Snail Family Transcriptional Repressor Research Articles

Dynamin 3 Inhibits the Proliferation of Non-small-Cell Lung Cancer Cells by Suppressing c-MET-GBR2-STAT3 Complex Formation.

Dynamin 3 (DNM3) has gained increased attention ever since its potential as a tumor suppressor was reported. However, its action in lung cancer (LC) is undefined. In this study, the role of DNM3 in LC development was investigated. DNM3 expression was found to be downregulated in tumors of patients with LC, especially those with metastasis. The DNM3 downregulation enhanced the proliferative and metastatic ability of LC cells, whereas its upregulation had the opposite effects. In vivo xenograft experiments confirmed that lung tumors with lower DNM3 expression had higher growth and metastatic abilities. Mechanistic studies revealed that DNM3 interacts with growth factor receptor-bound protein 2 (GBR2), thereby interrupting tyrosine-protein kinase Met (c-MET)–GBR2–signal transducer and activator of transcription 3 (STAT3) complex formation, which suppressed STAT3 activation. Therefore, the absence of DNM3 frees GBR2 to activate STAT3, which regulates the expression of genes related to LC proliferation and metastasis (e.g., cyclin D1 and Snail family transcriptional repressor 1). Additionally, the c-MET inhibitor crizotinib effectively suppressed LC cell proliferation and migration in vitro and in vivo, even with DNM3 depleted. Therefore, our study has demonstrated the antitumor effect of DNM3 in LC and suggests that the inhibition of c-MET might be a promising strategy for treating those LC patients with low DNM3 expression.

SPP1 and FN1 are significant gene biomarkers of tongue squamous cell carcinoma.

Tongue squamous cell carcinoma (TSCC) is one of the most common malignant tumor types in the oral and maxillofacial region. The etiology and pathogenesis behind TSCC is complicated. In the present study, three gene expression profiles, namely GSE31056, GSE13601 and GSE78060, were downloaded from the Gene Expression Omnibus (GEO). The GEO2R online tool was utilized to identify differentially expressed genes (DEGs) between TSCC and normal tissue samples. Furthermore, a protein-protein interaction (PPI) network was constructed and hub genes were validated and analyzed. A total of 83 common DEGs were obtained in three datasets, including 48 upregulated and 35 downregulated genes. Pathway enrichment analysis indicated that DEGs were primarily enriched in cell adhesion, extracellular matrix (ECM) organization, and proteolysis. A total of 63 nodes and 218 edges were included in the PPI network. The top 11 candidate hub genes were acquired, namely plasminogen activator urokinase (PLAU), signal transducer and activator of transcription 1, C-X-C motif chemokine ligand 12, matrix metallopeptidase (MMP) 13, secreted phosphoprotein 1 (SPP1), periostin, MMP1, MMP3, fibronectin 1 (FN1), serpin family E member 1 and snail family transcriptional repressor 2. Overall, 83 DEGs and 11 hub genes were screened from TSCC and normal individuals using bioinformatics and microarray technology. These genes may be used as diagnostic and therapeutic biomarkers for TSCC. In addition, SPP1 and FNl were identified as potential biomarkers for the progression of TSCC.

SNAI2-Induced CircMTO1 Promotes Cell Proliferation and Inhibits Apoptosis Through the miR-320b/MCL1 Axis in Human Granulosa-Like Tumor Cells.

Polycystic ovary syndrome (PCOS), one of the most common types of endocrine diseases, is characterized by a high prevalence among women of reproductive-age. However, its pathogenesis and molecular mechanisms remain unclear. CircMTO1 has been reported to participate in numerous biological processes, but, its role in PCOS progression remains unknown. In the current study, we elucidated the expression and circRNA characterization of circMTO1 in human granulosa-like tumor cells. We found that circMTO1 knockdown promoted human granulosa-like tumor cell proliferation and inhibited its apoptosis rate. Next, we explored the underlying molecular mechanisms by using a series of experiments. Our results revealed the effect of the novel circMTO1/miR-320b/MCL1 axis in human granulosa-like tumor cells. Furthermore, we found that the expression of circMTO1 was induced by Snail family transcriptional repressor 2 (SNAI2) in human granulosa-like tumor cells. Our results may provide potential targets for PCOS research and a novel direction for the diagnosis and treatment of PCOS.

Paramyxovirus replication induces the hexosamine biosynthetic pathway and mesenchymal transition via the IRE1α-XBP1s arm of the unfolded protein response.

