N-cadherin mRNA Expression Research Articles

ARHGAP17 Inhibits Hepatocellular Carcinoma Progression by Inactivation of Wnt/β-Catenin Signaling Pathway

ARHGAP17 Inhibits Hepatocellular Carcinoma Progression by Inactivation of Wnt/β-Catenin Signaling Pathway

Neurotensin modulates ovarian vascular permeability via adherens junctions.

Neurotensin (NTS) is a 13-amino acid peptide which is highly expressed in the mammalian ovary in response to the luteinizing hormone surge. Antibody neutralization of NTS in the ovulatory follicle of the cynomolgus macaque impairs ovulation and induces follicular vascular dysregulation, with excessive pooling of red blood cells in the follicle antrum. We hypothesize that NTS is an essential intrafollicular regulator of vascular permeability. In the present study, follicle injection of the NTS receptor antagonist SR142948 also resulted in vascular dysregulation. To measure vascular permeability changes invitro, primary macaque ovarian microvascular endothelial cells (mOMECs) were enriched from follicle aspirates and studied invitro. When treated with NTS, permeability of mOMECs decreased. RNA sequencing (RNA-Seq) of mOMECs revealed high mRNA expression of the permeability-regulating adherens junction proteins N-cadherin (CDH2) and K-cadherin (CDH6). Immunofluorescent detection of CDH2 and CDH6 confirmed expression and localized these cadherins to the cell-cell boundaries, consistent with function as components of adherens junctions. mOMECs did not express detectable levels of the typical vascular endothelial cadherin, VE-cadherin (CDH5) as determined by RNA-Seq, qPCR, western blot, and immunofluorescence. Knockdown of CDH2 or CDH6 via siRNA abrogated the NTS effect on mOMEC permeability. Collectively, these data suggest that NTS plays an ovulation-critical role in vascular permeability maintenance, and that CDH2 and CDH6 are involved in the permeability modulating effect of NTS on the ovarian microvasculature. NTS can be added to a growing number of angiogenic regulators which are critical for successful ovulation.

Nickel induces epithelial-mesenchymal transition in pulmonary fibrosis in mice via activation of the oxidative stress-mediated TGF-β1/Smad signaling pathway.

Nickel (Ni) is recognized as a carcinogenic metal, and its widespread use has led to severe environmental and health problems. Although the lung is among the main organs affected by Ni, the precise mechanisms behind this effect remain poorly understood. This study aimed to elucidate the physiological mechanisms underlying Ni-induced pulmonary fibrosis (PF), using various techniques including histopathological detection, biochemical analysis, immunohistochemistry, western blotting, and quantitative real-time PCR. Mice were treated with nickel chloride (NiCl2), which induced PF (detected by Masson staining), up-regulation of α-smooth muscle actin (α-SMA), and collagen-1 mRNA and protein expression. NiCl2 was found to induce PF by: activation of the epithelial-mesenchymal transition (EMT) and the transforming growth factor-β1 (TGF-β1)/Smad signaling pathway; up-regulation of protein and mRNA expression of TGF-β1, p-Smad2, p-Smad3, vimentin, and N-cadherin; and down-regulation of protein and mRNA expression of E-cadherin. In addition, NiCl2 treatment increased malondialdehyde content while inhibiting antioxidant activity, as indicated by decreased catalase, total antioxidant capacity, and superoxide dismutase activities, and glutathione content. Co-treatment with the effective antioxidant and free radical scavenger N-acetyl cysteine (NAC) plus NiCl2 was used to study the effects of oxidative stress in NiCl2-induced PF. The addition of NAC significantly mitigated NiCl2-induced PF, and reversed activation of the TGF-β1/Smad signaling pathway and EMT. NiCl2-induced PF was therefore shown to be due to EMT activation via the TGF-β1/Smad signaling pathway, mediated by oxidative stress.

INHBA gene silencing inhibits proliferation, migration, and invasion of osteosarcoma cells by repressing TGF-β signaling pathway activation

BackgroundOsteosarcoma (OS) is a refractory malignancy. This study aimed to explore the roles and mechanisms of Inhibin subunit beta A (INHBA) in OS.MethodsINHBA expression levels in OS tissues and cells were assessed using RT-qPCR and western blotting. The impact of INHBA silencing on OS development was then explored by transfecting the OS cell lines U2OS and MG63 with INHBA-small interfering RNA (siRNA). The influence of INHBA silencing on U2OS and MG63 cell proliferation, migration, and invasion was examined using MTT and Transwell assays. Epithelial–mesenchymal transition (EMT) markers (E-cadherin and N-cadherin) were analyzed by RT-qPCR. The expression of genes involved in cell proliferation, migration, invasion, and the TGF-β signaling pathway was evaluated by western blotting and RT-qPCR.ResultsINHBA levels were elevated in the OS tissues and cells. Furthermore, the transforming growth factor-β (TGF-β) signaling pathway of OS cells was suppressed in response to INHBA-siRNA, whereas proliferation, migration, and invasion of OS cells were inhibited. Besides, INHBA-siRNA significantly inhibited OS cell EMT, evidenced by enhanced E-cadherin mRNA expression and reduced N-cadherin mRNA expression. Further mechanistic studies revealed that the TGF-β1 agonist SRI-011381 hydrochloride increased OS cell proliferation, migration, and invasion after INHBA downregulation.ConclusionWe found that INHBA silencing could play a vital role in OS via TGF-β1-regulated proliferation, migration, and invasion.

