Expression Of Epithelial-mesenchymal Transition Research Articles

The effect of Annexin A5 overexpression on invasiveness and expression of the genes involved in epithelial-mesenchymal transition of HCT 116 cell line.

Epithelial-to-mesenchymal transition (EMT) plays a critical role in colorectal cancer (CRC) metastasis. In the present study, we evaluated the effects of annexin A5 (ANXA5) overexpression on invasiveness as well as the expression of genes involved in EMT of HCT 116 cell line. PCMV6-AC-IRES-GFP plasmid harboring ANXA5 cDNA was constructed. HCT 116 cell line was transfected with recombinant plasmids using Lipofectamine 3000. Fluorescent microscopy was used to determine the efficiency of plasmid transfection. Cell viability was determined using the MTT assay. HCT 116 cell migration was evaluated using wound healing assay and transwell migration assay. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to measure the expression of genes involved in EMT. The results of RT-qPCR showed overexpression of ANXA5 compared to the control group. ANXA5 overexpression had no significant effects on cell viability but significantly decreased the rate of wound closure in the wound healing assay as well as the number of migrated cells in transwell assay. Furthermore, ANXA5 overexpression decreased the expression of N-cadherin, Snail, Slug, MMP-2, and MMP-9 while the expression of E-cadherin increased following ANXA5 overexpression. However, VEGF expression did not significantly change after ANXA5 overexpression. Results of the present study suggest that ANXA5 overexpression might have inhibitory effects on the metastasis of CRC through modulating the expression of EMT- related genes.

Oxidized-LDL Deteriorated the Renal Residual Function and Parenchyma in CKD Rat through Upregulating Epithelial Mesenchymal Transition and Extracellular Matrix-Mediated Tubulointerstitial Fibrosis-Pharmacomodulation of Rosuvastatin.

This study tested the hypothesis that intrarenal arterial transfusion of oxidized low-density lipoprotein (ox-LDL) jeopardized the residual renal function and kidney architecture in rat chronic kidney disease ((CKD), i.e., induced by 5/6 nephrectomy) that was reversed by rosuvastatin. Cell culture was categorized into A1 (NRK-52E cells), A2 (NRK-52E + TGF-β), A3 (NRK-52E + TGF-β + ox-LDL) and A4 (NRK-52E + TGF-β + ox-LD). The result of in vitro study showed that cell viability (at 24, 48 and 72 h), NRK-52E ox-LDL-uptake, protein expressions of epithelial-mesenchymal-transition (EMT) markers (i.e., p-Smad2/snail/α-SMA/FSP1) and cell migratory and wound healing capacities were significantly progressively increased from A1 to A4 (all p < 0.001). SD rats were categorized into group 1 (sham-operated control), group 2 (CKD), group 3 (CKD + ox-LDL/0.2 mg/rat at day 14 after CKD induction) and group 4 (CKD + ox-LDL-treated as group 3+ rosuvastatin/10 mg/kg/day by days 20 to 42 after CKD induction) and kidneys were harvested at day 42. The circulatory levels of BUN and creatinine, ratio of urine-protein to urine-creatinine and the protein expressions of the above-mentioned EMT, apoptotic (cleaved-caspase3/cleaved-PARP/mitochondrial-Bax) and oxidative-stress (NOX-1/NOX-2/oxidized-protein) markers were lowest in group 1, highest in group 3 and significantly higher in group 4 than in group 2 (all p < 0.0001). Histopathological findings demonstrated that the kidney injury score, fibrotic area and kidney injury molecule-1 (KIM-1) displayed an identical pattern, whereas the cellular expression of podocyte components (ZO-1/synaptopodin) exhibited an opposite pattern of EMT markers (all p < 0.0001). In conclusion, ox-LDL damaged the residual renal function and kidney ultrastructure in CKD mainly through augmenting oxidative stress, EMT and fibrosis that was remarkably reversed by rosuvastatin.

Overexpression of KiSS1 Induces the Proliferation of Hepatocarcinoma and Increases Metastatic Potential by Increasing Migratory Ability and Angiogenic Capacity.