The paramyxoviridae, respiratory syncytial virus (RSV), and murine respirovirus are enveloped, negative-sense RNA viruses that are the etiological agents of vertebrate lower respiratory tract infections (LRTIs). We observed that RSV infection in human small airway epithelial cells induced accumulation of glycosylated proteins within the endoplasmic reticulum (ER), increased glutamine-fructose-6-phosphate transaminases (GFPT1/2) and accumulation of uridine diphosphate (UDP)-N-acetylglucosamine, indicating activation of the hexosamine biosynthetic pathway (HBP). RSV infection induces rapid formation of spliced X-box binding protein 1 (XBP1s) and processing of activating transcription factor 6 (ATF6). Using pathway selective inhibitors and shRNA silencing, we find that the inositol-requiring enzyme (IRE1α)-XBP1 arm of the unfolded protein response (UPR) is required not only for activation of the HBP, but also for expression of mesenchymal transition (EMT) through the Snail family transcriptional repressor 1 (SNAI1), extracellular matrix (ECM)-remodeling proteins fibronectin (FN1), and matrix metalloproteinase 9 (MMP9). Probing RSV-induced open chromatin domains by ChIP, we find XBP1 binds and recruits RNA polymerase II to the IL6, SNAI1, and MMP9 promoters and the intragenic superenhancer of glutamine-fructose-6-phosphate transaminase 2 (GFPT2). The UPR is sustained through RSV by an autoregulatory loop where XBP1 enhances Pol II binding to its own promoter. Similarly, we investigated the effects of murine respirovirus infection on its natural host (mouse). Murine respirovirus induces mucosal growth factor response, EMT, and the indicators of ECM remodeling in an IRE1α-dependent manner, which persists after viral clearance. These data suggest that IRE1α-XBP1s arm of the UPR pathway is responsible for paramyxovirus-induced metabolic adaptation and mucosal remodeling via EMT and ECM secretion.

Synergistic AHR Binding Pathway with EMT Effects on Serous Ovarian Tumors Recognized by Multidisciplinary Integrated Analysis.

Epithelial ovarian cancers (EOCs) are fatal and obstinate among gynecological malignancies in advanced stage or relapsed status, with serous carcinomas accounting for the vast majority. Unlike EOCs, borderline ovarian tumors (BOTs), including serous BOTs, maintain a semimalignant appearance. Using gene ontology (GO)-based integrative analysis, we analyzed gene set databases of serous BOTs and serous ovarian carcinomas for dysregulated GO terms and pathways and identified multiple differentially expressed genes (DEGs) in various aspects. The SRC (SRC proto-oncogene, non-receptor tyrosine kinase) gene and dysfunctional aryl hydrocarbon receptor (AHR) binding pathway consistently influenced progression-free survival and overall survival, and immunohistochemical staining revealed elevated expression of related biomarkers (SRC, ARNT, and TBP) in serous BOT and ovarian carcinoma samples. Epithelial–mesenchymal transition (EMT) is important during tumorigenesis, and we confirmed the SNAI2 (Snail family transcriptional repressor 2, SLUG) gene showing significantly high performance by immunohistochemistry. During serous ovarian tumor formation, activated AHR in the cytoplasm could cooperate with SRC, enter cell nuclei, bind to AHR nuclear translocator (ARNT) together with TATA-Box Binding Protein (TBP), and act on DNA to initiate AHR-responsive genes to cause tumor or cancer initiation. Additionally, SNAI2 in the tumor microenvironment can facilitate EMT accompanied by tumorigenesis. Although it has not been possible to classify serous BOTs and serous ovarian carcinomas as the same EOC subtype, the key determinants of relevant DEGs (SRC, ARNT, TBP, and SNAI2) found here had a crucial role in the pathogenetic mechanism of both tumor types, implying gradual evolutionary tendencies from serous BOTs to ovarian carcinomas. In the future, targeted therapy could focus on these revealed targets together with precise detection to improve therapeutic effects and patient survival rates.

A Novel Insight into Endothelial and Cardiac Cells Phenotype in Systemic Sclerosis Using Patient-Derived Induced Pluripotent Stem Cell.

Objective Systemic sclerosis (SSc) is a connective tissue disease associated with vascular damage and multi organ fibrotic changes with unknown pathogenesis. Most SSc patients suffer from defective angiogenesis/vasculogenesis and cardiac conditions leading to high mortality rates. We aimed to investigate the cardiovascular phenotype of SSc by cardiogenic differentiation of SSc induced pluripotent stem cells (iPSC). Materials and Methods In this experimental study, we generated iPSC from two diffuse SSc patients, followed by successful differentiation into endothelial cells (ECs) and cardiomyocytes (CMs). Results SSc-derived EC (SSc-EC) expressed KDR, a nearly EC marker, similar to healthy control-EC (C1-EC). After sorting and culturing KDR+ cells, the resulting EC expressed CD31, a late endothelial marker, but vascular endothelial (VE)-cadherin expression markedly dropped resulting in a functional defect as reflected in tube formation failure of SSc-EC. Interestingly, upregulation of SNAI1 (snail family transcriptional repressor 1) was observed in SSc-EC which might underlie VE-cadherin downregulation. Furthermore, SSc-derived CM (SSc-CM) successfully expressed cardiac- specific markers including ion channels, resulting in normal physiological behavior and responsiveness to cardioactive drugs. ConclusionThis study provides an insight into impaired angiogenesis observed in SSc patients by evaluating in vitro cardiovascular differentiation of SSc iPSC.

