Zinc Finger E-box Binding Homeobox Research Articles

CircPLOD2 knockdown suppresses endometriosis progression via the miR-216a-5p/ZEB1 axis

The present study aimed to identify the role of circPLOD2 in endometriosis and its underlying mechanisms. We determined circPLOD2 and miR-216a-5p expression in ectopic endometrial (EC) and eutopic endometrial (EU) samples as well as in endometrial samples from uterine fibroids of ectopic patients (EN) and embryonic stem cells (ESCs) using qRT-PCR. The association between circPLOD2 and miR-216a-5p or miR-216a-5p and zinc finger E-box binding homeobox 1 (ZEB1) expression was analyzed using Starbase, TargetScan, and dual-luciferase reporter gene assays. Cell viability, apoptosis, and migration and invasion were assessed using MTT, flow cytometry, and transwell assays, respectively. In addition, qRT-PCR and western blotting was used to measure circPLOD2, miR-216a-5p, E-cadherin, N-cadherin, and ZEB1 expression. circPLOD2 was upregulated and miR-216a-5p was downregulated in EC samples compared with that in EU samples. Similar trends were observed in ESCs. circPLOD2 interacted and negatively regulated miR-216a-5p expression in EC-ESCs. circPLOD2-siRNA significantly inhibited EC-ESC growth; promoted cellular apoptosis; and inhibited EC-ESC migration, invasion, and epithelial–mesenchymal transition; these effects could be reversed following miR-216a-5p inhibitor transfection. miR-216a-5p directly targeted and negatively regulated ZEB1 expression in EC-ESCs. In conclusion, circPLOD2 promotes the proliferation, migration, and invasion of EC-ESCs and inhibits their apoptosis by targeting miR-216a-5p. These findings indicate potential therapeutic targets for endometriosis.

Abstract 1480: ZEB1 dimerization is regulated by acetylation and promotes protein stability, interactions, EMT, and NSCLC metastasis

Abstract Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide due to the ability of cancer cells to metastasize. Therefore, it is essential to expand our current knowledge of the biological processes that contribute to metastasis to guide the discovery of novel therapeutic modalities. The Epithelial-to-mesenchymal transition (EMT) is a mechanism for the acquisition of malignant cells, which causes epithelial cells with specialized cell-cell contacts to gain mesenchymal migratory capacity. High expression of the Zinc finger E-box binding homeobox 1 (ZEB1) transcription factor is correlated with poor clinical outcomes of cancer, including therapeutic resistance and EMT-mediated metastasis. ZEB1 has a calculated molecular weight of 125kDa; however, numerous groups have reported discrepancies in the observed molecular weight (approximately 190-250kDa). This has been attributed to dimerization mediated by post-translational modifications (PTMs). Therefore, we performed mass spectrometry and identified a novel PTM - K811 acetylation - that may regulate ZEB1 dimerization and function. To define the role of ZEB1 acetylation, we generated ZEB1 acetyl mimetic (K811Q) and deficient (K811R) mutant-expressing NSCLC cell lines. We hypothesize that ZEB1 dimerization is regulated by K811 acetylation to promote protein stability and lung adenocarcinoma metastasis. We determined that the K811R ZEB1 (125 kDa) has a shorter half-life wild-type and high ubiquitination rate than (WT) ZEB1 and K811Q ZEB1 (~225 kDa), suggesting that disruption of ZEB1 acetylation hinders its dimerization and protein stability. Further, ZEB1 dimer recruits the nucleosome remodeling and deacetylase (NuRD) complex to bind the promoter of its target genes mir200c-141 and SEMA3F. RNA-sequencing revealed that unlike K811R ZEB1, WT ZEB1 and K811Q ZEB1 downregulate the expression of gene sets involved in tight junction, apical junction, and cell-cell junction formation. We also identified six novel ZEB1 acetylation-dependent gene candidates that function in signaling pathways and epithelial cell maintenance; CBLN3, STAP2, PLEKHG6, LAD1, RNF43, and LLGL2. In vitro 2D migration Boyden chamber, 3D invasion assays, and in vivo studies demonstrated that ZEB1 acetylation promotes the invasive and metastatic potential of the NSCLC cell line. Our findings suggest that K811 acetylation regulates ZEB1 dimerization and stability to promote NuRD interaction and lung adenocarcinoma via EMT. Future studies are focused on assessing changes in gene expression programs and chromatin accessibility that are ZEB1 dimerization dependent and that could contribute to NSCLC metastasis. Citation Format: Mabel Giselle Perez-Oquendo, Roxsan Manshouri, Yanhua Tian, Jared Fradette, Don Gibbons. ZEB1 dimerization is regulated by acetylation and promotes protein stability, interactions, EMT, and NSCLC metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1480.

