A positive feedback loop between TAZ and miR-942-3p modulates proliferation, angiogenesis, epithelial-mesenchymal transition process, glycometabolism and ROS homeostasis in human bladder cancer

Abstract

Background: Transcriptional coactivator with PDZ-binding motif (TAZ) has been reported to be involved in tumor progression, angiogenesis, epithelial-mesenchymal transition (EMT), glycometabolic modulation and reactive oxygen species (ROS) buildup. Herein, the underlying molecular mechanisms of the TAZ-induced biological effects in bladder cancer were discovered.

Methods: qRT-PCR, western blotting and immunohistochemistry were performed to determine the levels of TAZ in bladder cancer cells and tissues. CCK-8, colony formation, tube formation, wound healing and Transwell assays and flow cytometry were used to evaluate the biological functions of TAZ, miR-942-3p and growth arrest-specific 1 (GAS1). QRT-PCR and western blotting were used to determine the expression levels of related genes. Chromatin immunoprecipitation and a dual-luciferase reporter assay were performed to confirm the interaction between TAZ and miR-942. In vivo tumorigenesis and colorimetric glycolytic assays were also conducted.

Results: We confirmed the upregulation and vital roles of TAZ in bladder cancer. TAZ-induced upregulation of miR-942-3p expression amplified upstream signaling by inhibiting the expression of large tumor suppressor 2 (LATS2, a TAZ inhibitor). MiR-942-3p attenuated the impacts on cell proliferation, angiogenesis, EMT, glycolysis and ROS levels induced by TAZ knockdown. Furthermore, miR-942-3p restrained the expression of GAS1 to modulate biological behaviors.

Conclusion: Our study identified a novel positive feedback loop between TAZ and miR-942-3p that regulates biological functions in bladder cancer cells via GAS1 expression and illustrated that TAZ, miR-942-3p and GAS1 might be potential therapeutic targets for bladder cancer treatment.

Keywords: Angiogenesis; Bladder cancer; EMT; Glycolysis; Progression; Reactive oxygen species; TAZ; miR-942-3p.

Fig. 1

TAZ is overexpressed in bladder cancer cell lines and tissues. a-b Detection of TAZ expression in human bladder cancer tissues and adjacent normal tissues at the mRNA and protein levels (cohort 1, n = 20). c. TAZ expression levels in bladder cancer tissue and normal tissue in the TCGA database. d-e mRNA and protein expression levels of TAZ in SV-HUC-1 cells and different bladder cancer cell lines. f. The expression levels of TAZ in bladder cancer tissue specimens in cohort 2 were determined by IHC (n = 30). TAZ expression was significantly upregulated in bladder cancer tissue. Scale bar: 200 μm. Data are presented as the mean ± SD of three independent experiments. *P < 0.05 and **P < 0.01 vs. the control group

Fig. 2

TAZ is vital in cell growth, angiogenesis, migration, invasion, EMT, glycolysis and ROS homeostasis in bladder cancer cells. a. The knockdown efficiency of siRNAs specific for TAZ was determined by western blot analysis of T24 cells. b. A CCK-8 assay was performed to determine the effect of TAZ on cell viability. c. Depletion of TAZ inhibited the colony-forming abilities of T24 and EJ cells. d. The apoptotic rates of control and TAZ knockdown cells were detected by flow cytometry. e. A tube formation assay was performed to evaluate the effect of TAZ on tumor angiogenesis. f. A wound healing assay was used to evaluate the cell migration of control or TAZ-knockdown T24 and EJ cells. g. The cell migration and invasion of TAZ-deficient cells were evaluated by Transwell migration and invasion assays, respectively. h. EMT markers were detected in SV-HUC-1 cells and bladder cancer cell lines by western blotting. i-j The key role of TAZ in the EMT process was confirmed by western blotting and qRT-PCR. k. A schematic illustration of glycolysis is shown. l-m The uptake of glucose and production of lactate in TAZ-deficient and normal bladder cancer cells were determined. n. The alterations in PFKFB3, HK2 and GLUT1 at the protein level were confirmed by western blotting. o. qRT-PCR was performed to assess the expression levels of glycolysis-related genes (PFKFB3, LDHB, HK2, GLUT1, GLUT3 and GLUT4). p. Intracellular ROS levels were determined with DCFH–DA and analyzed by flow cytometry. Data are presented as the mean ± SD of three independent experiments. *P < 0.05 and **P < 0.01 vs. the control group

