Journal: Cells

Article Title: Proteoglycan SPOCK1 as a Poor Prognostic Marker Promotes Malignant Progression of Clear Cell Renal Cell Carcinoma via Triggering the Snail/Slug-MMP-2 Axis-Mediated Epithelial-to-Mesenchymal Transition

doi: 10.3390/cells12030352

Figure Lengend Snippet: Clinical significance of SPOCK1 in clear cell renal cell carcinoma (RCC; ccRCC). ( A ) SPOCK1 gene expression levels in RCC specimens ( n = 69), ccRCC specimens ( n = 32), and normal tissue samples ( n = 23) were measured by Affymetrix oligonucleotide arrays obtained from the GEO (GSE15641). ( B ) SPOCK1 gene expression levels in ccRCC samples from TCGA were compared according to the clinical stage, tumor size (T stage), lymph node metastasis (N stage), and distal metastasis (M stage). Statistical significance was analyzed by a t -test. * p < 0.05, *** p < 0.001. ( C ) Kaplan–Meier curves for survival of patients with ccRCC or papillary (p)RCC, as categorized according to high or low expression of SPOCK1. The p value indicates a comparison between patients with SPOCK1 high and SPOCK1 low . The ccRCC and pRCC datasets were retrieved from TCGA. ( D ) SPOCK1 protein expression levels in ccRCC specimens and adjacent normal tissue samples or benign tumor (oxyphilic adenoma) samples were measured by IHC staining. The right panels are the enlarged images of left panels. Scale bars of left panel and right panel are 200 and 100 µM, respectively.

Article Snippet: Human RCC cell lines (ccRCC: Caki-1 and 786-O; pRCC: ACHN and Caki-2) with wild-type (WT) or mutant Von Hippel-Lindau (VHL) were purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Gene Expression, Expressing, Comparison, Immunohistochemistry

Journal: Cells

Article Title: Proteoglycan SPOCK1 as a Poor Prognostic Marker Promotes Malignant Progression of Clear Cell Renal Cell Carcinoma via Triggering the Snail/Slug-MMP-2 Axis-Mediated Epithelial-to-Mesenchymal Transition

doi: 10.3390/cells12030352

Figure Lengend Snippet: SPOCK1 overexpression promotes the proliferation, clonogenicity, migration, and invasion of clear cell renal cell carcinoma (ccRCC) cells. ( A ) Endogenous SPOCK1 protein levels were detected using a Western blot analysis of ccRCC (Caki-1 and 786-O) and papillary (p)RCC (ACHN and Caki-2) cells. ( B ) The migratory ability of ccRCC and pRCC cells was examined by a transwell migration assay. ( C ) Knockdown efficiencies of three SPOCK1 shRNAs were determined by Western blotting in Caki-1 and 786-O cells. ( D ) Migratory abilities of SPOCK1-KD Caki-1 and 786-O cells were evaluated by a transwell migration assay. ( E ) SPOCK1 was overexpressed in Caki-1 and 786-O cells as determined by Western blotting. ( F ) Invasive abilities of SPOCK1-overexpressing Caki-1 and 786-O cells were determined by a Matrigel invasion assay. ( D , F ) Left panel: representative photomicrographs. Right panel: Data are presented as the mean ± SD of three independent experiments. *** p < 0.001, compared to control cells. ( G , H ) Proliferation rates and colony-forming abilities of SPOCK1-manipulated Caki-1 and 786-O cells were measured by performing MTS ( G ) and colony-formation ( H ) assays, respectively. Left panel of ( H ): representative photomicrographs. Data from ( G ) and ( H ) are presented as the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, compared to control cells.

Article Snippet: Human RCC cell lines (ccRCC: Caki-1 and 786-O; pRCC: ACHN and Caki-2) with wild-type (WT) or mutant Von Hippel-Lindau (VHL) were purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Over Expression, Migration, Western Blot, Transwell Migration Assay, Knockdown, Invasion Assay, Control

Journal: International Journal of General Medicine

Article Title: AK3 as a Hypoxia-Angiogenesis–Related Prognostic Biomarker and Therapeutic Target in Clear Cell Renal Cell Carcinoma

doi: 10.2147/IJGM.S552108

Figure Lengend Snippet: Identification and Validation of Hypoxia and Angiogenesis-Related Differentially Expressed Hub Genes (hHA-DEGs). ( A ) Correlation between hypoxia and angiogenesis gene sets in tumor samples from TCGA. ( B ) Expression scores of hypoxia and angiogenesis gene sets in ccRCC tumor tissues versus non-tumor tissues, based on single-cell dataset GSE14526 . ( C ) WGCNA of modules associated with tissue type, vascular proliferation, and hypoxia gene sets. Eighteen modules correlated with the three phenotypes were identified. ( D ) By intersecting WCGNA genes with differentially expressed genes of TCGA and hypoxia-angiogenesis gene set, 13 key differentially expressed genes (hHA-DEGs) were identified.