Liver cancer has a high prevalence, with majority of the cases presenting as hepatocellular carcinoma (HCC). The prognosis of metastatic HCC has hardly improved over the past decade, highlighting the necessity for liver cancer research. Studies have reported the ability of the KiSS1 gene to inhibit the growth or metastasis of liver cancer, but contradictory research results are also emerging. We, therefore, sought to investigate the effects of KiSS1 on growth and migration in human HCC cells. HepG2 human HCC cells were infected with lentivirus particles containing KiSS1. The overexpression of KiSS1 resulted in an increased proliferation rate of HCC cells. Quantitative polymerase chain reaction and immunoblotting revealed increased Akt activity, and downregulation of the G1/S phase cell cycle inhibitors. A significant increase in tumor spheroid formation with upregulation of β-catenin and CD133 was also observed. KiSS1 overexpression promoted the migratory, invasive ability, and metastatic capacity of the hepatocarcinoma cell line, and these effects were associated with changes in the expressions of epithelial mesenchymal transition (EMT)-related genes such as E-cadherin, N-cadherin, and slug. KiSS1 overexpression also resulted in dramatically increased tumor growth in the xenograft mouse model, and upregulation of proliferating cell nuclear antigen (PCNA) and Ki-67 in the HCC tumors. Furthermore, KiSS1 increased the angiogenic capacity by upregulation of the vascular endothelial growth factor A (VEGF-A) and CD31. Based on these observations, we infer that KiSS1 not only induces HCC proliferation, but also increases the metastatic potential by increasing the migratory ability and angiogenic capacity.

Prognostic biomarker MCP-4 triggers epithelial-mesenchymal transition via the p38 MAPK pathway in ovarian cancer.

Monocyte chemoattractant protein-4 (MCP-4/CCL13) is a proinflammatory factor that is overexpressed in various malignant tumors and may play an important role in tumor progression and metastasis. However, its role and mechanism of action in ovarian cancer remains unknown. Immunohistochemistry (IHC) was performed to detect the expression of MCP-4 in ovarian cancer tissues, and the effect of MCP-4 on patient survival and prognosis was analyzed. Overexpression and suppression of MCP-4 in ovarian cancer cell lines were then established, and their effects on cell invasion, migration, and apoptosis were studied. ES-2 cell lines were employed to establish a peritoneal dissemination model in nude mice. Western blotting was performed to detect the expression of epithelial mesenchymal transition (EMT) markers and the p38 mitogen-activated protein kinase (p38 MAPK) signaling pathway. MCP-4 was highly expressed in ovarian cancer tissues and its expression level was related to the prognosis of patients with ovarian cancer. MCP-4 overexpression promoted the migration and invasion of ovarian cancer cells but inhibited apoptosis. MCP-4 overexpression increased the expression of MMP-2, MMP-9, N-cadherin, vimentin and Bcl2/Bax and decreased the expression of E-cadherin. MCP-4 overexpression increased the phosphorylation of the p38 MAPK pathway. The inhibition of MCP-4 expression indicated an opposite trend. In vivo experiments have also confirmed that MCP-4 overexpression can promote metastasis of ovarian cancer. MCP-4 promotes ovarian cancer progression through the p38 MAPK signaling pathway, and may be a potential biomarker and therapeutic target for ovarian cancer.

Circ_0000799 promotes proliferation and invasion in colorectal cancer and epithelial-mesenchymal transition

The current study aimed to investigate the effect of circ_0000799 on the biological function of colorectal cancer (CRC) cells and its mechanism. First, quantitative reverse transcription polymerase chain reaction (qRT-PCR) was employed for detecting the expression of circ_0000799, miR-647, and miR-1243 in surgically resected specimens from hospitalized CRC patients, CRC-adjacent normal tissues (Normal group), human normal colon epithelial cells (FHC group), and CRC cell lines (HCT116, HT29, SW480, SW620). The cell proliferation, viability, and invasion were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), colony formation assay, transwell assay in HCT116 and SW480 cells with overexpression or inhibition of circ_0000799. The targeting relationship between circ_0000799 and miR-647 was verified by dual-luciferase reporter assay. The expression of epithelial-mesenchymal transition (EMT) proteins (E-cadherin, vimentin, and N-cadherin) was tested by western blot. The expression level of circ_0000799 was significantly increased in CRC tissues and cells. Overexpression of circ_0000799 significantly increased cell proliferation rate, viability, invasion, and the EMT process, whereas knockdown of circ_0000799 inhibited the biological performance of CRC cells. Bioinformatic analysis suggested that miR-647 was regulated by circ_0000799, and a dual-luciferase reporter assay further showed a targeting relationship between the two. In addition, circ_0000799 was negatively correlated with miR-647 expression in CRC. Our findings suggest that circ_0000799 promotes proliferation and invasion in CRC and EMT. These effects of circ_0000799 may be achieved by negatively regulating miR-62.