TRIM16 overexpression inhibits the metastasis of colorectal cancer through mediating Snail degradation

Tripartite motif containing 16 (TRIM16) is a member of the tripartite motif protein family and functions as a potential tumor suppressor in several cancers. However, the specific function and clinical significance of TRIM16 in colorectal cancer (CRC) remains unclear. In this study, we observed that low TRIM16 expression was detected frequently in primary colorectal cancer (CRC) tissues and was closely associated with a better prognosis. Functional studies demonstrate that TRIM16 overexpression notably inhibits the metastasis abilities of CRC in vivo and in vitro. Mechanistically, our results demonstrated that TRIM16 directly bound and ubiquitinated Snail family transcriptional repressor 1 (Snail), an important transcriptional factor of the epithelial-mesenchymal transition (EMT) process suppressing the EMT in CRC. Additionally, our data revealed that the inhibition effect of TRIM16 on cancer metastasis was dependent on Snail degradation. Collectively, our study is the first to report that TRIM16 plays a crucial anti-tumor role in CRC tumorigenesis. We also provided novel evidence that TRIM16 might act as a prognostic and therapeutic target to assess and inhibit CRC progression.

TMEM229A suppresses non‑small cell lung cancer progression via inactivating the ERK pathway.

Transmembrane protein 229A (TMEM229A) is a member of the TMEM family that plays an important role in tooth differentiation and development. However, the expression level and biological role of TMEM229A in cancer remains unknown. The present study aimed to investigate the expression level of TMEM229A in non-small cell lung cancer (NSCLC), as well as its effect and mechanism on NSCLC progression. Clinical specimens from patients with NSCLC were enrolled from the First People's Hospital of Huzhou (Huzhou, China). TMEM229A expression was detected using reverse transcription-quantitative PCR (RT-qPCR), western blotting and immunohistochemical analysis. The relationship between TMEM229A expression and the survival rate of patients with NSCLC was analyzed using Kaplan-Meier Plotter datasets. The effects of TMEM229A on cell proliferation, migration and invasion were detected using Cell Counting Kit-8, colony formation, soft agar, real-time cellular analysis and Transwell assays. The expression levels of epithelial-mesenchymal transition (EMT)-related proteins, as well as ERK and AKT phosphorylation were determined via RT-qPCR and western blot analysis. The results demonstrated that TMEM229A expression was significantly downregulated in human NSCLC tissues and in several cell lines compared with adjacent normal lung tissues and BEAS-2B cells, respectively. Survival analysis of lung adenocarcinoma and squamous cell lung carcinoma cases identified that low TMEM229A expression was associated with a poor prognosis. The in vitro assays indicated that overexpressing TMEM229A significantly inhibited cell proliferation, migration and invasion, while TMEM229A knockdown had the opposite effect. Mechanistically, TMEM229A overexpression effectively increased E-cadherin expression and reduced N-cadherin, snail family transcriptional repressor 1 and MMP2 expression, indicating that EMT was suppressed. In addition, overexpression of TMEM229A reduced the expression levels of phosphorylated (p)-ERK and p-AKT, and this effect was partially suppressed by the incorporation of specific ERK inhibitor PD98059. Collectively, the results of the present study demonstrated that the effects of TMEM229A on inhibiting cell proliferation, migration and invasion were partially mediated by inactivating the ERK signaling pathway, thereby providing a potential therapeutic target for the treatment of NSCLC.

CD9-positive cells in the intermediate lobe migrate into the anterior lobe to supply endocrine cells.