Fig. 3

TAZ-TEAD regulates the level of miR-942-3p by binding to its promoter. a. TAZ-related miRNAs in T24 cells were identified by RNA sequencing. A heatmap of these miRNAs was generated (N.C. vs shTAZ cells, each sample was mixed with three replicates). b-c qRT-PCR detection of miR-942-3p in TAZ-knockdown or TAZ-overexpressing cells. d-e Correlation between TAZ and miR-942-3p in bladder cancer in cohort 1 and the TCGA database. f-g The Hippo signaling pathway induces miR-942-3p expression. MiR-942-3p levels in LATS1/2-deficient or TEAD2-deficient cells were determined by qRT-PCR. h. Schematic illustration of the miR-942 promoter. Predicted TEAD binding site in the miR-942 promoter (indicated by the red arrow). The section of the promoter cloned for a luciferase reporter assay and the primers used for qRT-PCR validation in a ChIP assay are indicated. j. Chromatin immunoprecipitation followed by qRT-PCR using specific primers for different regions of the miR-942 promoter in 293 T cells demonstrated that TAZ could bind to the miR-942 promoter. k. The predicted TEAD binding site was mutated, and a luciferase reporter assay was performed to validate the binding site of TAZ-TEADs in the miR-942 promoter. Data are presented as the mean ± SD of three independent experiments. *P < 0.05 and **P < 0.01 vs. the control group

Fig. 4

MiR-942-3p is involved in the proliferation, angiogenesis, migration, invasion, EMT process and redox balance of bladder cancer cells. a. MiR-433-3p was detected in bladder cancer tissue and adjacent normal tissue (cohort 1, n = 20). b. TCGA database analysis indicated the upregulation of miR-942-3p expression in bladder cancer. c. qRT-PCR was performed to determine miR-942-3p levels in SV-HUC-1 cells and bladder cancer cell lines. d. Cell viability was measured to evaluate the biological effects of miR-942-3p. e. The effect of miR-942-3p on colony formation was evaluated with a colony formation assay. f. HUVECs were cultured with conditioned medium to assess angiogenesis mediated by miR-942-3p. g. A wound healing assay was performed to evaluate cell migration. h. MiR-942-3p influenced cell migration and invasion, as determined by Transwell migration and Matrigel invasion assays, respectively. i-j The protein and mRNA expression levels of EMT markers in T24 and EJ cells transfected with pre-miR-942 or a miR-942-3p sponge were detected by western blotting and qRT-PCR, respectively. k. ROS content was evaluated by flow cytometry in different cells. Data are presented as the mean ± SD of three independent experiments. *P < 0.05 and **P < 0.01 vs. the control group

Fig. 5

Validation of GAS1 and LATS2 as direct targets of miR-942-3p. a. MiR-942-3p target genes predicted by searching the TargetScan, miRDB, miRWalk and miRTarBase databases. b. The potential miR-942-3p binding sites in the 3′-UTRs of GAS1 and LATS2 mRNA transcripts are shown in the schematic illustration. Lowercase letters indicate the mutated binding sites in the same 3′-UTR. c. Luciferase reporter assay analysis of 293 T cells transfected with the indicated plasmids and miR-942-3p mimics (100 nM). d. TCGA database analysis indicated negative correlations among miR-942-3p, GAS1 and LATS2. e. The miR-942-3p level was negatively correlated with the protein levels of GAS1 and LATS2. Data are presented as the mean ± SD of three independent experiments. *P < 0.05 and **P < 0.01 vs. the control group

