Journal: Molecular Genetics & Genomic Medicine

Article Title: miR‐33a inhibits cell growth in renal cancer by downregulation of MDM4 expression

doi: 10.1002/mgg3.833

Figure Lengend Snippet: Inhibitory effects of miR‐33a on cell proliferation and cell cycle in renal cell cancer (RCC) cell lines. (a) Expression of miR‐33a in Caki‐1 and 786‐O cells after transfection with miR‐33a mimics and miR‐33a inhibitor or NC. NC represents negative control of miRNA. Results were expressed as of three independent experiments, with at least three replicates in each independent experiment, ** p < 0.01 versus NC. (b) CCK‐8 assays indicated that the effects of miR‐33a on the growth of RCC cell lines. Results were expressed as of three independent experiments, with at least three replicates in each independent experiment, * p < 0.05 versus NC. (c, d) The results of flow cytometry showed that upregulation of miR‐33a significantly increased the percentages of cells in the G0/G1 phase, which showed that the upregulation of miR‐33a could suppress RCC cells proliferation. * p < 0.05 versus Control

Article Snippet: Normal primary renal tubular HK‐2 cell lines and RCC cell lines (Caki‐1, ACHN and 786‐O) were purchased from China Center For Type Culture Collection (Wuhan, China).

Techniques: Expressing, Transfection, Negative Control, CCK-8 Assay, Flow Cytometry, Control

Journal: Molecular Genetics & Genomic Medicine

Article Title: miR‐33a inhibits cell growth in renal cancer by downregulation of MDM4 expression

doi: 10.1002/mgg3.833

Figure Lengend Snippet: miR‐33a directly targets Mouse double minute 4 (MDM4). (a) The predicted miR‐33a binding site within MDM4 3′ UTR and miR‐33a mutated version by site mutagenesis; (b) Caki‐1 cells were transfected with reporter constructs containing either wild type (WT) MDM4 , or MDM 4 3′ UTR with mutation (MUT), along with miR‐33a mimics, or negative control, respectively. Relative luciferase activity was measured. (c) qPCR analysis of MDM4 expression in renal cell cancer (RCC) Caki‐1 cells after overexpression or knockdown of miR‐33a . (d, e) Western blotting analysis of MDM4 protein expression in RCC Caki‐1 cells after overexpression or knockdown of miR‐33a . (f) Western blot analysis revealed that transfection of MDM4 siRNA into Caki‐1 cells resulted in decreased MDM4 expression compared to the cells transfected with scrambled siRNA. These effects of siRNA were attenuated by anti‐ miR‐33a inhibitor transfection. NC represents normal control, * p < 0.05, ** p < 0.01 versus NC

Article Snippet: Normal primary renal tubular HK‐2 cell lines and RCC cell lines (Caki‐1, ACHN and 786‐O) were purchased from China Center For Type Culture Collection (Wuhan, China).

Techniques: Binding Assay, Mutagenesis, Transfection, Construct, Negative Control, Luciferase, Activity Assay, Expressing, Over Expression, Knockdown, Western Blot, Control

Journal: Molecular Genetics & Genomic Medicine

Article Title: miR‐33a inhibits cell growth in renal cancer by downregulation of MDM4 expression

doi: 10.1002/mgg3.833

Figure Lengend Snippet: The effect of mouse double minute 4 ( MDM4 ) on cell growth in renal cell cancer (RCC). (a, b, c) RT‐qPCR and western blot analysis of MDM4 expression in RCC cells. (d) Kaplan–Meier analysis of overall survival in 30 RCC patients with low median ( n = 15) and high median ( n = 15) expression levels of MDM4 . (e) CCK‐8 assays indicated that the effects of MDM4 on growth of RCC cell lines. Results were expressed as ± x - s of three independent experiments, with at least three replicates in each independent experiment, * p < 0.05 versus NC. (f) Western blot analysis of p53 expression after miR‐33a mimics and ‐inhibitor transfection in RCC cells

Article Snippet: Normal primary renal tubular HK‐2 cell lines and RCC cell lines (Caki‐1, ACHN and 786‐O) were purchased from China Center For Type Culture Collection (Wuhan, China).