Reserve of Wnt/β-catenin Signaling Alleviates Mycoplasma pneumoniae P1-C-induced Inflammation in airway epithelial cells and lungs of mice

Mycoplasma pneumoniae (M. pneumoniae) is the most common pathogen of respiratory tract infections in both children and adults. M. pneumoniae P1 adhesin plays an important role in the pathogenesis of M. pneumoniae infection by mediating the attachment of pathogen to host cells. The inoculation of C-terminal residuals of P1 (P1-C) showed a protective role from M. pneumoniae infection. Accumulated evidence suggests that the Wnt/β-Catenin signaling is implicated in regulation of inflammatory responses to bacterial infections. However, mechanisms underlying the regulatory roles of Wnt signaling in host cells in response to M. pneumoniae infections are incompletely understood. In the present study, the impact and molecular mechanism of Wnt/β-catenin signaling in immune responses induced by M. pneumoniae P1-C were investigated. The results demonstrated that the P1-C could activate Wnt/β-catenin and Toll-like receptor (TLR) signaling in primary mouse airway epithelial cells cultured in an air-liquid interface (ALI) state. Interestingly, the inhibition of Wnt/β-catenin signaling by an adenovirus-mediated Wnt inhibitor Dickkopf-1 (Dkk1) gene transduction alleviated the P1-C induced inflammation fibrosis in mouse lung, accompanied by the reduced expression of epithelial mesenchymal transition (EMT) markers. Mechanistical analysis further demonstrated that the Dkk1 could suppress the expression of JAK2/STAT1-STAT3 and Caspase3, 8/Bax signaling in mouse lung tissues. In vitro study further revealed that XAV939, a small molecule of Wnt/β-catenin inhibitor, inhibited the P1-C-activated TLR4/MyD88 signaling and cytokine productions in primary mouse airway ALI epithelial cells. This study thus provides an insight into the function of Wnt/β-catenin signaling in regulation of the pathogenesis of M. pneumoniae infection, suggesting that targeting Wnt/β-catenin signaling by gene transduction of Dkk1, or pharmacological molecules of inhibitor may be a promised approach that worthy of further investigation in the treatment of M. pneumoniae pneumonia.

EMT-like Activation in CLL Provides Novel Therapeutic Target

Introduction: There is growing interest in the role of Epithelial to Mesenchymal Transition (EMT) regulators in immune cell development, but the functional role of EMT in B-cell biology and transformation remains obscured. The outcomes of the "classical" EMT activation of epithelial cells are known; committed migratory cells do not divide and become resistant to apoptotic stimuli. Importantly, EMT supports migratory cells' "stem cell-like" properties, allowing differentiation into more than one cell type and/or terminal differentiation and senescence. Chronic lymphocytic leukaemia (CLL) cells engage with antigen and microenvironment stimuli in tissue sites that lead to proliferation, followed by their egress from the tissue sites into the peripheral blood. This lifecycle implies functional changes altering CLL cell movement. Here we show EMT-like activation in CLL that correlates with disease progression and transformation. We identified a targetable candidate and show that it can be inhibited by small molecule ligands. Results: Our analysis of available expression datasets showed the expression of various EMT factors in healthy B cells. The single-cell analysis of lymph nodes confirms strong EMT-like activation in healthy B-cells (GSE159929), confirming the idea that genes involved in classical EMT would be expressed differently depending on the stage of B-cell differentiation. The following GENEVESTIGATOR analysis of B cell malignancies highlighted high EMT-like activation in CLL cases. However, differential expression (DE) analysis of peripheral blood (PB) samples from 44 CLL patients and 24 healthy B-cell donors identified six EMT factors aberrantly expressed in CLL cells compared to healthy B cells indicating significant changes in CLL EMT-like activation. At the transcription level, ZEB1 expression was higher and ZEB2 expression lower in PB CLL cells. The main stimulatory events in CLL cells appear restricted to affected tissue sites, primarily in lymph nodes (LN) where CLL cells migrate to reactivate and proliferate. Further, EMT factor protein expression was confirmed in CLL LN tissue microarrays. Strikingly, transformation into a more aggressive form (Richter syndrome) strongly correlates with an increase in ZEB2 but not ZEB1 protein expression. Interestingly, analysis of the published transcriptome data set of CLL cells co-cultured with nurse-like cells shows an increase in ZEB2 and a decrease in ZEB1 expression after 14 days (GSE13811). An apparent reliance of the EMT-like activation in CLL on the environmental stimuli has been demonstrated in CLL cells stimulated with BCR activation (anti-IgM) or mitogen (PMA), implying protection from cell death and activation of migration. Additionally, we observed higher S100A4 expression in CLL cells compared to mature B-cells from healthy individuals. Previously it has been shown that this calcium-dependent protein can control cell movement by direct interaction with non-muscle myosin and via regulation of stress fibre formation, thus facilitating cellular protrusions and increasing cell migration and invasion. Our analysis has shown the S100A4 positive correlation with CLL markers of poor prognosis, Zap70 and CD38. The S100A4 knockdown with siRNA dramatically impeded CLL migration in trans-well assay (Figure 1). Biochemical inhibitor screening for disruption of S100A4 interaction with myosin II identified several promising leads active in the low micromolar range in both biochemical and cell assays. Conclusions: Our findings improve the understanding of CLL pathobiology. Normally, healthy naïve B cells give rise to multiple B-cell subtypes with specific functions and locations. EMT-like activation likely contributes to B-cell development at different stages of differentiation and B-cell malignant transformation. Our investigation shows that the EMT-like activation is dysregulated in CLL cells compared to healthy B cells. The EMT expression signature changes upon B-cell receptor stimulation. These results suggest that targeting some of the aberrantly expressed EMT components such as S100A4 in CLL can be exploited, prolonging remission by, for example, impeding CLL returning to the tissue sites after an egress due to the treatment with Ibrutinib depriving these cells of vital microenvironmental support. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