Article Snippet: Human-derived ccRCC cell lines (Caki-2, OS-RC-2, 786-O) and a normal renal tubular epithelial cell line (HK-2) were used, all obtained from Servicebio.

Techniques: Biomarker Discovery, Expressing, Single Cell

Journal: International Journal of General Medicine

Article Title: AK3 as a Hypoxia-Angiogenesis–Related Prognostic Biomarker and Therapeutic Target in Clear Cell Renal Cell Carcinoma

doi: 10.2147/IJGM.S552108

Figure Lengend Snippet: Construction of a prognostic model related to hypoxia-angiogenesis. ( A ) The CI index of the top 18 algorithms for both the training and validation sets. ( B ) ROC curve and Kaplan Meier curve for the prognostic model in the TCGA-KIRC training set. ( C ) ROC curve and Kaplan Meier curve for the prognostic model in the E-MTAB-1980 validation set. ( D ) Nome plot displaying the prognostic significance of the six selected hHA-DEGs within the TCGA ccRCC cohorts. ( E ) Prognostic calibration curve assessing the agreement between predicted and actual risks for the six hHA-DEGs within the TCGA ccRCC cohor.

Article Snippet: Human-derived ccRCC cell lines (Caki-2, OS-RC-2, 786-O) and a normal renal tubular epithelial cell line (HK-2) were used, all obtained from Servicebio.

Techniques: Biomarker Discovery

Journal: International Journal of General Medicine

Article Title: AK3 as a Hypoxia-Angiogenesis–Related Prognostic Biomarker and Therapeutic Target in Clear Cell Renal Cell Carcinoma

doi: 10.2147/IJGM.S552108

Figure Lengend Snippet: Single cell analysis and RT-PCR validation. ( A ) Violin plots depicting the expression levels of RPL36A, AK3, LGALS1, TIMP1, and VIM across various cell types within ccRCC tissues, based on the single-cell dataset GSE14526 . ( B ) Comparative analysis of RNA expression levels of RPL36A, AK3, LGALS1, TIMP1, and VIM between normal and tumor tissues from ccRCC patients. ( C ) Forest plots summarizing the univariate and multivariate Cox proportional hazards regression analyses of the prognostic value of RPL36A, AK3, LGALS1, TIMP1, and VIM in the TCGA ccRCC cohort. The red box highlights AK3, which was identified as an independent protective prognostic factor in the multivariate analysis. Data were shown as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Human-derived ccRCC cell lines (Caki-2, OS-RC-2, 786-O) and a normal renal tubular epithelial cell line (HK-2) were used, all obtained from Servicebio.

Techniques: Single-cell Analysis, Reverse Transcription Polymerase Chain Reaction, Biomarker Discovery, Expressing, Single Cell, RNA Expression

Journal: International Journal of General Medicine

Article Title: AK3 as a Hypoxia-Angiogenesis–Related Prognostic Biomarker and Therapeutic Target in Clear Cell Renal Cell Carcinoma

doi: 10.2147/IJGM.S552108

Figure Lengend Snippet: Bioinformatics Analysis of AK3 Expression, Clinical Features, and Prognosis in ccRCC. ( A ) Boxplot analysis of AK3 mRNA expression levels across pan-cancers in TCGA. ( B ) Paired analysis of AK3 mRNA expression in normal versus tumor tissues in ccRCC. ( C ) Receiver Operating Characteristic (ROC) curve illustrating the diagnostic accuracy of AK3 expression for ccRCC. ( D ) Boxplot of AK3 protein expression levels in normal and primary tumor tissues from the CPTAC data. ( E ) Boxplots depicting AK3 expression correlation with clinical features, including pathologic M stage, histologic grade, and gender. ( F ) Kaplan-Meier survival curves showing the relationship between AK3 expression and OS, DSS and PFI in ccRCC patients. Data were shown as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Human-derived ccRCC cell lines (Caki-2, OS-RC-2, 786-O) and a normal renal tubular epithelial cell line (HK-2) were used, all obtained from Servicebio.