Fig. 6

A TAZ/miR-942-3p positive feedback loop modulates the oncogenic effects, glycolysis and redox state mediated by TAZ. a. Cells were transfected with a negative control vector, TAZ-specific shRNA or pre-miR-942. Cell viability was determined by a CCK-8 assay. b. MiR-942-3p promoted colony formation, as determined by a colony formation assay. c. A tube formation assay was used to verify the effect of the positive feedback loop on angiogenesis. d. To verify the effects on cell migration, a wound healing assay was performed. e. The migration and invasion of bladder cancer cells transfected with different vectors were evaluated with Transwell migration and invasion assays, respectively. f-g The expression levels of TAZ and EMT markers in T24 and EJ cells were evaluated by western blotting and qRT-PCR. h-i The uptake of glucose and production of lactate in cells under different conditions were determined to evaluate the glycolytic process. j-k The expression levels of glycolysis-related genes (PFKFB3, HK2 and GLUT1) were detected by qRT-PCR and western blotting in TAZ-deficient cells with or without supplementation with miR-942-3p. l. ROS levels were detected in cells transfected with a negative control vector, TAZ-specific shRNA or pre-miR-942. Data are presented as the mean ± SD of three independent experiments. *P < 0.05 and **P < 0.01 vs. the control group

Fig. 7

GAS1 overexpression impaired cell proliferation, angiogenesis, migration, invasion, EMT, glycolysis and ROS homeostasis in bladder cancer cells. a. The effect of GAS1 on cell viability was verified by a CCK-8 assay. b. A colony formation assay showed that GAS1 impaired the colony-forming ability of T24 and EJ cells. c. Conditioned medium was collected from control or GAS1-overexpressing cells and used in a tube formation assay to evaluate angiogenesis. d. A wound healing assay indicated the effect of GAS1 overexpression on migration. e. GAS1 overexpression suppressed the migration and invasion of T24 and EJ cells, as determined by Transwell migration and Matrigel invasion assays. f-g Western blot and qRT-PCR analyses showing the expression levels of EMT markers in GAS1-overexpressing bladder cancer cells at the protein and mRNA levels, respectively. h-i The effects of GAS1 on glycolysis were determined by glucose uptake and lactate production assays. j-k PFKFB3, HK2 and GLUT1 levels in GAS1-overexpressing cells were evaluated by western blotting and qRT-PCR. l. GAS1 remarkably upregulated intracellular ROS levels in T24 and EJ cells. m. TCGA database analysis showed a lower expression level of GAS1 expression in bladder cancer tissue than in normal tissue. n. Immunohistochemical detection of GAS1 in cohort 2 (n = 30) further confirmed the dysregulation of GAS1 in bladder cancer. Scale bar: 200 μm. Data are presented as the mean ± SD of three independent experiments. *P < 0.05 and **P < 0.01 vs. the control group

Fig. 8

TAZ and miR-942-3p enhance bladder cancer tumor growth in vivo. a. Equal numbers of different T24 cell populations (negative control, shTAZ or cotransfected with shTAZ and miR-942-3p) were used to establish subcutaneous xenograft tumors. The tumors were harvested and photographed (n = 4 each group). b. Tumor weight was determined when the mice were sacrificed. c. Tumor volume was measured and calculated after cells were injected into mice. d. MiR-942-3p levels were determined by qRT-PCR in tumors obtained from mice. e. TAZ, LATS2 and GAS1 were detected in mouse tumor tissues by western blotting. f. Immunohistochemical (IHC) detection of TAZ, LATS2 and GAS1 in tumors. Scale bars: 200 μm. g. Schematic diagram illustrating the novel positive feedback loop between TAZ and miR-942-3p that regulates GAS1 expression and modulates biological behaviors, EMT and glycometabolism in bladder cancer. Data are presented as the mean ± SD of three independent experiments. *P < 0.05 and **P < 0.01 vs. the control group