EPCO-09. TRANSCRIPTOMIC LANDSCAPE OF LOWER GRADE GLIOMA BASED ON AGE-RELATED NON-SILENT SOMATIC MUTATIONS

Abstract Glioma accounts for 80% of all malignant brain tumours and is the most common adult primary brain tumour. Age is an important factor affecting the development of cancer, as somatic mutations accumulate with age. Here, we aimed to analyse the significance of age-dependent non-silent somatic mutations in glioma prognosis. Histological tumour grade depends on age at diagnosis in patients with IDH1, TP53, ATRX, and EGFR mutations. Age of patients with wild-type IDH1 and EGFR increased with increase in tumour grade, while the age of patients with IDH1 or EGFR mutation remained constant. However, the age of patients with EGFR mutation was higher than that of patients with IDH1 mutation. The hierarchical clustering of patients was dominantly separated by IDH1 and EGFR mutations. Furthermore, patients with IDH1 mutation were dominantly separated by TP53 and ATRX double mutation and its double wild-type counterpart. The age of patients with ATRX and TP53 mutation was lower than that of patients with wild-type ATRX and TP53. Patients with the double mutation showed poorer prognosis than those with the double wild type genotype. Unlike IDH1 mutant, IDH1 wild-type showed upregulation of expression of epithelial mesenchymal transition associated genes.

Glycoprotein M6A Suppresses Lung Adenocarcinoma Progression via Inhibition of the PI3K/AKT Pathway.

Lung adenocarcinoma is the most common subtype of lung cancer and has high morbidity and mortality. Glycoprotein M6A (GPM6A) is a neuronal membrane glycoprotein reported to be related with cancer. However, studies on GPM6A in lung adenocarcinoma are rare. This study aimed to investigate the role of GPM6A in lung adenocarcinoma and its potential mechanism. GPM6A mRNA expression was analysed in 33 types of cancers using The Cancer Genome Atlas (TCGA) datasets. It was compared among normal lung tissues, lung adenocarcinoma tissues, and adjacent tissues using the Oncomine database. Real-time quantitative polymerase chain reaction (RT-qPCR) was performed to detect GPM6A expression in human lung adenocarcinoma cell lines (A549 and H1299) and normal pulmonary epithelial cells (BEAS-2B). When GPM6A was inhibited, cell proliferative capacity was detected by Cell Counting Kit 8 (CCK8), EdU, and colony formation assays. Cell migration ability was detected by wound healing and transwell assays. The expression of epithelial-mesenchymal transition (EMT) markers was detected by Western blotting (WB) and RT-qPCR. When GPM6A was overexpressed, cell proliferation and migration were detected again. Ten nude mice were subcutaneously injected with cells overexpressing GPM6A or empty vector, and the tumor size was recorded on day 14 and then measured every 3 days thereafter. The final tumor weight was measured on day 36. Furthermore, the expressions of phosphoinositide 3-kinase (PI3K), phosphorylated PI3K, AKT, and phosphorylated AKT were detected by WB. Results showed that GPM6A mRNA expression decreased in 15 types of tumors in TCGA dataset. GPM6A expression was lower in lung adenocarcinoma than in normal lung tissues or adjacent tissues in the Oncomine dataset. Similar results were found in lung adenocarcinoma cells. The function study showed that GPM6A downregulation enhanced the proliferation, migration, and EMT of lung adenocarcinoma cells, while GPM6A upregulation inhibited their development. The xenograft results suggested that GPM6A upregulation delayed tumor growth and reduced tumor weight. Moreover, WB showed that GPM6A knockdown activated the PI3K/AKT pathway, while GPM6A upregulation inhibited the activation of the PI3K/AKT pathway. In conclusion, GPM6A suppresses lung adenocarcinoma progression via inhibition of the PI3K/AKT pathway. Thus, GPM6A could be a possible treatment target for lung cancer therapy.