EMT-activated secretory and endocytic vesicular trafficking programs underlie a vulnerability to PI4K2A antagonism in lung cancer.

Hypersecretory malignant cells underlie therapeutic resistance, metastasis, and poor clinical outcomes. However, the molecular basis for malignant hypersecretion remains obscure. Here, we showed that epithelial-mesenchymal transition (EMT) initiates exocytic and endocytic vesicular trafficking programs in lung cancer. The EMT-activating transcription factor zinc finger E-box-binding homeobox 1 (ZEB1) executed a PI4KIIIβ-to-PI4KIIα (PI4K2A) dependency switch that drove PI4P synthesis in the Golgi and endosomes. EMT enhanced the vulnerability of lung cancer cells to PI4K2A small-molecule antagonists. PI4K2A formed a MYOIIA-containing protein complex that facilitated secretory vesicle biogenesis in the Golgi, thereby establishing a hypersecretory state involving osteopontin (SPP1) and other prometastatic ligands. In the endosomal compartment, PI4K2A accelerated recycling of SPP1 receptors to complete an SPP1-dependent autocrine loop and interacted with HSP90 to prevent lysosomal degradation of AXL receptor tyrosine kinase, a driver of cell migration. These results show that EMT coordinates exocytic and endocytic vesicular trafficking to establish a therapeutically actionable hypersecretory state that drives lung cancer progression.

Sertoli cells-derived exosomal miR-30a-5p regulates ubiquitin E3 ligase Zeb2 to affect the spermatogonial stem cells proliferation and differentiation.

The role of spermatogonial stem cells (SSCs) is crucial in spermatogenesis, and extracellular vesicles (EVs) have been the focus of research as an important intercellular communication mechanism. Various endogenous regulatory factors secreted by Sertoli cells (SCs) can affect the self-maintenance and regeneration of SSCs, but little is known about the roles of SCs-derived exosomal microRNAs (miRNAs) on SSCs. In this study, we aimed to explore the regulation of the SCs-derived exosomal miR-30a-5p on SSCs proliferation and differentiation. EVs from the SCs were detected by electron microscopy and nanoparticle tracking analysis (NTA). Subsequently, the SSCs were treated with the SCs-derived extracellular vesicles (SCs-EVs). CCK-8 assay and EdU staining was applied to detect the cell proliferation, and the results indicated that SCs-EVs promoted the SSCs proliferation. Western blot detection of the SSCs markers (Gfrα1, Plzf, Stra8, and C-kit) indicated that SCs-EVs promoted the SSCs differentiation. Additionally, we found that SCs-EVs secreted miR-30a-5p to show the promoting effects. Besides, we discovered that miR-30a-5p targeted zinc finger E-box binding homeobox 2 (Zeb2) to regulate the ubiquitination of fibroblast growth factor 9 (Fgf9) in SSCs. miR-30a-3p/Zeb2/Fgf9 promoted the SSCs proliferation and differentiation by activating the mitogen‑activated protein kinase (MAPK) signaling pathway. Taken together, our study showed that SCs-EVs can transport miR-30a-5p to SSCs and affect SSCs proliferation and differentiation by regulating the MAPK signaling pathway via Zeb2/Fgf9. This paper disclosed a novel molecular mechanism that regulates SSCs proliferation and differentiation, which could be valuable for the treatment of male infertility.

The Dual Role of ZEB2 in COAD Metastasis and Immunology

Objective: The zinc finger E-box binding homeobox (ZEB2), which can accelerate the nuclear DNA replication by inducing the activation of upstream transcription promoters, was widely considered as an oncogene. Recent study has found that the overexpression of ZEB2 is associated with a better prognosis in hepatocellular carcinoma. However, its roles in tumor growth, metastasis, and immunology are yet to be elucidated in COAD. Methods: The pan-cancer sequencing data was acquired from The Cancer Genome Atlas (TCGA)-Pan cancer cohort, normal human tissue data was acquired from the Genotype-tissue expression (GTEx) database, and Broad Institute Cancer Cell Line Encyclopedia (CCLE) were downloaded from UCSC Xena. We used the cBioPortal webtool to analyze and visualize the ZEB2 pan-cancer genomic alteration rate. GEO Expression Datasets were used to explore ZEB2 expression levels in COAD patients. UCSC Xena database was used to download prognostic information of COAD patients. The Cox regression and Kaplan–Meier analyses were used to assess the prognostic role of ZEB2 in COAD. Kyoto Encyclopedia of Genes and Genomes (KEGG) was performed to determine the biological pathways. Gene Ontology (GO) enrichment analysis was performed to determine the biological processes, molecular functions, and cellular components that were altered in a ZEB2-dependent manner in COAD. The module analysis of PPI interaction network was performed using the MCODE tool of Cytoscape software, and the characteristic molecules were selected by cytohHubba tool. CIBERSORTx database was used to analyze the ZEB2 expression in the presence of 22 types of immune infiltrating cells. Results: This study found that ZEB2 was aberrantly expressed in most cancer types, and it was significantly downregulated in COAD compared with normal tissue. In addition, our findings also show that overexpression of ZEB2 was associated with a better prognosis in COAD. Mechanistic analysis revealed that overexpression of ZEB2 was associated with the neutrophil extracellular trap formation in COAD. And the results show that ZEB2 expression was significantly correlated with several kinds of immune cell infiltration. Conclusion: This study demonstrates that overexpression of ZEB2 was associated with better prognoses in patients with COAD. ZEB2 has close relationship with ACTB, which was highly related to NETs. These findings suggest a dual role of ZEB2 in COAD growth, metastasis, and immunology.