Techniques: Quantitative RT-PCR, Western Blot, Expressing, CCK-8 Assay, Transfection

Journal: International journal of oncology

Article Title: Long non-coding RNA HOTTIP promotes renal cell carcinoma progression through the regulation of the miR-615/IGF-2 pathway.

doi: 10.3892/ijo.2018.4539

Figure Lengend Snippet: Figure 1. HOTTIP expression and its association with the clinical parameters in renal cancer tissues. (A) Expression of HOTTIP in RCC from our clinical specimens. (B) Results of RT-qPCR showing the expression levels of HOTTIP in RCC cell lines. (C) HOTTIP RNAseq expression data were retrieved from the TCGA database. The Wilcoxon signed-rank test was used for the comparison. (D) The overall survival (OS) and disease-free survival (DFS) curve for patients with RCC with low vs. high HOTTIP expression (Kaplan-Meier method). *P<0.05, **P<0.01. RCC, renal cell carcinoma.

Article Snippet: The human RCC cell lines (A-498, 786-O, Caki-1, Caki-2 and ACHN), the normal renal epithelial cells HK-2, 293T cell line were obtained from the American Tissue Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Expressing, Quantitative RT-PCR, Comparison

Journal: PLoS ONE

Article Title: WNT10A Plays an Oncogenic Role in Renal Cell Carcinoma by Activating WNT/β-catenin Pathway

doi: 10.1371/journal.pone.0047649

Figure Lengend Snippet: (A) mRNA expression profiles of WNT genes from kidney cell lines obtained using RT-PCR. mRNA expression profiles of 19 WNT genes from 5 RCC cell lines (786-O, Caki-1, RCC-1, A498, and ACHN) and 1 immortalized proximal tubule epithelial cell line from a normal adult human kidney (HK-2) were obtained using RT-PCR. Higher WNT10A expression was observed in RCC cell lines Caki-1, RCC-1, and ACHN, but lower expression was observed in RCC cell lines 786-O and A498. WNT10A expression was undetectable in the normal kidney cell line HK-2. (B) mRNA expression profile of WNT genes from BRD and RCC specimens obtained using RT-PCR. mRNA expression profiles of 19 WNT genes from 6 paired RCC (T1–T6) and paratumoral (N1–N6) tissues. mRNA expression profiles of WNT genes from other 4 BRD tissues (N7–N10) were also examined. Higher expression of WNT10A was observed in most RCC tissues than in paratumoral and BRD tissues. (C) Quantitative real-time PCR for detecting the expression of WNT genes. Expression of WNT genes in each cell line was determined using SYBR Green real-time PCR. An independent data set comprising 10 RCC and 10 normal kidney tissues was used. The results obtained were similar to those obtained for RT-PCR, i.e., higher expression of WNT10A was observed in most RCC cell lines and tissues than in normal controls. All experiments were performed in triplicate. Relative expression of each gene from each cell line was normalized with that of the gene from HK-2 normal kidney cell line; relative expression of each gene of tissues was normalized with the mean of 10 normal kidney tissues.

Article Snippet: Human RCC cell line A498 (derived from papillary RCC); ACHN, Caki-1, and 786-O (all derived from clear cell RCC); and immortalized proximal tubule epithelial cell line (HK-2) derived from normal adult human kidney were obtained from Bioresource Collection and Research Center (BCRC, Taiwan).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, SYBR Green Assay

Journal: PLoS ONE

Article Title: WNT10A Plays an Oncogenic Role in Renal Cell Carcinoma by Activating WNT/β-catenin Pathway