Novel plasma exosome biomarkers for prostate cancer progression in co-morbid metabolic disease

Comorbid Type 2 diabetes (T2D), a metabolic complication of obesity, associates with worse cancer outcomes for prostate, breast, head and neck, colorectal and several other solid tumors. However, the molecular mechanisms remain poorly understood. Emerging evidence shows that exosomes carry miRNAs in blood that encode the metabolic status of originating tissues and deliver their cargo to target tissues to modulate expression of critical genes. Exosomal communication potentially connects abnormal metabolism to cancer progression. Here, we hypothesized that T2D plasma exosomes induce epithelial-mesenchymal transition (EMT) and immune checkpoints in prostate cancer cells. We demonstrate that plasma exosomes from subjects with T2D induce EMT features in prostate cancer cells and upregulate the checkpoint genes CD274 and CD155. We demonstrate that specific exosomal miRNAs that are differentially abundant in plasma of T2D adults compared to nondiabetic controls (miR374a-5p, miR-93-5p and let-7b-3p) are delivered to cancer cells, thereby regulating critical target genes. We build on our previous reports showing BRD4 controls migration and dissemination of castration-resistant prostate cancer, and transcription of key EMT genes, to show that T2D exosomes require BRD4 to drive EMT and immune ligand expression. We validate our findings with gene set enrichment analysis of human prostate tumor tissue in TGCA genomic data. These results suggest novel, non-invasive approaches to evaluate and potentially block progression of prostate and other cancers in patients with comorbid T2D.

ODP562 ZnT5 Involvement in EMT in Breast Cancer

Abstract The zinc levels in breast cancer tissue are reportedly higher than in normal tissue. In addition, the expression levels of zinc transporters, including ZnT5 and ZnT6, in breast cancer tissue are higher than in normal breast tissue. Moreover, ZnT5 and ZnT6 contribute to the formation of heterodimers and are involved in different biological functions. In vitro studies using chicken DT40 cells showed that ZnT5 and ZnT6 heterodimers are required to activate several proteins that play important roles in tumorigenesis, infiltration, and metastasis. However, the functions of ZnT5 and ZnT6 heterodimers in breast cancer remain unknown. Therefore, in this study, we first immunolocalized ZnT5 and ZnT6 in 113 pathological specimens of breast cancer. We then analyzed the interaction between ZnT5 and ZnT6 in breast cancer tissue using proximity ligation assay (PLA). Further, we evaluated cell migration and the expression of epithelial mesenchymal transition (EMT) markers as well as vimentin and E-cadherin in ZnT5 knockdown MCF-7 cells using siRNA. Next, we utilized human phosphorylation multipathway profiling array to explore the underlying mechanism of ZnT5 knockdown-induced cell migration. Immunohistochemical analysis revealed that the number of ZnT5-positive breast cancer cells was significantly higher in patients with a low pathologic N factor, and the number of ZnT6-positive breast cancer cells was significantly higher in patients with a low histological grade. The number of ZnT5 and ZnT6 double-positive breast cancer cells was significantly higher in patients with low Ki-67 expression than those with ZnT5-positive and ZnT6-negative breast cancer cells. In addition, ZnT5 and ZnT6 heterodimers were detected using PLA in breast cancer tissues with high expression of both ZnT5 and ZnT6. Treatment with 100 µM ZnCl 2 inhibited migration of MCF-7 cells, but ZnT5 knockdown promoted cell migration. ZnT5 knockdown induced higher levels of vimentin and lower levels of E-cadherin and activated SMAD1 in the presence of 100 µM ZnCl 2 . These results indicate that the involvement of ZnT5 in the inhibition of EMT through SMAD1 inactivation. These results also indicate that the role of ZnT5 and ZnT6 heterodimers differs from that of the ZnT5 homodimer in cell proliferation. However, further studies are required to clarify the role of zinc transporters and zinc signaling in breast cancer. Presentation: No date and time listed

Obstructive sleep apnoea is related to melanoma aggressiveness through paraspeckle protein-1 upregulation.