Techniques: Expressing, Diagnostic Assay

Journal: International Journal of General Medicine

Article Title: AK3 as a Hypoxia-Angiogenesis–Related Prognostic Biomarker and Therapeutic Target in Clear Cell Renal Cell Carcinoma

doi: 10.2147/IJGM.S552108

Figure Lengend Snippet: Validation of AK3 Expression Levels in ccRCC Tissue Samples and Cell Lines. ( A ) Western blot analysis of AK3 protein expression in normal control (NC) and tumor tissues from ccRCC. ( B ) Immunohistochemical (IHC) analysis of AK3 expression in a tissue microarray containing normal (N) and tumor (T) samples from ccRCC patients. H-Score quantification of AK3 expression demonstrates significant downregulation in tumor tissues compared to normal tissues. ( C ) Western blot analysis of AK3 protein expression in ccRCC cell lines. ( D ) RT-PCR analysis of AK3 mRNA expression in ccRCC cell lines. ( E ) Immunofluorescence staining of AK3 protein in ccRCC cells. Labeling mitochondria with TOM20 antibody and AK3 protein with AK3 antibody. Data were shown as mean ± SD. * p < 0.05, ** p < 0.01.

Article Snippet: Human-derived ccRCC cell lines (Caki-2, OS-RC-2, 786-O) and a normal renal tubular epithelial cell line (HK-2) were used, all obtained from Servicebio.

Techniques: Biomarker Discovery, Expressing, Western Blot, Control, Immunohistochemical staining, Microarray, Reverse Transcription Polymerase Chain Reaction, Immunofluorescence, Staining, Labeling

Journal: International Journal of General Medicine

Article Title: AK3 as a Hypoxia-Angiogenesis–Related Prognostic Biomarker and Therapeutic Target in Clear Cell Renal Cell Carcinoma

doi: 10.2147/IJGM.S552108

Figure Lengend Snippet: Correlation Between AK3 Expression and Immune Infiltration in ccRCC. ( A ) Correlation analysis of AK3 expression with immune cell infiltration using the ssGSEA algorithm across 24 immune cell types. ( B ) Scatter plots depicting the correlation of AK3 expression with the enrichment scores of selected immune cell types: mast cells, T helper cells, Treg cells, and NK CD56bright cells. ( C ) Box plots comparing the expression levels of immune checkpoint genes (CD274, CTLA4, TIGIT, LAG3, PDCD1) between high AK3 (G1), low AK3 (G2), and normal groups. ( D ) Comparison of mutation frequencies of selected genes between high and low AK3 expression groups in the TCGA-KIRC cohort. ( E ) Correlation analysis of AK3 mRNA expression with drug sensitivity using the GSCA database. Data were shown as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Human-derived ccRCC cell lines (Caki-2, OS-RC-2, 786-O) and a normal renal tubular epithelial cell line (HK-2) were used, all obtained from Servicebio.

Techniques: Expressing, Comparison, Mutagenesis

Journal: International Journal of General Medicine

Article Title: AK3 as a Hypoxia-Angiogenesis–Related Prognostic Biomarker and Therapeutic Target in Clear Cell Renal Cell Carcinoma

doi: 10.2147/IJGM.S552108

Figure Lengend Snippet: AK3 Overexpression Inhibits Growth and Migration of ccRCC Cells. ( A ) Western blot analysis showing the successful overexpression of AK3 protein in 786O and OS ccRCC cell lines. ( B ) Relative RNA expression levels of AK3 in 786O and OS cells following transfection with AK3 overexpression plasmids, as determined by RT-PCR. ( C ) Cell growth curves for 786O and OS cells, illustrating a significant decrease in proliferation rates in the AK3 overexpression group compared to controls over a 5-day period. ( D ) Representative images and quantification of colony formation assays in 786O and OS cell lines. ( E ) Wound healing assay images and quantification demonstrating reduced migration of 786O and OS cells following AK3 overexpression. ( F ) Transwell migration assay results showing a significant decrease in the number of migrating 786O and OS cells upon AK3 overexpression. Data were shown as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Human-derived ccRCC cell lines (Caki-2, OS-RC-2, 786-O) and a normal renal tubular epithelial cell line (HK-2) were used, all obtained from Servicebio.

