Zinc Finger E-box Binding Homeobox Research Articles

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.

TRIM9 Interacts with ZEB1 to Suppress Esophageal Cancer by Promoting ZEB1 Protein Degradation via the UPP Pathway.

Esophageal cancer remains one of the most lethal malignant diseases globally. Previous studies indicated that TRIM9 (Tripartite Motif Containing 9) is a potential marker in breast cancer patients. Therefore, in the current research, we intended to clarify the regulatory network of TRIM9 and its relative role in esophageal cancer patients. We aimed to elucidate the regulatory role of TRIM9 in esophageal cancer. Clinical tumor tissue samples combined with cancer cell line models were utilized to explore the TRIM9 expression pattern. Functional experiments including transwell assay, cell viability assay, and ubiquitination blocking experiments were performed to evaluate the role of the TRIM9/ZEB1 (zinc finger E-box binding homeobox 1) axis and UPP pathway in esophageal cancer progression and exacerbation. Both esophageal cancer samples and cell line models showed significantly suppressed levels of TRIM9. Functional experiments confirmed that TRIM9 overexpression inhibited the cell viability, invasiveness, and stem-like phenotype of cancer cells. Subsequent investigations suggested that TRIM9-ZEB1 interaction accelerated ZEB1 protein degradation through the modulation of the UPP pathway, which confirmed the protective role of TRIM9 in esophageal cancer progression and metastasis. This study concluded that TRIM9 was a tumor suppressor that interacted with ZEB1 and accelerated ZEB1 protein degradation via the ubiquitin-proteasome pathway (UPP). Our research emphasized TRIM9-ZEB1 interaction as a valuable target for esophageal cancer treatment in future development. More detailed studies are needed to further consolidate our findings.

Genetic Engineered Ultrasound-Triggered Injectable Hydrogels for Promoting Bone Reconstruction

Genetic engineering technology can achieve specific gene therapy for a variety of diseases, but the current strategy still has some flaws, such as a complex system, single treatment, and large implantation trauma. Herein, the genetic engineering injectable hydrogels were constructed by ultrasonic technology for the first time to realize in vivo ultrasound-triggered in situ cross-linking and cell gene transfection, and finally complete in situ gene therapy to promote bone reconstruction. First, ultrasound-triggered calcium release was used to activate transglutaminase and catalyze the transamidation between fibrinogen. Simultaneously, liposome loaded with Zinc-finger E-box-binding homeobox 1 (ZEB1) gene plasmid (Lip-ZEB1) was combined to construct an ultrasound-triggered in situ cross-linked hydrogels that can deliver Lip-ZEB1. Second, ultrasound-triggered injectable hydrogel introduced ZEB1 gene plasmid into endothelial cell genome through Lip-ZEB1 sustained release, and then acted on the ZEB1/Notch signal pathway of cells, promoting angiogenesis and local bone reconstruction of osteoporosis through genetic engineering. Overall, this strategy provides an advanced gene delivery system through genetic engineered ultrasound-triggered injectable hydrogels.

Transcription factor ZEB1 regulates PLK1-mediated SKA3 phosphorylation to promote lung cancer cell proliferation, migration and cell cycle.

Lung cancer (LC) is one of the most common malignancies worldwide with low 5-year survival rate. The mechanism of spindle and kinetochore-associated complex subunit 3 (SKA3) in LC tumorgenesis remains largely unclear. The expression of SKA3 in LC cells was detected by quantitative PCR. Cell proliferation, migration and cell cycle were evaluated by functional assays including 5-ethynyl-2'-deoxyuridine, wound healing, transwell assays and flow cytometry analysis. Bioinformatics analysis, chromatin immunoprecipitation, luciferase reporter, co-immunoprecipitation and in vitro phosphorylation assays were applied to explore the interactions between zinc finger E-box binding homeobox 1 (ZEB1) and SKA3/polo-like kinase 1 (PLK1). SKA3 is highly expressed in LC cell lines and drives LC cell proliferation, migration and cell cycle. PLK1 also enhances the malignancy of LC cells. PLK1 can mediate SKA3 phosphorylation and enhance the stability of SKA3 protein, thus promoting LC progression. Besides, we found that transcription factor ZEB1 transcriptionally activates SKA3/PLK1 expression, contributing to LC cell malignancy. This study demonstrated that transcription factor ZEB1 modulates PLK1-mediated SKA3 phosphorylation to accelerate LC cell growth, migration and cycle, which might offer novel insight into LC treatment.