In patients with obstructive sleep apnoea (OSA), intermittent hypoxia induces overexpression of paraspeckle component (PSPC)1, a master modulator of transforming growth factor (TGF)-β signalling, which promotes cell cancer progression through epithelial-mesenchymal transition (EMT) and acquisition of cancer stem cell (CSC)-like features. However, the persistence of intermittent hypoxia-induced effects on PSPC1, and their consequences in cancer patients are not known. To this effect, circulating PSPC1 levels were compared in patients with cutaneous melanoma with or without OSA, and their relationship with tumour aggressiveness along with the in vitro effects of soluble PSPC1 and intermittent hypoxia on melanoma cell aggressiveness mechanisms were assessed. In 292 cutaneous melanoma patients, sleep studies and serum levels of PSPC1 and TGF-β were evaluated. The effect of PSPC1 on expression of EMT and CSC transcription factors was assessed using melanoma cell lines with patient sera under both normoxia and intermittent hypoxia conditions. PSPC1 levels were higher in patients with moderate-severe OSA compared with mild OSA or non-OSA patients. Serum levels of PSPC1 were associated with several cutaneous melanoma clinical aggressiveness indicators. Both intermittent hypoxia exposures and serum from OSA patients upregulated TGF-β expression and amplified the expression of transcription factors associated with EMT activation and acquisition of CSC characteristics. In cutaneous melanoma patients, OSA severity is associated with higher PSPC1 serum levels, which jointly with intermittent hypoxia would enhance the self-reprogramming capabilities of EMT and CSC feature acquisition of melanoma cells, promoting their intrinsic aggressiveness.

Oct4 promoted proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) in colon cancer cells by activating the SCF/c-Kit signaling pathway

ABSTRACT Octamer-binding transcription factor 4 (Oct4) is closely related to the occurrence and development of cancer. In the present study, we paid a special interest in exploring the effect of Oct4 on colon cancer (CC) proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) and its molecular mechanism. Immunohistochemistry (IHC) was used to detect the expression level of Oct4 in colon tissue of patients with colon cancer. Oct4 overexpression vector pcDNA-Oct4 was used to stably express Oct4 in human colon cancer cells HT29 and SW480. Cell counting kit-8 (CCK-8) assay was used to detect the cell proliferation. The invasion and migration abilities were observed by transwell and wound healing assays. The expression of EMT relate genes were observed by Western blot. We found that Oct4 was up-regulated in human colon cancer tissues than that in paracancerous tissues. The proliferation, migration, and invasion of HT29 and SW480 cells was significantly induced by transfection of pcDNA-Oct4. Furthermore, Oct4 overexpression enhanced EMT of CC cells, characterized by the increased expression of vimentin, Twist, and Snail, as well as decreased expression of E-cadherin. Mechanistically, Oct4 overexpression activated stem cell factor (SCF)/c-Kit signaling pathway in CC cells, and the SCF/c-Kit signaling inhibitor imatinib reversed pro-oncogenic effects of Oct4. These finding provide an insight into the potential of Oct4 for CC diagnosis and therapy.

Matrine induces hepatocellular carcinoma apoptosis and represses EMT and stemness through microRNA-299-3p/PGAM1 axis

This study explored the impacts of matrine on hepatocellular carcinoma (HCC) cell growth, metastasis, epithelial-mesenchymal transition (EMT), and stemness through regulating the microRNA (miR)-299-3p/phosphoglycerate mutase 1 (PGAM1) axis. The association between miR-299-3p expression with the prognosis of HCC patients was studied. miR-299-3p and PGAM1 sequences were transfected into matrine-treated HCC cells, and cell proliferation, invasion, apoptosis, and stemness were detected, as well as protein expression of EMT- and stemness-related makers. The targeting relationship between miR-299-3p and PGAM1 was identified. Matrine elevated miR-299-3p expression, repressed proliferation, invasion, and anti-apoptosis of HCC cells, and constrained EMT and stemness in vitro. PGAM1 was a target of miR-299-3p. Repression of PGAM1 rescued the effects of miR-299-3p downregulation on HCC cells. Matrine stimulates HCC cell apoptosis and represses the process of EMT and stemness through the miR-299-3p/PGAM1 axis.

MiR-23b-3p suppresses epithelial-mesenchymal transition, migration, and invasion of hepatocellular carcinoma cells by targeting c-MET

BackgroundAberrant expression of c-MET is known to be associated with tumor recurrence and metastasis by promoting cell proliferation, epithelial-mesenchymal transition (EMT), and migration in hepatocellular carcinoma (HCC). Recently, miR-23b-3p has been identified as a tumor suppressor, but detailed role of miR-23b-3p in HCC is still unclear. Our study aimed to investigate how miR-23b-3p is associated with the malignant potential of HCC cells. MethodsHCC tissues and their adjacent non-tumor tissues were acquired from 30 patients with HCC. Expression of EMT- or stemness-related genes were examined in the two HCC cell lines. Migration of HCC cells was analyzed using transwell and wound healing assays. Resultsc-MET was overexpressed in HCC tissues compared to the adjacent non-tumor tissues. c-MET knockdown inhibited EMT and reduced migration and invasion of HCC cells. Furthermore, c-MET was a target of miR-23b-3p, and miR-23b-3p expression was decreased in HCC tissues compared to non-tumor tissues. Treatment of miR-23b-3p inhibitor in HCC cells promoted EMT, cell migration, and invasion. In contrast, miR-23b-3p overexpression suppressed EMT, cell migration, and invasion, concomitantly reducing c-MET expression. Transfection of miR-23b-3p inhibitor with concomitant c-MET knockdown mitigated the effects of miR-23b-3p inhibitor on EMT in HCC cells. In addition, transforming growth factor beta1 (TGF-β1) stimulation after miR-23b-3p overexpression induced neither the mesenchymal phenotype nor migratory property of HCC cells. ConclusionIn this study, we confirmed that miR-23b-3p downregulation significantly increased EMT, migration, and invasion of HCC cells. In addition, c-MET was confirmed to be a target of miR-23b-3p in HCC cells and regulated the functional effects of miR-23b-3p. These results suggest that miR-23b-3p can be used as a prognostic biomarker and candidate target for HCC treatment.