The adenohypophysis is composed of the anterior and intermediate lobes (AL and IL), and secretes important hormones for growth, sexual development, metabolism, and reproduction. In the marginal cell layer (MCL) facing Rathke's cleft between the IL and AL, cluster of differentiation (CD) 9-, CD81-, S100β-, and SOX2-quadruple positive (CD9/CD81/S100β/SOX2-positive) cells in the adult IL are settled as tissue-resident stem/progenitor cells supplying hormone-producing cells to the AL. However, it is unclear how CD9/CD81/S100β/SOX2-positive cells in the IL-side MCL migrate into the AL across Rathke's cleft. In the present study, we performed chimeric pituitary tissue culture using S100β/GFP-transgenic rats and Wistar rats, and traced the footprint of S100β/GFP-expressing cells. We detected IL-side S100β/GFP-expressing cells in the AL tissue, demonstrating that these cells migrate from the IL to the AL. However, the cells failed to migrate in the opposite direction. Consistently, scanning electron microscopic analysis revealed well-developed cytoplasmic protrusions in the IL-side MCL, but not in the AL-side MCL, suggesting that IL-side CD9/CD81/S100β/SOX2-positive cells had higher migratory activity. We also searched for a specific marker for IL-side CD9/CD81/S100β/SOX2-positive cells and identified tetraspanin 1 (TSPAN1) from microarray analysis. Downregulation of Tspan1 by specific siRNA impaired cell migration and significantly reduced expression of snail family transcriptional repressor 2 (Slug), a marker of epithelial-mesenchymal transition (EMT). Therefore, CD9/CD81/S100β/SOX2-positive cells in the IL-side MCL can be stem/progenitor cells that provide stem/progenitor cells to the AL-side MCL via SLUG-mediated EMT and cell migration.

Mechanism underlying long non‑coding RNA ILF3‑AS1‑mediated inhibition of cervical cancer cell proliferation, invasion and migration, and promotion of apoptosis.

Long non-coding RNA ILF3 divergent transcript (ILF3-AS1) displays a tumor-suppressing effect. StarBase predicted that the potential target microRNA (miR) of ILF3-AS1 was miR-454-3p; therefore, the present study investigated the effect of ILF3-AS1 and its target miR-454-3p on cervical cancer (CC). Gene Expression Profiling Interactive Analysis was used to predict the expression of ILF3-AS1 in CC and the overall survival rate of patients. The present study demonstrated that ILF3-AS1 expression was significantly downregulated in human CC tissues and cells compared with adjacent tissues (ANTs) and normal cervical epithelial cells (NCEs), respectively. Patients with CC with high ILF3-AS1 expression displayed higher survival rates compared with patients with low ILF3-AS1 expression. Cell viability, apoptosis, migration and invasion were detected by performing Cell Counting Kit-8, flow cytometry, wound healing and Transwell assays, respectively. Compared with the negative control (NC) group, ILF3-AS1 overexpression significantly inhibited CC cell viability and migration, but significantly increased CC cell apoptosis. Moreover, ILF3-AS1 overexpression significantly upregulated E-Cadherin expression levels, but significantly downregulated N-Cadherin and snail family transcriptional repressor 1 expression levels compared with the NC group. miR-454-3p expression was negatively correlated with ILF3-AS1, and highly expressed in CC tissues and cells compared with ANTs and NCEs, respectively. PTEN, which was predicted and verified as the target gene for miR-454-3p, was significantly downregulated in CC tissues and cells compared with ANTs and NCEs, respectively. ILF3-AS1 expression was positively correlated with PTEN expression, and ILF3-AS1 overexpression partially reversed the inhibitory effect of miR-454-3p on PTEN expression. In conclusion, the present study indicated that ILF3-AS1 inhibited CC cell proliferation and migration, and promoted CC cell apoptosis by inhibiting epithelial-mesenchymal transition, and ILF3-AS1 overexpression partially reversed the inhibitory effect of miR-454-3p on PTEN expression.

SMARCC2 combined with c‑Myc inhibits themigration and invasion of glioma cells via modulation of the Wnt/β‑catenin signaling pathway.