Magnolol regulates miR-200c-3p to inhibit epithelial–mesenchymal transition and retinoblastoma progression by modulating the ZEB1/E-cadherin axis in vitro and in vivo

BackgroundRetinoblastoma, the most common pediatric intraocular malignancy, can develop during embryogenesis, with most children being diagnosed at 3–4 years of age. Multimodal therapies are typically associated with high levels of cytotoxicity and side effects. Therefore, the development of novel treatments with minimal side effects is crucial. Magnolol has a significant anti-tumor effect on various cancers. However, its antitumor effect on retinoblastoma remains unclear. PurposeThe study aimed to determine the effects of magnolol on the regulation of EMT, migration, invasion, and cancer progression in retinoblastoma and the modulation of miR-200c-3p expression and the Wnt/ zinc finger E-box binding homeobox 1 (ZEB1)/E-cadherin axis in vivo and in vitro. MethodsThe 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) assay was used to evaluate magnolol-induced cell toxicity in the Y79 retinoblastoma cell line. Flow cytometry and immunostaining assays were performed to investigate the magnolol-regulated mitochondrial membrane potential and the intracellular and mitochondrial reactive oxygen species levels in Y79 retinoblastoma cells. Orthotopic and subcutaneous xenograft experiments were performed in eight-week-old male null mice to study retinoblastoma progression and metastasis. In situ hybridization and quantitative reverse transcription polymerase chain reaction (RT-qPCR) assays were performed to evaluate the level of the anti-cancer miRNA miR-200c-3p. The mRNA and protein levels of E-cadherin, β-catenin, α-smooth muscle actin (α-SMA), fibronectin-1, and ZEB1 were analyzed using RT-qPCR, immunoblot, immunocytochemistry, and immunohistochemistry assays in vitro and in vivo. ResultsMagnolol increased E-cadherin levels and reduced the activation of the EMT signaling pathway, EMT, tumor growth, metastasis, and cancer progression in the Y79 retinoblastoma cell line as well as in the orthotopic and subcutaneous xenograft animal models. Furthermore, magnolol increased the expression of miR-200c-3p. Our results demonstrate that miRNA-200c-3p inhibits EMT progression through the Wnt16/β-catenin/ZEB1/E-cadherin axis, and the ZEB1 silencing response shows that miR-200c-3p regulates ZEB1-mediated EMT in retinoblastoma. ConclusionMagnolol has an antitumor effect by increasing E-cadherin and miRNA-200c-3p expression to regulate ZEB1-mediated EMT and cancer progression in retinoblastoma. The anti-tumor effect of magnolol by increasing E-cadherin and miRNA-200c-3p expression to regulate ZEB1-mediated EMT and cancer progression in retinoblastoma has been elucidated for the first time.

Immunohistochemical Markers of the Epithelial-to-Mesenchymal Transition (EMT) Are Related to Extensive Lymph Nodal Spread, Peritoneal Dissemination, and Poor Prognosis in the Microsatellite-Stable Diffuse Histotype of Gastric Cancer.

Although the prognostic value of the epithelial-to-mesenchymal transition (EMT) in gastric cancer has been reported in several studies, the strong association with the diffuse type may represent a confounding factor. Our aim is to investigate potential correlations among EMT status, tumor advancement, and prognosis in diffuse gastric cancer. Between 1997 and 2012, 84 patients with microsatellite-stable (MSS) diffuse-type tumors underwent surgery. The EMT phenotype was assessed with the E-cadherin, CD44, and zinc finger E-box binding homeobox 1 (ZEB-1) immunohistochemical markers. Forty-five out of 84 cases (54%) were EMT-positive; more advanced nodal status (p = 0.010), pTNM stage (p = 0.032), and vascular invasion (p = 0.037) were observed in this group. The median numbers of positive nodes (13 vs. 5) and involved nodal stations (4 vs. 2) were higher in the EMT-positive group. The cancer-related survival time was 26 months in EMT-positive cases vs. 51 in negative cases, with five-year survival rates of 17% vs. 51%, respectively (p = 0.001). The EMT status had an impact on the prognosis of patients with &lt;70 years, R0 resections, or treatment with adjuvant chemotherapy. Tumor relapses after surgery and peritoneal spread were significantly higher in the EMT-positive tumors. EMT status, when assessed through immunohistochemistry, identified an aggressive phenotype of MSS diffuse-type tumors with extensive lymph nodal spread, peritoneal dissemination, and worse long-term outcomes.