doi: 10.1371/journal.pone.0047649

Figure Lengend Snippet: (A) Cell growth curve of pcDNA-WNT10A-transfected cells and WNT10A siRNA knockdown cells. WNT10A gain-of-function was achieved by transfecting pcDNA-WNT10A in normal kidney cell line HK-2 and RCC cell lines 786-O and A498, which had relatively lower endogenous WNT10A expression. Cotransfection of pcDNA-WNT10A and β-catenin siRNA was also performed. Conversely, WNT10A loss-of-function was achieved by WNT10A siRNA knockdown in RCC-1 and Caki-1, which have higher endogenous WNT10A. After transient transfection for 48 h, cell viability was determined using the MTT assay and was compared with that of cells transfected with Lipofectamine only (reagent control), pcDNA3.1 vector only (vector), and pcDNA-WNT10A and with that of cells cotransfected with pcDNA-WNT10A and β-catenin siRNA at 12, 24, 48, and 72 h. HK-2, 786-O, and A498 transfected with pcDNA-WNT10A showed significant increase in cell proliferation after 48 h; however, cotransfection of pcDNA-WNT10A and β-catenin siRNA reduced WNT10A-induced cell proliferation. Conversely, RCC-1 and Caki-1 transfected with WNT10A siRNA showed significant decrease in cell proliferation as compared to cells transfected with reagent and scrambled siRNA controls. (** P <0.001, * P <0.05; Student’s t -test). Western blot analysis of pcDNA-WNT10A- and WNT10A siRNA-transfected cells from each cell line was also performed. HK-2, 786-O, and A498 were transfected with 3 µg of pcDNA-WNT10A, pcDNA3.1 vector alone, and cotransfected with β-catenin siRNA for 48 h. Conversely, RCC-1 and Caki-1 were transfected with 1 µg WNT10A siRNA or scrambled siRNA controls for 72 h. Twenty micrograms of total protein extract from each cell line was loaded onto SDS-polyacrylamide gel and western blot analysis was performed. Forced WNT10A expression in HK-2, 786-O, and A498 remarkably increased the concentration of WNT10A than that of the vector control in these cells. Forced WNT10A expression also upregulated nuclear β-catenin, cyclin D1, and c-myc levels, and cotransfection with β-catenin siRNA reduced cyclin D1 and c-myc expression levels. Conversely, WNT10A siRNA knockdown in RCC-1 and Caki-1 markedly reduced endogenous WNT10A levels, thus reducing nuclear β-catenin, cyclin D1, and c-myc levels. (B) Immunocytochemical analysis of pcDNA-WNT10A-transfected cells and WNT10A siRNA knockdown cells. After pcDNA-WNT10A was transfected in HK-2 and 786-O, expression of WNT10A, β-catenin, and cyclin D1 was determined by immunocytochemistry. WNT10A levels were significantly increased in the transfected cells. β-catenin showed high intracellular accumulation in WNT10A-transfected cells and low membranous expression in vector- and reagent-transfected controls. Moreover, cyclin D1 was upregulated in the nucleus of pcDNA-WNT10A-transfected cells but showed low cytoplasmic expression in vector- and reagent-transfected controls. Upregulated nuclear β-catenin and cyclin D1 were also observed in the same pcDNA-WNT10A-transfected cells (red staining, β-catenin; brown staining, cyclin D1). Conversely, WNT10A siRNA-transfected RCC-1 and Caki-1 showed an obvious reduction in endogenous WNT10A expression and reduced intracellular β-catenin accumulation and cyclin D1 expression. (C) TCF/LEF reporter assay. Forced WNT10A expression in 786-O for 48 h significantly induced luciferase activity in these cells than that in vector- and reagent-transfected controls. Conversely, WNT10A siRNA-transfected Caki-1 showed significantly reduced luciferase activity after 72 h as compared to that in vector- and reagent-transfected controls.

Article Snippet: Human RCC cell line A498 (derived from papillary RCC); ACHN, Caki-1, and 786-O (all derived from clear cell RCC); and immortalized proximal tubule epithelial cell line (HK-2) derived from normal adult human kidney were obtained from Bioresource Collection and Research Center (BCRC, Taiwan).