Glioma is the most common type of central nervous system tumor. SWItch/sucrose non-fermentable (SWI/SNF) is a tumor suppressor that serves an important role in epithelial-mesenchymal transition (EMT). The present study aimed to identify key molecules involved in the EMT process. SWI/SNF related, matrix associated, actin dependent regulator of chromatin subfamily c member 2 (SMARCC2) is mutated in and its expression is low in multiple types of cancer. SMARCC2 is the core subunit of the chromatin-remodeling complex, SWI/SNF. Relative mRNA SMARCC2 expression levels in human glioma tissue were analyzed via reverse transcription-quantitative PCR, whereas the protein expression levels were determined via immunohistochemistry staining. SMARCC2 expression was knocked down in glioma cells using small interfering RNA (si) and overexpressed by infection with adenovirus vectors carrying SMARCC2 cDNA. Wound healing and Transwell assays were performed to assess cell migration and invasion, respectively. Subsequently, immunofluorescence and western blotting were performed to analyze the expression levels of the oncogene c-Myc, which is associated with SMARCC2. SMARCC2 combines with C-MYC to downregulate its expression. Consistent with the results of the bioinformatics analysis, which revealed that the upregulated expression levels of SMARCC2 were associated with a more favorable prognosis in patients with glioma, the mRNA and protein expression levels of SMARCC2 were significantly upregulated in low-grade glioma tissues compared with high-grade glioma tissues. The results of the wound healing assay demonstrated that cell migration was significantly increased in the siSMARCC2-1/3 groups compared with the negative control (NC) group. By contrast, the migratory ability of cells was significantly reduced following transduction with adenovirus overexpressing SMARCC2, which upregulated the expression of SMARCC2, compared with the lentiviral vector-non-specific control (LVS-NC) group. The Transwell assay results further showed that SMARCC2 overexpression significantly inhibited the migratory and invasive abilities of U87MG and LN229 cells compared with the LVS-NC group. Co-immunoprecipitation assays were subsequently conducted to validate the binding of SMARCC2 and c-Myc; the results demonstrated that the expression of c-Myc was downregulated in adenovirus-transfected cells compared with LVS-NC-transfected cells. The results of the western blotting experiments demonstrated that the expression levels of N-cadherin, vimentin, snail family transcriptional repressor 1 and β-catenin were notably downregulated, whereas the expression levels of T-cadherin were markedly upregulated in cell lines stably overexpressing SMARCC2 compared with the LVS-NC group. In conclusion, the results of the present study suggested that SMARCC2 may inhibit Wnt/β-catenin signaling by regulating c-Myc expression in glioma. SMARCC2 regulates the EMT status of the glioblastoma cell line by mediating the expression of the oncogene C-MYC to inhibit its migration and invasion ability. Thus, SMARCC2 may function as a tumor suppressor or oncogene by regulating associated oncogenes or tumor suppressor genes.

Changes in SLIT2 expression are associated with the migration of human ovarian clear cell carcinoma cells.

Ovarian clear cell carcinoma (OCCC) is characterized by a poor survival of patients, which is mainly due to metastasis and treatment failure. Slit guidance ligand 2 (SLIT2), a secreted protein, has been reported to modulate the migration of neural cells and human cancer cells. However, the effect of changes in SLIT2 expression on the regulation of cell migration in OCCC remains unknown. The present study examined alterations in SLIT2 expression using OCCC cell models, including low- and high-mobility SKOV3 cells, as well as OCCC tissues. DNA methylation analysis suggested that promoter hypermethylation was responsible for the low expression levels of SLIT2 in OCCC cells. The demethylating agent 5-Aza-deoxycytosine was able to restore SLIT2 expression at both the mRNA and protein levels in high-mobility SKOV3 cells that harbored the relevant methylated promoter. Overexpression of SLIT2 inhibited the migration of high-mobility OCCC cells, as well as decreased the protein expression levels of β-catenin, phosphorylated (p)AKT and snail family transcriptional repressor 1 (SNAI1). On the other hand, knockdown of SLIT2 increased the migration of low-mobility OCCC cells, and enhanced the protein expression levels of β-catenin, pAKT and SNAI1. Overall, the results of the present study provided evidence that low expression levels of SLIT2 were associated with increased OCCC cell migration, and that SLIT2 may act as a suppressor gene of cancer cell migration.

GTSE1 promotes SNAIL1 degradation by facilitating its nuclear export in hepatocellular carcinoma cells.

Snail family transcriptional repressor 1 (SNAIL1) is a master inducer of the epithelial-to-mesenchymal transition (EMT) process, contributing to tumor metastasis and recurrence. Our previous study reported that G2 and S phase-expressed-1 (GTSE1) served a role in regulating SNAIL1 expression in hepatocellular carcinoma (HCC). However, the underlying mechanism remains unknown. Therefore, the present study aimed to reveal the regulatory mechanism of GTSE1 on SNAIL1 expression using in vitro assays performed in HCC cell models. It was demonstrated that endogenous SNAIL1 expression was downregulated and upregulated by GTSE1 overexpression or small interfering RNA-mediated knockdown, respectively. Via cycloheximide chase experiments, it was identified that GTSE1 overexpression increased the protein turnover of SNAIL1, while knockdown of GTSE1 reduced its degradation rate. Furthermore, it was demonstrated that GTSE1 overexpression induced the cytoplasmic expression of SNAIL1 using immunofluorescence and subcellular fractionation methods. The nuclear export inhibitor leptomycin B was able to decrease the cytoplasmic retention of SNAIL1 caused by GTSE1 overexpression. In addition, TGF-βI treatment increased both the mRNA and protein expression levels of GTSE1, and decreased the protein expression level of SNAIL1 without affecting its mRNA transcription in Huh7 cells. It was also found that TGF-β signaling could upregulate the transcription of GTSE1 expression by transactivating the Smad binding elements in the GTSE1 promoter. Moreover, the TGF-βI-induced decrease in SNAIL1 protein expression was GTSE1-dependent in Huh7 cells. In conclusion, the current study provides a novel mechanism via which GTSE1 affects the stability of SNAIL1 by regulating its subcellular localization in HCC cells.