MiR-145-5p Targets Sp1 in Non-Small Cell Lung Cancer Cells and Links to BMI1 Induced Pemetrexed Resistance and Epithelial–Mesenchymal Transition

Pemetrexed is a folic acid inhibitor used as a second-line chemotherapeutic agent for the treatment of locally advanced or metastatic non-small cell lung cancer (NSCLC), which accounts for 85% of lung cancers. However, prolonged treatment with pemetrexed may cause cancer cells to develop resistance. In this study, we found increased expressions of BMI1 (B Lymphoma Mo-MLV insertion region 1 homolog) and Sp1 and a decreased expression of miR-145-5p was found in pemetrexed-resistant A400 cells than in A549 cells. Direct Sp1 targeting activity of miR-145-5p was demonstrated by a luciferase based Sp1 3'-UTR reporter. Changed expression of miR-145-5p in A400 or A549 cells by transfection of miR-145-5p mimic or inhibitor affected the sensitivity of the cells to pemetrexed. On the other hand, the overexpression of Sp1 in A549 cells caused the decreased sensitivity to pemetrexed, induced cell migratory capability, and epithelial-mesenchymal transition (EMT) related transcription factors such as Snail Family Transcriptional Repressor 1 and Zinc Finger E-Box Binding Homeobox 1. In addition, the overexpression of BMI1 in the A549 cells resulted in an increase in Sp1 and a decrease in miR-145-5p accompanied by the elevations of cell proliferation and EMT transcription factors, which could be reduced by the overexpression of miR-145-5p or by treatment with the Sp1 inhibitor of mithramycin A. In conclusion, the results of this study suggest that the downregulation of miR-145-5p by BMI1 overexpression could lead to the enhanced expression of Sp1 to induce the EMT process in pemetrexed-resistant NSCLC cells. These results suggest that increasing miR-145-5p expression by delivering RNA drugs may serve as a sensitizing agent for pemetrexed-resistant NSCLC patients.

Glomerular Endothelial Cell-Derived miR-200c Impairs Glomerular Homeostasis by Targeting Podocyte VEGF-A.

Deciphering the pathophysiological mechanisms of primary podocytopathies that can lead to end-stage renal disease and increased mortality is an unmet need. Studying how microRNAs (miRs) interfere with various signaling pathways enables identification of pathomechanisms, novel biomarkers and potential therapeutic options. We investigated the expression of miR-200c in urine from patients with different renal diseases as a potential candidate involved in podocytopathies. The role of miR-200c for the glomerulus and its potential targets were studied in cultured human podocytes, human glomerular endothelial cells and in the zebrafish model. miR-200c was upregulated in urine from patients with minimal change disease, membranous glomerulonephritis and focal segmental glomerulosclerosis and also in transforming growth factor beta (TGF-β) stressed glomerular endothelial cells, but not in podocytes. In zebrafish, miR-200c overexpression caused proteinuria, edema, podocyte foot process effacement and glomerular endotheliosis. Although zinc finger E-Box binding homeobox 1/2 (ZEB1/2), important in epithelial to mesenchymal transition (EMT), are prominent targets of miR-200c, their downregulation did not explain our zebrafish phenotype. We detected decreased vegfaa/bb in zebrafish overexpressing miR-200c and could further prove that miR-200c decreased VEGF-A expression and secretion in cultured human podocytes. We hypothesize that miR-200c is released from glomerular endothelial cells during cell stress and acts in a paracrine, autocrine, as well as context-dependent manner in the glomerulus. MiR-200c can cause glomerular damage most likely due to the reduction of podocyte VEGF-A. In contrast, miR-200c might also influence ZEB expression and therefore EMT, which might be important in other conditions. Therefore, we propose that miR-200c-mediated effects in the glomerulus are context-sensitive.