Effect of Co-Cultured Bone Marrow Mesenchymal Stem Cells (BMSC) and Neuropilin 1 on the Migration of Endometrial Stromal Cells and Epithelial-Mesenchymal Transition in Adenomyosis

Our study intends to assess the effect of co-cultured bone marrow mesenchymal stem cells (BMSC) and Neuropilin 1 on the migration of endometrial stromal cells and epithelial-mesenchymal transition in adenomyosis. qRT-PCR was utilized for measuring Neuropilin 1 level in adenomyosis tissues and cells. Endometrial stromal cells were treated with BMSC, si-Neuropilin 1, and pc-DNANeuropilin followed by analysis of cell proliferation and migration, as well as the expression of epithelial-mesenchymal transition (EMT)-related proteins. Neuropilin 1 expression was significantly upregulated in 77 adenomyosis patients. Neuropilin 1 expression showed a positive correlation with the diagnostic sensitivity of adenomyosis. BMSC+pc-DNA-Neuropilin 1 treatment significantly promoted cell viability, migration, and EMT which were reduced by BMSC+si-Neuropilin 1. In conclusion, our study demonstrates that co-culture of BMSCs and Neuropilin 1 increases cell viability, migration, and EMT in adenomyosis.

Consecutive dosing of UVB irradiation induces loss of ABCB5 expression and activation of EMT and fibrosis proteins in limbal epithelial cells similar to pterygium epithelium

Pterygium pathogenesis is often attributed to a population of altered limbal stem cells, which initiate corneal invasion and drive the hyperproliferation and fibrosis associated with the disease. These cells are thought to undergo epithelial to mesenchymal transition (EMT) and to contribute to subepithelial stromal fibrosis. In this study, the presence of the novel limbal stem cell marker ABCB5 in clusters of basal epithelial pterygium cells co-expressing with P63α and P40 is reported. ABCB5-positive pterygium cells also express EMT-associated fibrosis markers including vimentin and α-SMA while their β-catenin expression is reduced. By using a novel in vitro model of two-dose UV-induced EMT activation on limbal epithelial cells, we could observe the dysregulation of EMT-related proteins including an increase of vimentin and α-SMA as well as downregulation of β-catenin in epithelial cells correlating to downregulation of ABCB5. The sequential irradiation of limbal fibroblasts also induced an increase in vimentin and α-SMA. Taken together, these data demonstrate for the first time the expression of ABCB5 in pterygium stem cell activity and EMT-related events while the involvement of limbal stem cells in pterygium pathogenesis is exhibited via sequential irradiation of limbal epithelial cells. The later in vitro approach can be used to further study the involvement of limbal epithelium UV-induced EMT in pterygium pathogenesis and help identify novel treatments against pterygium growth and recurrence.

Regulation of MMP9 transcription by ETS1 in immortalized salivary gland epithelial cells of patients with salivary hypofunction and primary Sjögren’s syndrome

Primary Sjögren’s syndrome (pSS) patients exhibit enhanced degradation of the salivary epithelium initially through MMP9 overexpression. We assessed the expression of MMP9 and an associated transcription factor, ETS1, in primary salivary gland epithelial cells (SGECs) and investigated potential regulatory mechanism(s) in immortalized SGECs. SGECs and iSGECs were derived from pSS and/or xerostomic “sicca” patients. siRNA knockdown of ETS1 in iSGECs was performed to determine MMP9 mRNA (qRT-PCR) and protein expression (ELISA). ETS1 binding to MMP9 promoter was assessed by luciferase activity and binding confirmed by mutagenesis and ChIP. Effects of ETS1 overexpression on progenitor and Epithelial-Mesenchymal transition (EMT) associated markers were determined by Western blot. Expression of ETS1 and its phosphorylated form in iSGECs was determined by immunofluorescence microscopy. ETS1 and MMP9 were overexpressed in SGECs of pSS and non-pSS sicca patients with salivary gland lymphocytic infiltration compared to non-pSS sicca patients without infiltration. ETS1 siRNA knockdown reduced both MMP9 mRNA and protein levels. ETS1 overexpression affected the expression of EMT and progenitor cell markers. Lastly, ETS1 bound the MMP9 promoter within the DNA region of −296 bp to −339 bp. ETS1 may impair salivary function through direct transcriptional control of the MMP9 promoter. ETS1 upregulation may also affect other factors involved in repair of the dysfunctional pSS salivary epithelium.