Techniques: Over Expression, Migration, Western Blot, RNA Expression, Transfection, Reverse Transcription Polymerase Chain Reaction, Wound Healing Assay, Transwell Migration Assay

Journal: International Journal of General Medicine

Article Title: AK3 as a Hypoxia-Angiogenesis–Related Prognostic Biomarker and Therapeutic Target in Clear Cell Renal Cell Carcinoma

doi: 10.2147/IJGM.S552108

Figure Lengend Snippet: AK3 Overexpression Inhibits the PI3K-AKT/GSK3β Signaling Pathway in ccRCC Cells. ( A ) Volcano plot depicting differential gene expression in 786O cells following transfection with AK3 overexpression plasmids. ( B ) KEGG pathway enrichment analysis highlighting downregulated pathways upon AK3 overexpression. ( C ) KEGG enrichment analysis of upregulated pathways in AK3-overexpressing cells. ( D ) Western blot analysis of key signaling proteins in the PI3K/AKT pathway. ( E ) In vivo analysis of tumor growth volume and weight in nude mice. ( F ) Immunohistochemical analysis of AK3 and Ki67 expression in tumor tissues. Data were shown as mean ± SD. * p < 0.05, ** p < 0.01.

Article Snippet: Human-derived ccRCC cell lines (Caki-2, OS-RC-2, 786-O) and a normal renal tubular epithelial cell line (HK-2) were used, all obtained from Servicebio.

Techniques: Over Expression, Gene Expression, Transfection, Western Blot, In Vivo, Immunohistochemical staining, Expressing

Journal: Journal of translational medicine

Article Title: ADAM12 promotes clear cell renal cell carcinoma progression and triggers EMT via EGFR/ERK signaling pathway.

doi: 10.1186/s12967-023-03913-1

Figure Lengend Snippet: Fig. 2 ADAM12 promoted the proliferation of ccRCC cells. A, B ADAM12 was significantly downregulated in CAKI-2 and 786-O cells at both the transcriptional and translational levels after infection with shADAM12 lentivirus. C, D ADAM12 was markedly overexpressed in ACHN and 786-O cells at both the transcriptional and translational levels after infection with OE-ADAM12 lentivirus. E–F CCK-8 assays showed the proliferation capacity of ccRCC cells infected with the indicated lentivirus. G, H Colony formation assay revealed the colony number of ccRCC cells infected with the indicated lentivirus

Article Snippet: ccRCC cell culture and tissue specimens Human ccRCC cell lines (769-P, 786-O, Caki-1, Caki-2, ACHN) and the human proximal tubular epithelial cell line HK-2 were acquired from the American Type Culture Collection (ATCC).

Techniques: Infection, CCK-8 Assay, Colony Assay

Journal: Journal of translational medicine

Article Title: ADAM12 promotes clear cell renal cell carcinoma progression and triggers EMT via EGFR/ERK signaling pathway.

doi: 10.1186/s12967-023-03913-1

Figure Lengend Snippet: Fig. 3 ADAM12 facilitated migration and invasion and induced EMT in ccRCC cells. A, B Wound healing assay showed the migration capacity of ccRCC cells infected with the indicated lentivirus. C, D Transwell assay revealed that the invasive capacity of ccRCC cells was obviously inhibited or strengthened after depletion or restoration of ADAM12 in ccRCC cells, respectively. E The effect of ADAM12 knockdown on EMT marker expression was assessed by western blotting. F The effect of ADAM12 overexpression on EMT marker expression in ACHN and CAKI-2 cells

Article Snippet: ccRCC cell culture and tissue specimens Human ccRCC cell lines (769-P, 786-O, Caki-1, Caki-2, ACHN) and the human proximal tubular epithelial cell line HK-2 were acquired from the American Type Culture Collection (ATCC).

Techniques: Migration, Wound Healing Assay, Infection, Transwell Assay, Knockdown, Marker, Expressing, Western Blot, Over Expression

Journal: Journal of translational medicine

Article Title: ADAM12 promotes clear cell renal cell carcinoma progression and triggers EMT via EGFR/ERK signaling pathway.

doi: 10.1186/s12967-023-03913-1

Figure Lengend Snippet: Fig. 7 ADAM12 promoted the growth of ccRCC cells in vivo. A, B ACHN cells stably infected with shADAM12 or OE-ADAM12 were injected subcutaneously into the axilla of nude mice (n = 6 for each group) to create a xenograft tumor model. The tumor growth curves were documented according to the measurement of tumor volume every 3 days, and the tumor weight was measured. C, D Representative immunohistochemistry images of xenograft tumor tissues for HE, ADAM12, c-Myc, E-cadherin, N-cadherin and Snail staining

Article Snippet: ccRCC cell culture and tissue specimens Human ccRCC cell lines (769-P, 786-O, Caki-1, Caki-2, ACHN) and the human proximal tubular epithelial cell line HK-2 were acquired from the American Type Culture Collection (ATCC).

Techniques: In Vivo, Stable Transfection, Infection, Injection, Immunohistochemistry, Staining