Overexpression of the telomerase holoenzyme induces EMT and tumorigenesis of HPV-immortalized keratinocytes.

Cervical cancer is the most frequent malignancy of the female genital tract and is associated with persistent infection of the uterine cervix with high-risk human papillomaviruses (HPV). The two HPV oncoproteins, E6 and E7, cooperatively immortalize cervical cells and are essential but insufficient for inducing tumorigenicity. During the progression of HPV-associated cervical dysplasia to carcinoma, the cellular telomerase reverse transcriptase (TERT) gene is activated and the TERC gene amplified. We questioned whether these increases in telomerase components might mediate the acquisition of the tumorigenic phenotype. We therefore transduced the TERT and TERC genes into E6/E7 immortalized keratinocytes that were anchorage-dependent and nontumorigenic. The resultant cells showed a profound morphological change characteristic of epithelial-mesenchymal transitionas well as a corresponding increase in expression of vimentin, N-cadherin, Zinc finger E-Box binding homeobox 1, snail family transcriptional repressor 1and matrix Metallopeptidase 2 and decrease in keratin and E-cadherin. More important, the transduced cells were now anchorage-independent and formed tumors in immunodeficient mice. Our findings indicate that overexpression of the telomerase holoenzyme in HPV-immortalized cells is sufficient to induce the complete transformed phenotype.

MiR-147a targets ZEB2 to regulate ox-LDL-induced monocyte adherence to HUVECs, atherosclerotic plaque formation, and stability in atherosclerosis

The mechanisms underlying atherosclerosis (AS) that seriously affect human health, such as those involved in endothelial cell injury and monocyte/macrophage aggregation and infiltration, have not been fully elucidated. To investigate these processes, we established human umbilical vein endothelial cells (HUVECs) injured by oxidized low-density lipoprotein (ox-LDL) to mimic AS invitro. Apolipoprotein E knockout (ApoE-/-) C57BL/6 mice were fed with a high-cholesterol diet to establish an AS model invivo. We detected HUVEC apoptosis, and apoptosis-related proteins by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide and lactate dehydrogenase, flow cytometry, and Western blot assays, respectively, and we observed monocytes (THP-1cells) adhering to HUVECs. Furthermore, miR-147a and its downstream target gene ZEB2 (zinc finger E-box binding homeobox 2) were predicted by bioinformatics analysis to be involved in AS, and their correlation was confirmed by several experiments. We determined the localization of miR-147a and ZEB2 within macrophages of AS mice by in situ hybridization and immunofluorescence. Atherosclerotic plaques in whole aortas were detected by histology observation. miR-147a attenuated adherence of monocytes to HUVECs and the upregulation of mononuclear chemotactic adhesion receptors in THP-1cells induced by ox-LDL-injured HUVEC supernatants through directly downregulating ZEB2 levels. Moreover, miR-147a influenced M1/M2 macrophage polarization from THP-1cells and the roles of their supernatants (THP-1cells) in HUVEC apoptosis. miR-147a targeted ZEB2 to impact lipid accumulation and atherosclerotic plaque formation through regulating M1/M2 polarization and macrophage adhesion in AS mice. In summary, miR-147a attenuates ox-LDL-induced adherence of monocytes to HUVECs and modulates atherosclerotic plaque formation and stability through targeting ZEB2 during AS.

Insulin-like growth factor 1 ameliorates pre-eclampsia by inhibiting zinc finger E-box binding homeobox 1 by up-regulation of microRNA-183.