Techniques: Transfection, Expressing, Cotransfection, MTT Assay, Plasmid Preparation, Western Blot, Concentration Assay, Immunocytochemistry, Staining, Reporter Assay, Luciferase, Activity Assay

Journal: PLoS ONE

Article Title: WNT10A Plays an Oncogenic Role in Renal Cell Carcinoma by Activating WNT/β-catenin Pathway

doi: 10.1371/journal.pone.0047649

Figure Lengend Snippet: Effects of WNT10A on cell survival were analyzed using PI staining with flow cytometry. pcDNA-WNT10A-transfected 786-O showed slight increment in G1 phase as compared to that observed in vector-transfected controls after 48 h of solvent (DMSO) treatment. Moreover, cotransfection of pcDNA-WNT10A and β-catenin siRNA showed an obvious G1 arrest (left lane of 786-O). However, pcDNA-WNT10A-transfected 786-O showed lower sub-G1 cell population than vector-transfected controls after 48 h of treatment with 2 µM epirubicin (middle lane of 786-O) or 10 µM cisplatin (right lane of 786-O). Besides, cotransfection of pcDNA-WNT10A and β-catenin siRNA increased the chemosensitivity of 786-O. Conversely, WNT10A siRNA-transfected Caki-1 showed minor sub-G1 and G1 phase modifications as compared to those observed in scrambled siRNA-transfected controls. However, β-catenin siRNA induced both higher sub-G1 and G1 arrest (left lane of Caki-1). WNT10A siRNA- and β-catenin siRNA-transfected Caki-1 showed significantly increase sub-G1 cell population than scrambled siRNA-transfected controls after 48 h of treatment with 2 µM epirubicin (middle lane of Caki-1) or 10 µM cisplatin (right lane of Caki-1). The proportion of each cell cycle phase belonging to both cell lines are shown using bar charts.

Article Snippet: Human RCC cell line A498 (derived from papillary RCC); ACHN, Caki-1, and 786-O (all derived from clear cell RCC); and immortalized proximal tubule epithelial cell line (HK-2) derived from normal adult human kidney were obtained from Bioresource Collection and Research Center (BCRC, Taiwan).

Techniques: Staining, Flow Cytometry, Transfection, Plasmid Preparation, Cotransfection

Journal: PLoS ONE

Article Title: WNT10A Plays an Oncogenic Role in Renal Cell Carcinoma by Activating WNT/β-catenin Pathway

doi: 10.1371/journal.pone.0047649

Figure Lengend Snippet: Effects of WNT10A on cell survival were evaluated by western blot analysis of apoptotic markers. pcDNA-WNT10A-transfected 786-O showed a slightly reduced basal level of cleaved caspase 3 and PARP as compared to that observed in vector-transfected controls after 48 h of solvent (DMSO) treatment. However, β-catenin siRNA-transfected 786-O showed higher levels of cleaved caspase 3 and PARP than those observed in other groups of cells. After 48 h of treatment with 2 µM epirubicin or 10 µM cisplatin, pcDNA-WNT10A-transfected 786-O showed lower levels of cleaved caspase 3 and PARP than those observed in vector-transfected controls or cells cotransfected with pcDNA-WNT10A and β-catenin siRNA. Conversely, both WNT10A siRNA- and β-catenin siRNA-transfected Caki-1 showed higher levels of cleaved caspase 3 and PARP than those observed in scrambled siRNA-transfected controls. β-catenin silencing seemed to induce the production of cleaved caspase 3 and PARP. Both WNT10A siRNA- and β-catenin siRNA-transfected Caki-1 showed significantly higher levels of cleaved caspase 3 and PARP than those observed in scrambled siRNA-transfected controls after 48 h of treatment with 2 µM epirubicin or 10 µM cisplatin.

Article Snippet: Human RCC cell line A498 (derived from papillary RCC); ACHN, Caki-1, and 786-O (all derived from clear cell RCC); and immortalized proximal tubule epithelial cell line (HK-2) derived from normal adult human kidney were obtained from Bioresource Collection and Research Center (BCRC, Taiwan).

Techniques: Western Blot, Transfection, Plasmid Preparation

Journal: PLoS ONE

Article Title: WNT10A Plays an Oncogenic Role in Renal Cell Carcinoma by Activating WNT/β-catenin Pathway

doi: 10.1371/journal.pone.0047649

Figure Lengend Snippet: After 48 h of transfection with 3 µg pcDNA-WNT10A or WNT10A siRNA, cells were scraped with p200 tip (time 0) and photographed each day. Forced WNT10A expression in 786-O and A498 increased the migration ability of these cells in the wound healing assay, especially 48 h after scraping. Moreover, cotransfection of pcDNA-WNT10A and β-catenin siRNA reduced WNT10A promotive effect on cell migration ( P <0.001, Student’s t -test between pcDNA-WNT10A-transfected cells and other cell groups). Conversely, each WNT10A siRNA knockdown in Caki-1 reduced the migration ability of these cells in the wound healing assay ( P <0.001 at 48 h after scraping, Student’s t -test between WNT10A siRNA-transfected cells and scrambled siRNA-transfected controls or between WNT10A siRNA-transfected cells and reagent-transfected controls). Wound repair percentage of each cell line is shown using bar charts.