Salivary Fusobacterium nucleatum serves as a potential diagnostic biomarker for gastric cancer.

BACKGROUNDAs one of the most common tumors, gastric cancer (GC) has a high mortality rate, since current examination approaches cannot achieve early diagnosis. Fusobacterium nucleatum (Fn) primarily colonized in the oral cavity, has been reported to be involved in the development of gastrointestinal tumor. Until now, little is known about the relationship between salivary Fn and GC.AIMTo determine whether salivary Fn could be a biomarker to diagnose GC and explore the influence of Fn on GC cells.METHODSThe abundance of Fn in saliva was quantified by droplet digital polymerase chain reaction in 120 GC patients, 31 atrophic gastritis (AG) patients, 35 non-AG (NAG) patients, 26 gastric polyp (GP) patients, and 20 normal controls (NC) from Qilu Hospital of Shandong University from January 2019 to December 2020. Receiver operating characteristic (ROC) curve analysis was performed to evaluate the diagnostic value of Fn as well as traditional serum tumor markers, including carcinoembryonic antigen (CEA), carbohydrate antigen (CA) 19-9, and CA72-4. Transwell assay and wound-healing assay were conducted to assess the influence of Fn infection on GC cells. The expression of epithelial-mesenchymal transition (EMT) markers was detected using western blot assay.RESULTSWe found that the level of salivary Fn in GC patients was significantly increased compared with those in AG, NAG, and GP patients and NC (P < 0.001). ROC curve analysis showed a favorable capability of Fn (73.33% sensitivity; 82.14% specificity; area under the curve: 0.813) in GC diagnosis, which was superior to that of CEA, CA19-9, CA72-4, ferritin, and sialic acid. The Fn level in saliva of GC patients was increased as the TNM stage increased. GC patients with lymph node metastasis had higher Fn levels than those without metastasis. Both transwell and wound-healing assays indicated that Fn infection promoted the migration and invasion of GC cells. Western blot analysis showed that Fn infection decreased the expression of E-cadherin and increased the expressions of N-cadherin, vimentin, and snail.CONCLUSIONFn abundance in saliva could be used as a promising biomarker to diagnose GC, and Fn infection could promote GC metastasis by accelerating the EMT process.

PEBP4 Directs the Malignant Behavior of Hepatocellular Carcinoma Cells via Regulating mTORC1 and mTORC2.

Phosphatidylethanolamine binding protein 4 (PEBP4) is an understudied multifunctional small protein. Previous studies have shown that the expression of PEBP4 is increased in many cancer specimens, which correlates to cancer progression. The present study explored the mechanism by which PEBP4 regulates the growth and progression of hepatocellular carcinoma cells. Thus, we showed that knockdown of PEBP4 in MHCC97H cells, where its expression was relatively high, diminished activities of serine/threonine protein kinase B (PKB, also known as Akt), mammalian target of rapamycin complex 1(mTORC1), and mTORC2, events that were not restored by insulin-like growth factor 1 (IGF-1). Conversely, overexpression of PEBP4 in MHCC97L cells with the low endogenous level yielded opposite effects. Furthermore, physical association of PEBP4 with Akt, mTORC1, and mTORC2 was observed. Interestingly, introduction of AktS473D mutant, bypassing phosphorylation by mTORC2, rescued mTORC1 activity, but without effects on mTORC2 signaling. In contrast, the effect of PEBP4 overexpression on the activity of mTORC1 but not that of mTORC2 was suppressed by MK2206, a specific inhibitor of Akt. In conjunction, PEBP4 knockdown-engendered reduction of cell proliferation, migration and invasion was partially rescued by Akt S473D while increases in these parameters induced by overexpression of PEBP4 were completely abolished by MK2206, although the expression of epithelial mesenchymal transition (EMT) markers appeared to be fully regulated by the active mutant of Akt. Finally, knockdown of PEBP4 diminished the growth of tumor and metastasis, whereas they were enhanced by overexpression of PEBP4. Altogether, our study suggests that increased expression of PEBP4 exacerbates malignant behaviors of hepatocellular cancer cells through cooperative participation of mTORC1 and mTORC2.