As a common hypertensive complication of pregnancy, preeclampsia (PE) remains one of the leading causes of maternal and fetal with high morbidity and mortality worldwide. Much research has identified the vital functions of insulin-like growth factor 1 (IGF-1) in PE treatment. However, the combined roles and molecular mechanism of IGF-1 and microRNAs (miRNAs) underlying PE remain unclear. Therefore, we first measured the expression of IGF-1, zinc finger E-box binding homeobox 1 (ZEB1) and microRNA-183 (miR-183) expression in the placenta tissues of patients with PE by Western blot analysis and RT-qPCR. Interactions among IGF-1, ZEB1 and miR-183 were assessed by Western blot analysis, ChIP-PCR and dual-luciferase reporter gene assay. The effect of IGF-1 on the biological characteristics of trophoblast cells was investigated by CCK-8, colony formation assay and in vitro angiogenesis experiments after cells were transfected with si-IGF-1. Finally, a mouse eclampsia model induced by knockdown of IGF-1 was established to confirm the in vitro effect of IGF-1 on PE. We found that IGF-1, ZEB1 and miR-183 were highly expressed in the placental tissues of patients with PE. The knockdown of IGF-1 resulted in reduced proliferation and invasion of trophoblast cells and was accompanied by inhibited angiogenesis. ZEB1 was positively regulated by IGF-1 via ERK/MAPK pathway, which in turn inhibited miR-153 expression by binding to the miR-183 promoter. The in vitro experiments further confirmed that IGF-1 knockdown could induce PE. To sum up, IGF-1 knockdown elevated expression of miR-183 by downregulating ZEB1, thereby promoting deterioration of PE.

Endothelial HDAC1-ZEB2-NuRD Complex Drives Aortic Aneurysm and Dissection Through Regulation of Protein S-Sulfhydration.

Aortic aneurysm and aortic dissection (AAD) are life-threatening vascular diseases, with endothelium being the primary target for AAD treatment. Protein S-sulfhydration is a newly discovered posttranslational modification whose role in AAD has not yet been defined. This study aims to investigate whether protein S-sulfhydration in the endothelium regulates AAD and its underlying mechanism. Protein S-sulfhydration in endothelial cells (ECs) during AAD was detected and hub genes regulating homeostasis of the endothelium were identified. Clinical data of patients with AAD and healthy controls were collected, and the level of the cystathionine γ lyase (CSE)/hydrogen sulfide (H2S) system in plasma and aortic tissue were determined. Mice with EC-specific CSE deletion or overexpression were generated, and the progression of AAD was determined. Unbiased proteomics and coimmunoprecipitation combined with mass spectrometry analysis were conducted to determine the upstream regulators of the CSE/H2S system and the findings were confirmed in transgenic mice. Higher plasma H2S levels were associated with a lower risk of AAD, after adjustment for common risk factors. CSE was reduced in the endothelium of AAD mouse and aorta of patients with AAD. Protein S-sulfhydration was reduced in the endothelium during AAD and protein disulfide isomerase (PDI) was the main target. S-sulfhydration of PDI at Cys343 and Cys400 enhanced PDI activity and mitigated endoplasmic reticulum stress. EC-specific CSE deletion was exacerbated, and EC-specific overexpression of CSE alleviated the progression of AAD through regulating the S-sulfhydration of PDI. ZEB2 (zinc finger E-box binding homeobox 2) recruited the HDAC1-NuRD complex (histone deacetylase 1-nucleosome remodeling and deacetylase) to repress the transcription of CTH, the gene encoding CSE, and inhibited PDI S-sulfhydration. EC-specific HDAC1 deletion increased PDI S-sulfhydration and alleviated AAD. Increasing PDI S-sulfhydration with the H2S donor GYY4137 or pharmacologically inhibiting HDAC1 activity with entinostat alleviated the progression of AAD. Decreased plasma H2S levels are associated with an increased risk of aortic dissection. The endothelial ZEB2-HDAC1-NuRD complex transcriptionally represses CTH, impairs PDI S-sulfhydration, and drives AAD. The regulation of this pathway effectively prevents AAD progression.