Article Snippet: Human RCC cell line A498 (derived from papillary RCC); ACHN, Caki-1, and 786-O (all derived from clear cell RCC); and immortalized proximal tubule epithelial cell line (HK-2) derived from normal adult human kidney were obtained from Bioresource Collection and Research Center (BCRC, Taiwan).

Techniques: Transfection, Expressing, Migration, Wound Healing Assay, Cotransfection

Journal: PLoS ONE

Article Title: WNT10A Plays an Oncogenic Role in Renal Cell Carcinoma by Activating WNT/β-catenin Pathway

doi: 10.1371/journal.pone.0047649

Figure Lengend Snippet: Transwell assay was used to evaluate the effects of WNT10A on cell migration. After 48 h of transfection, 1×10 4 cells were transferred into 8-µm inserts and incubated for another 48 h. Forced WNT10A expression in 786-O significantly increased the migration ability of these cells through the transwell; however, cotransfection with β-catenin siRNA reduced the WNT10A promotive effect on cell migration. Conversely, WNT10A siRNA knockdown in Caki-1 significantly reduced the migration ability of these cells. Bar charts show the cell migration ratio; significances were analyzed using the Student’s t -test. Invasion assay took advantage of the Matrigel-coated transwell assay. After 48 h of transfection, 2×10 4 cells were transferred into 1 mg/mL Matrigel-coated insert and incubated for another 48 h. Forced WNT10A expression in 786-O significantly increased the invasive ability of these cells, but cotransfection with β-catenin siRNA reduced the WNT10A promotive effect on cell invasion. Conversely, WNT10A siRNA knockdown in Caki-1 significantly reduced the invasive ability of these cells. Bar charts show the cell invasion ratio; and significances were analyzed as mentioned above.

Article Snippet: Human RCC cell line A498 (derived from papillary RCC); ACHN, Caki-1, and 786-O (all derived from clear cell RCC); and immortalized proximal tubule epithelial cell line (HK-2) derived from normal adult human kidney were obtained from Bioresource Collection and Research Center (BCRC, Taiwan).

Techniques: Transwell Assay, Migration, Transfection, Incubation, Expressing, Cotransfection, Invasion Assay

Journal: PLoS ONE

Article Title: WNT10A Plays an Oncogenic Role in Renal Cell Carcinoma by Activating WNT/β-catenin Pathway

doi: 10.1371/journal.pone.0047649

Figure Lengend Snippet: Colony formation assay was used to evaluate the effect of WNT10A on cell transformation. After 48 h of transfection, 2×10 4 cells were mixed with 0.3% agarose in 1× complete RPMI and transferred into a coated 6-cm dish, as described in . To prolong the effects of transfection, pcDNA-WNT10A-transfected cells were maintained in 200 µg/mL G418, and WNT10A siRNA or β-catenin siRNA knockdown cells were maintained in 10 nM of WNT10A siRNA for 15 days. Forced WNT10A expression in 786-O significantly increased the number of colonies of these cells; cotransfection with β-catenin siRNA reduced the WNT10A promotive effect on colony formation ability. Moreover, each WNT10A siRNA knockdown in Caki-1 reduced the number of colonies of these cells. Bar charts show the number of colonies; significances were analyzed by using the Student’s t -test.

Article Snippet: Human RCC cell line A498 (derived from papillary RCC); ACHN, Caki-1, and 786-O (all derived from clear cell RCC); and immortalized proximal tubule epithelial cell line (HK-2) derived from normal adult human kidney were obtained from Bioresource Collection and Research Center (BCRC, Taiwan).

Techniques: Colony Assay, Transformation Assay, Transfection, Expressing, Cotransfection