Analysis of crucial genes, pathways and construction of the molecular regulatory networks in vascular smooth muscle cell calcification.

Vascular calcification (VC) accompanies the trans-differentiation of vascular smooth muscle cells (VSMCs) into osteo/chondrocyte-like cells and resembles physiological bone mineralization. However, the molecular mechanisms underlying VC initiation and progression have remained largely elusive. The aim of the present study was to identify the genes and pathways common to VSMC and osteoblast calcification and construct a regulatory network of non-coding RNAs and transcription factors (TFs). To this end, the Gene Expression Omnibus dataset GSE37558 including mRNA microarray data of calcifying VSMCs (CVSMCs) and calcifying osteoblasts (COs) was analyzed. The differentially expressed genes (DEGs) were screened and functionally annotated and the microRNA (miRNA/mRNA)-mRNA, TF-miRNA and long non-coding RNA (lncRNA)-TF regulatory networks were constructed. A total of 318 DEGs were identified in the CVSMCs relative to the non-calcified VSMCs, of which 43 were shared with the COs. The CVSMC-related DEGs were mainly enriched in the functional terms cell cycle, extracellular matrix (ECM), inflammation and chemotaxis-mediated signaling pathways, of which ECM was enriched by the DEGs for the COs as well. The protein-protein interaction network of CVSMCs consisted of 281 genes and 3,650 edges. There were 30 hub genes in this network, including maternal embryonic leucine zipper kinase (MELK), which potentially regulates the differentially expressed TF (DETF) forkhead box (FOX)M1 and is a potential target gene of Homo sapiens miR-485-3p and miR-181d. The TF-miRNA network included 251 TFs and 60 miRNAs, including 10 DETFs such as FOXO1 and snail family transcriptional repressor 2 (SNAI2). Furthermore, the lncRNAs H19 imprinted maternally expressed transcript (H19) and differentiation antagonizing non-protein coding RNA (DANCR) were predicted as the upstream regulators of FOXO1 and SNAI2 in the lncRNA-TF regulatory network. DANCR, MELK and FOXM1 were downregulated, and H19, FOXO1 and SNAI2 were upregulated in the CVSMCs. Taken together, the CVSMCs and COs exhibited similar molecular changes in the ECM. In addition, the MELK-FOXM1, H19/DANCR-FOXO1 and SNAI2 regulatory pathways likely mediate VSMC calcification.

Calycosin Induces Gastric Cancer Cell Apoptosis via the ROS-Mediated MAPK/STAT3/NF-κB Pathway.

BackgroundCalycosin, an active compound in plants, can promote the apoptosis of various cancer cells; however, the mechanism by which it regulates reactive oxygen species (ROS) in gastric cancer (GC) cells remains unclear.PurposeIn this study, we investigated the effects of calycosin on apoptosis, the cell cycle, and migration in GC cells under ROS regulation.ResultsThe results of the Cell Counting Kit-8 assay suggested that calycosin had significant cytotoxic effects on 12 gastric cancer cells, but no significant cytotoxic effects on normal cells. Hoechst 33342/propidium iodide (PI) double staining and flow cytometry showed that calycosin had clear pro-apoptotic effects on AGS cells. Western blotting revealed that the expression of cytochrome C and pro-apoptotic proteins B-cell lymphoma 2 (Bcl-2)-associated agonist of cell death (Bad), cleaved (cle)-caspase-3, and cle-poly (ADP-ribose) polymerase gradually increased, and the expression of anti-apoptotic protein Bcl-2 gradually decreased. Calycosin also decreased the expression of extracellular signal-regulated kinase, nuclear factor kappa B (NF-κB), and signal transducer and activator of transcription 3 (STAT3), and increased the phosphorylation levels of p38, c-Jun N-terminal kinase, and inhibitor of NF-κB. In addition, calycosin markedly increased ROS accumulation, and pretreatment with active oxygen scavenger n-acetyl-l-cysteine (NAC) clearly inhibited apoptosis. Calycosin downregulated the cell cycle proteins cyclin-dependent kinase 2 (CDK2), CDK4, CDK6, cyclin D1, and cyclin E; upregulated p21 and p27; and arrested cells in the G0/G1 phase. Similarly, calycosin also downregulated Snail family transcriptional repressor 1, E-cadherin, and β-catenin and inhibited cell migration. However, pretreatment with NAC inhibited the calycosin-induced effects of cycle arrest and migration.ConclusionIn summary, calycosin induces apoptosis via ROS-mediated MAPK/STAT3/NF-κB pathways, thereby exerting its anti-carcinogenic functions in GC cells.