Effect of ZEB1 Associated with microRNAs on Tumor Stem Cells in Head and Neck Cancer

Cancer biologists have focused on studying cancer stem cells (CSCs) because of their ability to self-renew and recapitulate tumor heterogeneity, which increases their resistance to chemotherapy and is associated with cancer relapse. Here, we used two approaches to isolate CSCs: the first involved the metabolic enzyme aldehyde dehydrogenase ALDH, and the second involved the three cell surface markers CD44, CD117, and CD133. ALDH cells showed a higher zinc finger E-box binding homeobox 1 (ZEB1) microRNA (miRNA) expression than CD44/CD117/133 triple-positive cells, which overexpressed miRNA 200c-3p: a well-known microRNA ZEB1 inhibitor. We found that ZEB1 inhibition was driven by miR-101-3p, miR-139-5p, miR-144-3p, miR-199b-5p, and miR-200c-3p and that the FaDu Cell Line inhibition occurred at the mRNA level, whereas HN13 did not affect mRNA expression but decreased protein levels. Furthermore, we demonstrated the ability of the ZEB1 inhibitor miRNAs to modulate CSC-related genes, such as TrkB, ALDH, NANOG, and HIF1A, using transfection technology. We showed that ALDH was upregulated upon ZEB1-suppressed miRNA transfection (Mann-Whitney ** p101 = 0.009, t-test ** p139 = 0.009, t-test ** p144 = 0.002, and t-test *** p199 = 0.0006). Overall, our study enabled an improved understanding of the role of ZEB1-suppressed miRNAs in CSC biology.

Ploidy-stratified single cardiomyocyte transcriptomics map Zinc Finger E-Box Binding Homeobox 1 to underly cardiomyocyte proliferation before birth

Whereas cardiomyocytes (CMs) in the fetal heart divide, postnatal CMs fail to undergo karyokinesis and/or cytokinesis and therefore become polyploid or binucleated, a key process in terminal CM differentiation. This switch from a diploid proliferative CM to a terminally differentiated polyploid CM remains an enigma and seems an obstacle for heart regeneration. Here, we set out to identify the transcriptional landscape of CMs around birth using single cell RNA sequencing (scRNA-seq) to predict transcription factors (TFs) involved in CM proliferation and terminal differentiation. To this end, we established an approach combining fluorescence activated cell sorting (FACS) with scRNA-seq of fixed CMs from developing (E16.5, P1, and P5) mouse hearts, and generated high-resolution single-cell transcriptomic maps of in vivo diploid and tetraploid CMs, increasing the CM resolution. We identified TF-networks regulating the G2/M phases of developing CMs around birth. ZEB1 (Zinc Finger E-Box Binding Homeobox 1), a hereto unknown TF in CM cell cycling, was found to regulate the highest number of cell cycle genes in cycling CMs at E16.5 but was downregulated around birth. CM ZEB1-knockdown reduced proliferation of E16.5 CMs, while ZEB1 overexpression at P0 after birth resulted in CM endoreplication. These data thus provide a ploidy stratified transcriptomic map of developing CMs and bring new insight to CM proliferation and endoreplication identifying ZEB1 as a key player in these processes.

Knockdown of PLAGL2 in hibits oral squamous cell carcinoma cell growth and cisplatin resistance via ZEB1/PD L1 pathway

Purpose: To investigate the effects of pleomorphic adenoma gene like-2 (PLAGL2) on oral squamous cell carcinoma (OSCC).
 Methods: The effects of PLAGL2 on OSCC (CAL-27 and HSC-3) were conducted with loss- and gain-functional assays. Cell proliferation was determined by Cell Counting Kit-8 (CCK8) and colony formation assays. Cell metastasis was assessed by Transwell and wound healing assays. Cell apoptosis of cisplatin-resistant OSCC was evaluated by flow cytometry.
 Results: PLAGL2 expression was significantly elevated in OSCC (p < 0.001), while silencing of PLAGL2 significantly reduced cell proliferation and metastasis of OSCC (p < 0.001). However, overexpression of PLAGL2 promoted OSCC progression through increase in cell proliferation, invasion, and migration. Knockdown of PLAGL2 promoted cell apoptosis of -resistant OSCC, but significantly downregulated protein expression of programmed cell death ligand 1(PD-L1) and zinc finger E-box-binding homeobox 1 (ZEB1) in OSCC (p < 0.01). Moreover, loss of ZEB1 attenuated the PLAGL2 overexpression-induced increase in ZEB1 and PD-L1. Loss of PLAGL2 also reduced in vivo tumor growth of OSCC via regulation of cluster of differentiation 4 protein (CD4) and CD8.
 Conclusion: Knockdown of PLAGL2 exerts anti-tumor effects, improves cisplatin resistance, and enhances anti-tumor immunity in OSCC through suppression of ZEB1/PD-L1 pathway. Thus, PLAGL2 might be a potential therapeutic target for the treatment of OSCC.

