Fig 1: Molecular mechanism of NEDD4 and the KLF8/miR-132/NRF2 axis in the progression of bladder cancer.In bladder cancer cells, NEDD4 stabilizes the expression of KLF8 and promotes the transcriptional activity of KLF8 by promoting the ubiquitination of KLF8. KLF8 restricts the expression of miR-132. Downregulation of miR-132 weakens the targeted inhibition of NRF2. Elevated expression of NRF2 regulates the expression of downstream genes related to viability and migratory ability, leading to increased viability and migratory ability of bladder cancer cells.
Fig 2: NEDD4 promotes binding between KLF8 and the miR-132 promoter region.a Network diagram of TransmiR v2.0 (http://www.cuilab.cn/transmir) predicting downstream regulatory miRNAs of transcription factor KLF8. b Differential expression analysis of GSE40355 (with | logFoldChange | > 1, P value <0.05 as threshold) shows that miR-132 is poorly expressed in bladder cancer. c RT-qPCR detection of the expression of miR-132 in clinical samples and different cell lines. d RT-qPCR was used to examine the expressions of KLF8 and miR-132 after overexpressing KLF8 in T24 cells. e ChIP assay was used to examine the binding of KLF8 in the miR-132 promoter region. f Dual-luciferase reporter gene assay was used to verify the regulation of KLF8 to miR-132. g RT-qPCR was used to examine the expression of miR-132 in response to oe-NEDD4 and siKLF8. h ChIP assay to KLF8 binding in the miR-132 promoter region in response to NEDD4 and siKLF8. *P < 0.05 versus adjacent normal tissues, SVHUC-1 cell line, the oe-NC or oe-NC + si-NC group; #P < 0.05 versus the IgG or oe-NC + si-NC group. The experimental results are measurement data and are expressed as the mean ± standard deviation. Unpaired data with a normal distribution and homogeneity between two groups were compared using an unpaired t test. Comparisons among multiple groups were conducted by one-way ANOVA with Tukey’s post hoc test. Cell experiments were independently repeated three times (n = 45).
Fig 3: NEDD4 potentiates malignant phenotypes of bladder cancer cells by promoting the stability and transcriptional activity of KLF8 through ubiquitination.a IP assay of ubiquitination of KLF8 by NEDD4. b IP assay of ubiquitination of KLF8 by NEDD4 knockdown. c Analysis of KLF8 protein stability after CHX intervention. d Dual-luciferase assay to examine cyclin D1 promoter activity. e RT-qPCR and western blot assay to examine KLF8 silencing efficiency. f RT-qPCR to examine expressions of NEDD4 and KLF8 in response to oe-NEDD4 and siKLF8. g Western blot assay to examine the expression of NEDD4 and KLF8 normalized to GAPDH in response to oe-NEDD4 and siKLF8. h MTT assay to examine cell viability in response to oe-NEDD4 and siKLF8. i Flow cytometry analysis for apoptosis in response to oe-NEDD4 and siKLF8 under the induction of etoposide. j Transwell assay to examine cell migration in response to oe-NEDD4 and siKLF8 (scale bar: 50 µm). k Western blot assay to examine apoptosis-related proteins Bax and BCl-2, tumor suppressor proteins p53, p21, and metastasis-related proteins vimentin, N-cadherin, and E-cadherin normalized to GAPDH in response to oe-NEDD4 and siKLF8. *P < 0.05 versus the NC, oe-NC or oe-NC + si-NC group; #P < 0.05 versus the siKLF8 + oe-NC group. The experimental results are measurement data and are expressed as the mean ± standard deviation. Unpaired data with a normal distribution and homogeneity between two groups was compared using an unpaired t test. Comparisons among multiple groups were conducted by one-way ANOVA with Tukey’s post hoc test. Statistical analysis in relation to time-based measurements within each group was performed using repeated-measures ANOVA, followed by Bonferroni’s post hoc test. Cell experiments were independently repeated three times.
Fig 4: NEDD4 facilitates tumorigenesis of bladder cancer by mediating the KLF8/miR-132/NRF2 axis in vivo.a Images of xenograft tumors from nude mice. b Quantification of tumor volume in mice in response to NEDD4 overexpression and NRF2 silencing. c Quantification of tumor weight in mice in response to NEDD4 overexpression and NRF2 silencing. d RT-qPCR was used to examine the expression of NEDD4, KLF8, miR-132, and NRF2 in tumors in response to NEDD4 overexpression and NRF2 silencing. e Western blot assay was used to examine expression of NEDD4, KLF8, miR-132, and NRF2 in tumors normalized to GAPDH in response to NEDD4 overexpression and NRF2 silencing. f H&E staining (×200) of mouse lung metastatic nodules in response to NEDD4 overexpression and NRF2 silencing. g The number of lung metastatic nodules in response to NEDD4 overexpression and NRF2 silencing. *P < 0.05 versus the vector + sh-Ctrl group; #P < 0.05 versus the NEDD4 + sh-Ctrl group. The experimental results are the measurement data and are expressed as the mean ± standard deviation. Comparisons among multiple groups were conducted by one-way ANOVA with Tukey’s post hoc test. Statistical analysis in relation to time-based measurements within each group was performed using repeated-measures ANOVA, followed by Bonferroni’s post hoc test. n = 10.
Fig 5: NEDD4 promotes bladder cell viability and migratory ability via the NRF2/KLF8/miR-132 axis.a RT-qPCR and western blot assay was used to examine the silencing efficiency of siNRF2-1 and siNRF2-2. b RT-qPCR was used to examine the expression of NEDD4, KLF8, miR-132, and NRF2 in response to oe-NEDD4 and siNRF2. c Western blot assay was used to examine the expression of NEDD4, KLF8, and NRF2 normalized to GAPDH in response to oe-NEDD4 and siNRF2. d MTT assay was used to examine cell viability in response to oe-NEDD4 and siNRF2. e Flow cytometry was used to examine apoptosis in response to oe-NEDD4 and siNRF2 under etoposide induction. f Transwell assay was used to examine cell migration in response to oe-NEDD4 and siNRF2 (×200). g Western blot assay was used to examine expression of the apoptosis-related proteins Bax and BCl-2, tumor suppressor proteins p53 and p21, and metastasis-related proteins vimentin, N-cadherin, and E-cadherin normalized to GAPDH in response to oe-NEDD4 and siNRF2. *P < 0.05 versus the si-NC or oe-NC + si-NC group; #P < 0.05 versus the oe-NEDD4 + si-NC group. The experimental results are the measurement data and are expressed as the mean ± standard deviation. Comparisons among multiple groups were conducted by one-way ANOVA with Tukey’s post hoc test. Statistical analysis in relation to time-based measurements within each group was performed using repeated-measures ANOVA, followed by Bonferroni’s post hoc test. Cell experiments were independently repeated three times.
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