YWHAZ amplification/overexpression defines aggressive bladder cancer and contributes to chemo-/radio-resistance by suppressing caspase-mediated apoptosis

The objective of this study was to characterize the oncogenic actions of a recently identified cancer-associated gene YWHAZ (also named as 14-3-3 ζ/δ) in urothelial carcinomas of the urinary bladder (UCUB). A genome-wide study revealed YWHAZ to be involved in the amplicon at 8q22.3, and its genetic amplification was detected predominantly in muscle-invasive bladder cancer (MIBC). Immunohistochemical staining confirmed the association of YWHAZ overexpression with higher tumor stages, lymph node/vascular invasion, and mitotic activity. Univariate and multivariate analyses further indicated the prognostic potential of YWHAZ for more aggressive cancer types. Both gene set enrichment analysis and STRING network studies suggested involvement of YWHAZ in regulating caspase-mediated apoptosis. Ectopic expression of YWHAZ in bladder cells with low endogenous YWHAZ levels boosted cell resistance to doxorubicin and cisplatin, as well as to ionizing radiation. Conversely, YWHAZ-knockdown using specific shRNA in cells with high endogenous YWHAZ levels diminished survival activity, suppressing cell growth and increasing cell death. Our findings confirm the essential role played by YWHAZ in sustaining cell proliferation during chemo/radiotherapy. Treatments based on anti-YWHAZ strategies may thus be beneficial for UCUB patients overexpressing YWHAZ. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

YWHAZ Binds to TRIM21 but Is Not Involved in TRIM21-stimulated Osteosarcoma Cell Proliferation

Objective Osteosarcoma is the most common type of malignant bone tumor in children and adolescents. The role of E3 ligases in tumorigenesis is currently a focus in tumor research. In the present study, we investigated the role of the E3 ligase tripartite motif 21(TRIM21) in osteosarcoma cell proliferation.Methods 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide(MTT) assays were used to assess osteosarcoma cell viability. U2-OS cells stably carrying a recombinant lentivirus expressing tetracycline-regulated TRIM21 were screened. Co-immunoprecipitation was coupled with LCMS/MS analysis to identify novel interacting partners of TRIM21. Co-immunoprecipitation and bimolecular fluorescence complementation(BIFC) were performed to validate the interactions between TRIM21 and its novel partner YWHAZ. A TRIM21-ΔRING construct was generated to test the effects of TRIM21 ligase activity on YWHAZ.Results TRIM21 positively regulated osteosarcoma cell proliferation. Overexpression of TRIM21 enhanced osteosarcoma cell tolerance toward various stresses. YWHAZ protein was identified as a novel interacting partner of TRIM21 and its expression levels were negatively regulated by TRIM21. The RING domain of TRIM21 was required for TRIM21 negative regulation of YWHAZ expression. However,overexpression of YWHAZ did not affect positive regulation of osteosarcoma cell proliferation by TRIM21.Conclusion Our results further clarify the molecular mechanisms underlying the pathogenesis of osteosarcoma.     

Involvement of miR-30c in resistance to doxorubicin by regulating YWHAZ in breast cancer cells

MicroRNAs (miRNAs) are small RNA molecules that modulate gene expression implicated in cancer, which play crucial roles in diverse biological processes, such as development, differentiation, apoptosis, and proliferation. The aim of this study was to investigate whether miR-30c mediated the resistance of breast cancer cells to the chemotherapeutic agent doxorubicin (ADR) by targeting tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta (YWHAZ). miR-30c was downregulated in the doxorubicin-resistant human breast cancer cell lines MCF-7/ADR and MDA-MB-231/ADR compared with their parental MCF-7 and MDA-MB-231 cell lines, respectively. Furthermore, we observed that transfection of an miR-30c mimic significantly suppressed the ability of MCF-7/ADR to resist doxorubicin. Moreover, the anti-apoptotic gene YWHAZ was confirmed as a target of miR-30c by luciferase reporter assay, and further studies indicated that the mechanism for miR-30c on the sensitivity of breast cancer cells involved YWHAZ and its downstream p38 mitogen-activated protein kinase (p38MAPK) pathway. Together, our findings provided evidence that miR-30c was one of the important miRNAs in doxorubicin resistance by regulating YWHAZ in the breast cancer cell line MCF-7/ADR.     

miR‐375‐3p/YWHAZ/β‐catenin axis regulates migration,invasion, EMT in gastric cancer cells

YWHAZ (14‐3‐3ζ) plays crucial roles in regulating proliferation, apoptosis, migration, and invasion of gastric cancer (GC) cells. However, its extensive roles and potential mechanisms in GC cells remain unknown, and need to be researched deeply. In this study, we focus on the role of miR‐375/YWHAZ axis in migration, invasion and epithelial‐to‐mesenchymal transition (EMT) of GC cells. YWHAZ level was assessed by western blot and qPCR assays in GC cells. Scratch and transwell assays were used to determine the migration and invasion of GC cells. The protein levels of correlative molecules were detected by western blot. The regulation of miR‐375 on the expression of its target gene YWHAZ was verified by dual‐luciferase report system. According to the results, knockdown of YWHAZ inhibited the migration, invasion and EMT of GC cells. Moreover, silencing of YWHAZ restrained the activation of wnt/β‐catenin signalling pathway. YWHAZ was confirmed to be a target gene of miR‐375, and its expression was regulated by miR‐375 in GC cells. Transfection of miR‐375 inhibitor promoted the migration, invasion, EMT and activation of wnt/β‐catenin pathway in GC cells, which was suppressed by inhibition of YWHAZ. Taken together, this study suggests that miR‐375/YWHAZ axis may be served as a novel therapeutic target for GC patients.     