The aryl sulfonamide indisulam inhibits gastric cancer cell migration by promoting the ubiquitination and degradation of the transcription factor ZEB1

Gastric cancer is one of the cancers with high morbidity and mortality worldwide. The aryl sulfonamide indisulam inhibits the proliferation of several types of cancer cells through its function as a molecular glue to promote the ubiquitination and degradation of RNA-binding motif protein 39 (RBM39). However, it is unknown whether and how indisulam regulates the migration of cancer cells. In this work, using label-free quantitative proteomics, we discover that indisulam significantly attenuates N-cadherin, a marker for epithelial to mesenchymal transition and migration of cancer cells. Our bioinformatics analysis and biochemical experiments reveal that indisulam promotes the interaction between the zinc finger E-box-binding homeobox 1 (ZEB1), a transcription factor of N-cadherin, and DCAF15, a substrate receptor of CRL4 E3 ubiquitin ligase, and enhances ZEB1 ubiquitination and proteasomal degradation. In addition, our cell line-based experiments demonstrate that indisulam inhibits the migration of gastric cancer cells in a ZEB1-dependent manner. Analyses of patient samples and datasets in public databases reveal that tumor tissues from patients with gastric cancer express high ZEB1 mRNA and this high expression reduces patient survival rate. Finally, we show that treatment of gastric tumor samples with indisulam significantly reduces ZEB1 protein levels. Therefore, this work discloses a new mechanism by which indisulam inhibits the migration of gastric cancer cells, indicating that indisulam exhibits different biological functions through distinct signaling molecules.

Exploration and validation of the influence of angiogenesis-related factors in aortic valve calcification.

Over the years, bioinformatics tools have been used to identify functional genes. In the present study, bioinformatics analyses were conducted to explore the underlying molecular mechanisms of angiogenic factors in calcific aortic valve disease (CAVD). The raw gene expression profiles were from datasets GSE153555, GSE83453, and GSE51472, and the angiogenesis-related gene set was from the Gene Set Enrichment Analysis database (GSEA). In this study, R was used to screen for differentially expressed genes (DEGs) and co-expressed genes. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genome (KEGG) Pathway enrichment analysis were performed on DEGs and validated in clinical samples. DEGs in CAVD were significantly enriched in numerous immune response pathways, inflammatory response pathways and angiogenesis-related pathways. Nine highly expressed angiogenesis-related genes were identified, of which secretogranin II (SCG2) was the most critical gene. MiRNA and transcription factors (TFs) networks were established centered on five DEGs, and zinc finger E-box binding homeobox 1 (ZEB1) was the most important transcription factor, verified by PCR, immunohistochemical staining and western blotting experiments. Overall, this study identified key genes and TFs that may be involved in the pathogenesis of CAVD and may have promising applications in the treatment of CAVD.

Bilateral varicocele leads to ferroptosis, pyroptosis and necroptosis of human spermatozoa and affects semen quality in infertile men.

Purpose: This study explored the effects of bilateral varicocele on male semen quality in infertile men and the molecular mechanisms involving ferroptosis, pyroptosis and necroptosis signaling pathways. Methods: Totally, 20 healthy males and 26 patients with bilateral varicocele receiving infertility treatment were enrolled. Semen samples were collected. Basic semen parameters, acrosome integrity and membrane integrity, mitochondrial membrane potential (MMP) and apoptosis rate were compared. Levels of reactive oxygen species (ROS), iron, glutathione (GSH), total superoxide dismutase (T-SOD), and, Catalase (CAT), were detected in human seminal plasma. Relative mRNA expression of Ca 2+-independent phospholipases A2 beta (iPLA 2β), P53, Zinc finger E-box binding homeobox 1 (ZEB1) and GSH-dependent peroxidase 4 (GPX4) were evaluated. Relative protein expression was determined for GPX4, receptor interacting serine/threonine kinase 1 (RIPK1) and receptor interacting serine/threonine kinase 3 (RIPK3), as well as pyroptosis markers of Gasdermin E (GSDME) and heat shock protein 90 (HSP 90). Results: The results revealed that the bilateral varicocele group had significantly higher abnormalities (sperm progressive rate and sperm motility) compared to the control group. Meanwhile, compared to control group, GSH, T-SOD, and CAT levels were reduced in the bilateral varicocele group (p < 0.05). However, the level of ROS and iron were significantly increased (p < 0.05). Relative mRNA expression of P53, iPLA 2β, ZEB1, and GPX4 were reduced. In addition, ROS exposure activated ferroptosis-related signal pathways. RIPK1, RIPK3, GSDME and HSP 90 were increased in bilateral varicocele group. ROS exposure affected signaling pathways related to ferroptosis, necrosis and pyroptosis in human spermatozoa. Conclusion: Bilateral varicocele leads to ferroptosis, pyroptosis and necroptosis of human spermatozoa and affects semen quality in infertile men.

