signaling. D and E qRT-PCR detection of the expression of NFκB signaling-related factors in cells overexpressing or knocked down for hsa_circ_0021727. F Dual-luciferase assay detecting the luciferase activity related to NFκB signaling in cells overexpressing or knocked down for hsa_circ_0021727. G In cells overexpressing or knocked down for hsa_circ_0021727, western blotting was used to detect the protein levels of TAB1 and NFκB pathway factors. H TAB1 mutant and wild-type sequences were designed. I Dual-luciferase assays showing the luciferase activity after co-transfection of miR-23b-5p overexpression vector or NC vector, TAB1 wild-type or TAB1 mutant vector. J qRT-PCR and western blotting analyses of the expression of hsa_circ_0021727, miR-23b-5p, and TAB1 in ESCC patient tissues. K Analysis of the correlation between miR-23b-5p and TAB1. L Analysis of the correlation between hsa_circ_0021727 and TAB1. M In cells overexpressing hsa_circ_0021727 and knocked down for TAB1 , western blotting examined the protein level of TAB1 and downstream related factors. N In cells overexpressing hsa_circ_0021727 and miR-23b-5p, western blotting examined the protein level of TAB1 and downstream related factors. Western blot detection of the protein level of TAB1 and downstream related factors in KYSE510 and TE-1 cells overexpressing hsa_circ_0021727 and treated with INH14. Values are expressed as the means ± SD; * P < 0.05.

Article Snippet: CircRNA overexpression and short hairpin RNA (shRNA) lentiviral vectors, miRNA lentiviral vectors, and matching negative control vectors were designed and synthesized by Synbio Technologies (Suzhou, China).

Techniques: Over Expression, Plasmid Preparation, Quantitative RT-PCR, Expressing, Luciferase, Activity Assay, Western Blot, Mutagenesis, Cotransfection