Long noncoding RNA H19 acts as a miR‐340‐3p sponge to promote epithelial‐mesenchymal transition by regulating YWHAZ expression in paclitaxel‐resistant breast cancer cells

Breast cancer (BC) is the leading cause of cancer‐related death in women worldwide and one of the most prevalent malignancy. In recent years, increasing evidence had illuminated that long noncoding RNAs (lncRNAs) serve as critical factors in multiple tumor progression, including BC. Emerging references had indicated that the lncRNA H19 acts as significant roles in tumor progression and epithelial‐mesenchymal transition (EMT). However, the underlying molecular mechanisms and biological roles of H19 in BC invasion, metastasis and EMT are still unclear. In this study, it was detected that the expression level of H19 was increased in BC paclitaxel‐resistant (PR) cells subline (MCF‐7/PR) in comparison with MCF‐7 parental cells. In vitro, there were demonstrated that H19 overexpression promoted BC cells proliferation, metastasis, invasion and EMT procedures, and suppressed cells apoptosis. Whereas, H19 suppression resulted in the contrary biological effects. Besides, bioinformatics tools and dual‐luciferase reporters assays indicated that miR‐340‐3p could act as a potential target gene of H19, the underlying mechanism studies proved that H19 could act as a competing endogenous RNA (ceRNA) via competitively binding miR‐340‐3p to promote BC cell proliferation, metastasis and EMT by regulating tyrosine 3‐monooxygenase/tryptophan 5‐monooxygenase activation protein zeta (YWHAZ) and potentiate the Wnt/β‐catenin signaling in BC cells. In summary, our findings demonstrated that H19 could act as a ceRNA in BC progression, metastasis and EMT through modulating miR‐340‐3p/YWHAZ axis and activating the canonical Wnt/β‐catenin signaling pathway, indicating that H19 might act as an underlying therapeutic target and prognostic biomarker for BC therapy.     

LncRNA SNHG14 promotes the progression of cervical cancer by regulating miR-206/YWHAZ

Accumulating evidence suggests that lncRNAs play key roles in many cancers. It has been reported that long non‐coding RNA SNHG14 promotes cell proliferation and metastasis in multiple cancers. However, the role and underlying molecular mechanism of SNHG14 in cervical cancer (CC) remain largely unclear. In this study, we discovered that the relative expression of SNHG14 was significantly upregulated in CC tissues and cells, and associated with the overall survival of CC patients. Moreover, knockdown of SNHG14 significantly inhibited cell proliferation, migration and invasion, and promoted cell apoptosis in CC. Molecular mechanism explorations revealed that SNHG14 acted as a sponge of miR-206 and that YWHAZ was a downstream target gene of miR-206 in CC. Spearman’s correlation analysis uncovered a significantly negative correlation between SNHG14 (or YWHAZ) and miR-206 expression, while a significantly positive correlation between SNHG14 and YWHAZ expression in CC tissues. We also found that the effect of SNHG14 knockdown on the CC progression could be partly rescued by overexpression of YWHAZ at the same time. Our findings revealed that SNHG14 acted as a sponge of miR-206 to regulate the expression of YWHAZ in CC, hinting the promising therapeutic target role of SNHG4 for CC patients.     

RNA干扰YWHAZ基因对结肠癌HCT-8细胞增殖的影响

目的:探讨中RNA干扰YWHAZ基因对结肠癌HCT-8细胞增殖的影响,阐明其作用机制。方法:构建靶向基因YWHAZ的siRNA载体重组质粒pGPU6/GFP/Neo-YWHAZ-792、pGPU6/GFP/Neo-YWHAZ-733、pGPU6/GFP/Neo-YWHAZ-612和pGPU6/GFP/Neo-YWHAZ-505作为RNA干扰质粒组,同时设立阴性对照质粒组(pGPU6/GFP/Neo-shNC质粒);采用荧光定量PCR检测YWHAZ mRNA的表达水平和表达抑制率;采用MTT法检测HCT-8细胞的增殖率。结果:倒置荧光显微镜下观察,细胞转染率为60%。荧光定量PCR检测,RNA干扰组YWHAZ mRNA表达水平低于阴性对照组(P<0.01),平均抑制率为52.86%。MTT检测,与阴性对照组比较,RNA干扰质粒组细胞增殖率明显降低(P<0.01),平均细胞增殖率为68.75%。结论:干扰YWHAZ可抑制该基因的转录,并抑制HCT-8细胞的增殖。