CSTF2T up-regulates IGHG1 by binding to ZEB1 to promote melanoma cell proliferation, migration, and invasion

ObjectivesMelanoma progression involves multiple molecular pathways. In this study, we explored the effect of immunoglobulin heavy constant gamma 1 (IGHG1) on melanoma progression. MethodsIGHG1, zinc finger E-box binding homeobox 1 (ZEB1), and cleavage stimulation factor subunit 2 tau variant (CSTF2T) expression levels were measured by quantitative reverse-transcription polymerase chain reaction and western blot. BALB/c nude mice were subcutaneously injected with A375 cells to develop a transplantation tumor model, 4 weeks after which the tumor tissues were collected. Cell proliferation, cell invasion, and cell migration of A375 and SK-MEL-28 cells were measured after gain- and loss-of-function of IGHG1, ZEB1, and CSTF2T. Binding of ZEB1 to the IGHG1 promoter was assayed by chromatin immunoprecipitation and dual-luciferase reporter gene assays, while binding of CSTF2T to ZEB1 mRNA was investigated by RNA immunoprecipitation and RNA pull-down assays. ResultsIGHG1, ZEB1, and CSTF2T were all highly expressed in human melanoma cell lines. In A375 and SK-MEL-28 cells, ZEB1 binds directly to the IGHG1 promoter. ZEB1 silencing reduced IGHG1 expression and melanoma cell proliferative, invasive, and migratory properties, and these cellular effects were nullified by overexpression of IGHG1. CSTF2T binds directly to ZEB1 mRNA. Silencing of CSTF2T diminished ZEB1 expression and restrained melanoma cell proliferative, invasive, and migratory capabilities. The impacts of CSTF2T silencing on melanoma cells were counteracted by concomitant overexpression of ZEB1. CSTF2T knockdown reduced tumor volume and weight in nude mice. ConclusionsThe CSTF2T–ZEB1–IGHG1 axis promotes melanoma cell proliferation and invasion.

PLAC8 contributes to the malignant behaviors of cervical cancer cells by activating the SOX4-mediated AKT pathway.

Cervical cancer (CC) is the primary cancer-related cause of morbidity and mortality in women. Previous studies have shown that placenta-specific 8 (PLAC8) has different functions in multiple malignancies. This study aimed to explore the function and regulatory mechanism of PLAC8 in CC. Bioinformatics and immunohistochemical analyses demonstrated that PLAC8 was significantly upregulated in CC tissues compared with normal tissues. Gain/loss-of-function experiments showed that siRNA-mediated knockdown of PLAC8 suppressed cell migration and invasion, while PLAC8 overexpression promoted cell motility. Moreover, PLAC8 was revealed to affect the epithelial-mesenchymal transition (EMT) process by upregulating epithelial (E)-cadherin and decreasing the expression of mesenchymal markers of EMT, including vimentin, zinc finger E-box binding homeobox 1 (ZEB1), neural (N)-cadherin, matrix metalloproteinase-9 (MMP-9), and MMP-2 in PLAC8-silenced cells. PLAC8 activated the AKT pathway, as proven by the downregulation of p-AKTSer473 and p-AKTThr308 expression after PLAC8 knockdown. Furthermore, PLAC8 overexpression upregulated the expression of sex-determining region Y-related high-mobility group box transcription factor 4 (SOX4), which is reported to mediate the activation of the AKT pathway, and SOX4 deficiency reversed the cellular functions caused by PLAC8 overexpression. Overall, the present study indicates that PLAC8 may facilitate CC development by activating the SOX4-mediated AKT pathway, suggesting that PLAC8 may serve as a potential biomarker for CC treatment.

Long noncoding RNA MALAT1 inhibition attenuates sepsis-induced acute lung injury through modulating the miR-129-5p/PAX6/ZEB2 axis.

This research aimed to investigate the role of the long noncoding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1)/microRNA-129-5p (miR-129-5p)/paired box gene 6 (PAX6) axis in sepsis-induced acute lung injury (ALI). MLE-12 cells and C57BL/6 mice were induced by LPS to establish lung injury in in vitro and in vivo models. Cell viability and apoptosis were measured by cell counting kit-8 assay and TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) staining, respectively. Levels of inflammatory cytokines in cell supernatants and bronchoalveolar lavage fluid (BALF) were detected by ELISA. Lung injury was evaluated by lung wet weight-to-dry weight ratio and hematoxylin-eosin staining. MALAT1, PAX6, and zinc finger E-box-binding homeobox 2 (ZEB2) expression was elevated and miR-129-5p expression was reduced in the serum of patients with sepsis-induced ALI, LPS-induced MLE-12 cells, and lung tissues of ALI mice. MALAT1 interference delayed the LPS-induced cell proliferation decrease, apoptosis increase, and inflammatory factor increase. miR-129-5p inhibition could reverse the delaying effect of MALAT1 interference on LPS-induced lung cell injury. PAX6 overexpression (oe) reversed the inhibitory effect of miR-129-5p oe on LPS-induced lung cell injury. Downregulating MALAT1 reduced pulmonary edema, inflammatory cytokine levels, lung injury, and apoptosis in ALI mice. Moreover, miR-129-5p suppression or PAX6 oe reversed the delaying effect of MALAT1 interference on sepsis-induced ALI. MALAT1 aggravates sepsis-induced ALI via the miR-129-5p/PAX6/ZEB2 axis.