A novel anthraquinone‑quinazoline hybrid7B blocks breast cancer metastasis and EMT via targeting EGFR and Rac1.

At present, effective therapeutic drugs for triple‑negative breast cancer (TNBC) are lacking due to the absence of identified or available targets. Therefore, the present study aimed to identify key molecular targets and a specific targeted therapeutic drug to aid with the development of novel therapeutic strategies for TNBC. Based on the high expression of EGFR and Rac1 in TNBC and inspired by a novel antitumor strategy termed combi‑targeting, novel anthraquinone‑quinazoline hybrid7B was synthesized to simultaneously target EGFR and Rac1. It was hypothesized that hybrid7B may possess enhanced potency compared with its parent compounds. Breast cancer cell viability was detected by performing MTT assays. Flow cytometry was conducted to detect the effects of hybrid7B on the cell cycle, apoptosis and the mitochondrial outer membrane potential. Ultrastructural alterations were observed by transmission electron microscopy. Cell invasion and migration were assessed by performing Transwell and wound‑healing assays, respectively. The expression levels of epithelial‑mesenchymal transition (EMT) markers and metastasis‑related proteins were detected by western blotting. Compared with Rhein and gefitinib, hybrid7B displayed superior antiproliferative activity in MDA‑MB‑231 cells with an IC50 value of 2.31µM, which was 14‑fold higher compared with the EGFR tyrosine kinase inhibitor gefitinib. Further experiments demonstrated that hybrid7B significantly reduced the mitochondrial membrane potential, enhanced MDA‑MB‑231 cell apoptosis and induced cell cycle arrest at the G2/Mphase compared with the control group. Typical morphological alterations of apoptotic cells were observed in hybrid7B‑treated MDA‑MB‑231 and MCF‑7 cells. Compared with the control group, hybrid7B significantly inhibited MDA‑MB‑231 cell invasion and migration by downregulating Rac1, EGFR, matrix metalloproteinases, snail family transcriptional repressor1, Vimentin and β‑catenin protein expression levels, and upregulating E‑cadherin protein expression levels. The present study demonstrated that hybrid7B inhibited TNBC cell migration and invasion by reversing EMT and targeting EGFR and Rac1; therefore, hybrid7B may serve as a promising therapeutic agent for TNBC.

MiR-34a-5p might have an important role for inducing apoptosis by down-regulation of SNAI1 in apigenin-treated lung cancer cells

Apigenin is a flavonoid with antioxidant and anticancer effects. It has been reported that apigenin inhibits proliferation, migration, and invasion and induces apoptosis in cultured lung cancer cells. However, there is little information on the involvement of microRNAs (miRNAs) in its effects. miRNA microarray analysis and polymerase-chain-reaction analysis of miRNAs revealed that treatment of human lung cancer A549 cells with apigenin up-regulated the level of miR-34a-5p. Furthermore, mRNA microarray analysis and the results of three microRNA target prediction tools showed that Snail Family Transcriptional Repressor 1 (SNAI1), which inhibits the induction of apoptosis, had its mRNA expression down-regulated in A549 cells treated with apigenin. Our findings suggest that apigenin might induce apoptosis by down-regulation of SNAI1 through up-regulation of miR-34a-5p in A549 cells.

Lycorine inhibits cell proliferation, migration and invasion, and primarily exerts invitro cytostatic effects in human colorectal cancer via activating the ROS/p38 and AKT signaling pathways.

Colorectal cancer (CRC) is a life-threatening malignant tumor of the digestive tract. Diverse gene mutations and complicated alterations to the signaling pathways in CRC lead to heterogeneity in response to chemotherapy. Moreover, anticancer drugs for CRC chemotherapy are limited due to adverse events. Therefore, developing more effective, tolerable and safe drugs for the treatment of CRC is important. The present study aimed to investigate the effect of lycorine on human CRC cell proliferation, migration, invasion, apoptosis, cell cycle distribution, as well as the underlying molecular mechanism. The crystal violet staining and MTT assay results demonstrated that lycorine suppressed cell proliferation in a dose- and time-dependent manner in the three CRC cell lines, HCT116, LoVo and SW480. Similarly, verified by performing wound healing and Transwell assays, lycorine significantly inhibited HCT116 and LoVo cell migration and invasion in vitro compared with the control group. In LoVo cells, the protein expression levels of matrix metallopeptidases, snail family transcriptional repressor 1, Vimentin and N-cadherin were significantly downregulated, whereas the protein expression levels of E-cadherin were significantly upregulated by lycorine treatment compared with the control group. The Hoechst 33258 staining and flow cytometry assay results indicated that lycorine mediated its cytostatic effect on CRC cells potentially via inducing cell cycle arrest, but not apoptosis. Compared with the control group, lycorine significantly induced HCT116 cell cycle arrest at the G2/M phase, but significantly induced LoVo cell cycle arrest at the S and G2/M phases. Furthermore, lycorine significantly downregulated the protein expression levels of cyclin D1 and cyclin E1, but significantly increased p21 and Smad4 protein expression levels in HCT116 and LoVo cells compared with the control group. The intracellular reactive oxygen species (ROS) measurement results also indicated that compared with the control group, lycorine significantly induced ROS accumulation, and increased phosphorylated-p38 expression levels and AKT phosphorylation. Collectively, the present study suggested that lycorine might induce cell cycle arrest and exert cytostatic effects potentially via activating ROS/p38 and AKT signaling pathways in CRC cells.