The rat Downunder (Du) coat color mutation is associated with eye anomalies and embryonic lethality and maps to a 3.9-Mb region on chromosome 3.

Rodent coat color genes have been studied as a bioresource to understand developmental and cellular processes. The Downunder rat is a fancy variety with a marking on its belly that runs from the neck to the breech and appears to mirror the dorsal hooded marking. Here, we established a congenic strain carrying the Downunder (Du) gene in an F344 genetic background. In addition to the ventral marking, Du/+ rats exhibit anophthalmia or microphthalmia with incomplete penetrance. Du/Du embryos die in the early stages of organogenesis. Genetic linkage analysis mapped the Du gene to rat chromosome 3 and haplotype mapping with congenic rats localized the Du locus to a 3.9-Mb region. The Du locus includes two functional genes, glycosyltransferase-like domain-containing 1 (Gtdc1) and zinc finger E-box binding homeobox 2 (Zeb2). Although we found no functional variation within any of Zeb2's exons or intron-exon boundaries, Zeb2 mRNA levels were significantly lower in Du/+ rats compared with wild-type rats. It is known that melanocyte-specific Zeb2 deletion results in the congenital loss of hair pigmentation in mice. Taken together, our results indicate that the Du mutation exerts pleiotropic effects on hair pigmentation, eye morphology, and development. Moreover, the Zeb2 gene is a strong candidate for the Du mutation.

ZEB1 Transcriptionally Activates PHGDH to Facilitate Carcinogenesis and Progression of HCC

Phosphoglycerate dehydrogenase (PHGDH), the rate-limiting enzyme of the de novo serine synthesis pathway (SSP), has been implicated in the carcinogenesis and metastasis of hepatocellular carcinoma (HCC) because of its excessive expression and promotion of SSP. In previous experiments we found that SSP flux was diminished by knockdown of zinc finger E-box binding homeobox 1 (ZEB1), a stimulator of HCC metastasis, but the underlying mechanism remains largely unknown. Here, we aimed to determine how SSP flux is regulated by ZEB1 and the contribution of such regulation to carcinogenesis and progression of HCC. We used genetic mice with Zeb1 knockout in liver specifically to determine whether Zeb1 deficiency impacts HCC induced by the carcinogen diethylnitrosamine plus CCl4. We explored the regulatory mechanism of ZEB1 in SSP flux using uniformly-labeled [13C]-glucose tracing analyses, liquid chromatography-mass spectrometry, real-time quantitative polymerase chain reaction, luciferase report assay, and chromatin immunoprecipitation assay. We determined the contribution of the ZEB1-PHGDH regulatory axis to carcinogenesis and metastasis of HCC by cell counting assay, methyl thiazolyl tetrazolium (MTT) assay, scratch wound assay, Transwell assay, and soft agar assay invitro, orthotopic xenograft, bioluminescence, and H&E assays invivo. We investigated the clinical relevance of ZEB1 and PHGDH by analyzing publicly available data sets and 48 pairs of HCC clinical specimens. We identified that ZEB1 activates PHGDH transcription by binding to a nonclassic binding site within its promoter region. Up-regulated PHGDH augments SSP flux to enable HCC cells to be more invasive, proliferative, and resistant to reactive oxygen species and sorafenib. Orthotopic xenograft and bioluminescence assays have shown that ZEB1 deficiency significantly impairs the tumorigenesis and metastasis of HCC, and such impairments can be rescued to a large extent by exogenous expression of PHGDH. These results were confirmed by the observation that conditional knockout of ZEB1 in mouse liver dramatically impedes carcinogenesis and progression of HCC induced by diethylnitrosamine/CCl4, as well as PHGDH expression. In addition, analysis of The Cancer Genome Atlas database and clinical HCC samples showed that the ZEB1-PHGDH regulatory axis predicts poor prognosis of HCC. ZEB1 plays a crucial role in stimulating carcinogenesis and progression of HCC by activating PHGDH transcription and subsequent SSP flux, deepening our knowledge of ZEB1 as a transcriptional factor in fostering the development of HCC via reprogramming the metabolic pathway.