SNHG3 cooperates with ELAVL2 to modulate cell apoptosis and extracellular matrix accumulation by stabilizing SNAI2 in human trabecular meshwork cells under oxidative stress

Glaucoma is the main reason for irreversible blindness, and pathological increased intraocular pressure is the leading risk factor for glaucoma. It is reported that trabecular meshwork cell injury is closely associated with the elevated intraocular pressure. The current study aimed to investigate the role of small nucleolar RNA host gene 3 (SNHG3) in human trabecular meshwork (HTM) cells under oxidative stress. A series of experiments including real-time quantitative polymerase chain reaction, subcellular fractionation assay, western blot analysis, cell counting kit-8 assay, RNA pull down, flow cytometry analysis, and RNA immunoprecipitation assay were used to explore the biological function and regulatory mechanism of SNHG3 in HTM cells under oxidative stress. First, we observed that H2 O2 induced SNHG3 upregulation in HTM cells. Then, we found that SNHG3 silencing alleviated H2 O2 -induced oxidative damage in HTM cells. Moreover, snail family transcriptional repressor 2 (SNAI2) knockdown alleviated the oxidative damage induced by H2 O2 in HTM cells. Mechanistically, SNHG3 bound with ELAV like RNA binding protein 2 (ELAVL2) to stabilize SNAI2. Finally, SNAI2 overexpression counteracted the effect of SNHG3 silencing on H2 O2 -treated HTM cells. In conclusion, our results demonstrated that SNHG3 cooperated with ELAVL2 to modulate cell apoptosis and extracellular matrix accumulation by stabilizing SNAI2 in HTM cells under oxidative stress.

Hypoxia promotes the metastasis of pancreatic cancer through regulating NOX4/KDM5A-mediated histone methylation modification changes in a HIF1A-independent manner

BackgroundHypoxia is a characteristic of the tumor microenvironments within pancreatic cancer (PC), which has been linked to its malignancy. Recently, hypoxia has been reported to regulate the activity of important carcinogenic pathways by changing the status of histone modification. NOX4, a member of NADPH oxidase (NOX), has been found to be activated by hypoxia and promote cancer progression in several cancers. But whether it is involved in the epigenetic changes of tumor cells induced by hypoxia is still unclear, and its biological roles in PC also need to be explored.MethodsA hypoxic-related gene signature and its associated pathways in PC were identified by analyzing the pancreatic cancer gene expression data from GEO and TCGA database. Candidate downstream gene (NOX4), responding to hypoxia, was validated by RT-PCR and western blot. Then, we evaluated the relationship between NOX4 expression and clinicopathologic parameters in 56 PC patients from our center. In vitro and in vivo assays were preformed to explore the phenotype of NOX4 in PC. Immunofluorescence, western blot and chromatin immunoprecipitation assays were further applied to search for a detailed mechanism.ResultsWe quantified hypoxia and developed a hypoxia signature, which was associated with worse prognosis and elevated malignant potential in PC. Furthermore, we found that NADPH oxidase 4 (NOX4), which was induced by hypoxia and upregulated in PC in a HIF1A-independent manner, caused inactivation of lysine demethylase 5A (KDM5A), increased the methylation modification of histone H3 and regulated the transcription of EMT-associated gene_ snail family transcriptional repressor 1 (SNAIL1). This served to promote the invasion and metastasis of PC. NOX4 deficiency repressed hypoxia-induced EMT, reduced expression of H3K4ME3 and impaired the invasion and metastasis of PC cells; however, knockdown of KDM5A reversed the poor expression of H3KEME3 induced by NOX4 deficiency, thereby promoting EMT.ConclusionsThis study highlights the prognostic role of hypoxia-related genes in PC and strong correlation with EMT pathway. Our results also creatively discovered that NOX4 was an essential mediator for hypoxia-induced histone methylation modification and EMT in PC cells.