Effect of acetylalkannin from Arnebia euchroma on proliferation, migration, and invasion of human melanoma A375 cells

This study aimed to explore the effect and mechanism of acetylalkannin from Arnebia euchroma on the proliferation, migration, and invasion of human melanoma A375 cells. A375 cells were divided into a blank group, and low-, medium-, and high-dose acetylalkannin groups(0.5, 1.0, and 2.0 μmol·L~(-1)). The MTT assay was used to detect cell proliferation. Cell scratch and transwell migration assays were used to detect cell migration ability, and the transwell invasion assay was used to detect cell invasion ability. Western blot was used to detect the protein expression of migration and invasion-related N-cadherin, vimentin, matrix metalloproteina-se-9(MMP-9), and Wnt/β-catenin pathway-related Wnt1, Axin2, glycogen synthase kinase-3β(GSK-3β), phosphorylated GSK-3β(p-GSK-3β), β-catenin, cell cycle protein D_1(cyclin D_1), and p21. Real-time fluorescence-based quantitative polymerase chain reaction(real-time PCR) was used to detect the mRNA expression of E-cadherin, matrix metalloproteinase-2(MMP-2), N-cadherin, vimentin, β-catenin, snail-1, and CD44. MTT results showed that the cell inhibition rates in the acetylalkannin groups significantly increased as compared with that in the blank group(P<0.01). The results of cell scratch and transwell assays showed that compared with the blank group, the acetylalkannin groups showed reduced cell migration and invasion, and migration and invasion rates(P<0.05, P<0.01) and weakened horizontal and vertical migration and invasion abilities. Western blot results showed that compared with the blank group, the high-dose acetylalkannin group showed increased expression of Axin2 protein(P<0.05), and decreased expression of N-cadherin, vimentin, MMP-9, Wnt1, p-GSK-3β, β-catenin, cyclin D_1, and p21 proteins(P<0.05, P<0.01). The expression of GSK-3β protein did not change significantly. PCR results showed that the overall trend of MMP-2, N-cadherin, vimentin, β-catenin, snail-1, and CD44 mRNA expression was down-regulated(P<0.01), and the expression of E-cadherin mRNA increased(P<0.01). Acetylalkannin can inhibit the proliferation, migration, and invasion of human melanoma A375 cells, and its mechanism of action may be related to the regulation of Wnt/β-catenin signaling pathway.

Hsa-miR-497-3p impedes the proliferation and invasion of triple-negative breast cancer cells by controlling epithelial-mesenchymal transition through ZEB1 targeting.

This study aimed to examine the hsa-miR-497-3p effect and mechanism on the behavior of triple-negative breast cancer (TNBC) cells. We evaluated the expression of Hsa-miR-497-3p in tissue samples obtained from patients diagnosed with TNBC or luminal breast cancer (BrCa), utilizing the quantitative fluorescence polymerase chain reaction (PCR) method. We transfected hsa-miR-497-3p mimics and NC into MDA-MB-231 cells, whilehsa-miR-497-3p inhibitor and NC into TNBC cells, respectively. To examine the impact of hsa-miR-497-3p expression level on TNBC cell proliferation, invasion, and migration, we employed MTT, clone formation, Transwell, and wound healing assays. We utilized both q-PCR and western blot to validate the role of hsa-miR-497-3p in the epithelial-mesenchymal transition (EMT) of TNBC cells. To confirm the targeting relationship between hsa-miR-497-3p and ZEB1, we performed luciferase assays, q-PCR, and western blot analysis. We found that the hsa-miR-497-3p expression was down-regulated in both TNBC tissues and cell lines in comparison to luminal BrCa tissues and cell lines. Furthermore, hsa-miR-497-3p overexpression hindered the cell function of TNBC cells MDA-MB-231, while downregulating the mRNA and protein expression of vimentin and N-cadherin, while simultaneously upregulating E-cadherin expression. Our results demonstrate that hsa-miR-497-3p regulates EMT in TNBC cells through ZEB1 targeting, as evidenced by the modulation of the expression of vimentin, N-cadherin, and E-cadherin via ZEB1 inhibition. Overall, our study suggests that hsa-miR-497-3p inhibits the proliferation and invasion of TNBC cells through the modulation of EMT via ZEB1 targeting.

Downregulation of FHL2 suppressed trophoblast migration, invasion and epithelial–mesenchymal transition in recurrent miscarriage

Research questionIs four and a half LIM domain 2 (FHL2) involved in trophoblast migration, invasion and epithelial–mesenchymal transition (EMT) in recurrent miscarriage? DesignVillus tissue was collected from 24 patients who had experienced recurrent miscarriage and 24 healthy controls. FHL2 mRNA and protein expression in villus specimens were observed by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. Small interfering RNA and overexpression plasmid were used to change the FHL2 expression. JAR and HTR8/SVneo cell lines were used to conduct scratch-wound assay and transwell assay to detect trophoblast migration and invasion of FHL2. Downstream molecule expression of mRNA and protein and EMT markers were verified by qRT-PCR and Western blot. ResultsSignificantly lower FHL2 mRNA (P = 0.019) and protein (P = 0.0014) expression was found in trophoblasts from the recurrent miscarriage group compared with healthy controls. FHL2 knockdown repressed migration (P = 0.0046), invasion (P < 0.001) and EMT, as shown by significant differences in mRNA and protein expression of the EMT markers N-cadherin, E-cadherin, Vimentin and Snail (all P < 0.05) of extravillus trophoblasts. FHL2 overexpression enhanced migration (P = 0.025), invasion (P < 0.001) and EMT of extravillus trophoblasts (all EMT markers P < 0.05). The positive upstream factor FHL2 in the extracellular signal-related kinase pathway induced JunD expression, thereby promoting trophoblast migration and invasion via matrix metalloproteinase 2. ConclusionsFHL2 is involved in a regulatory pathway of trophoblast migration, invasion and EMT during early pregnancy, and may have a role in recurrent miscarriage pathogenesis, which can serve as a possible target for novel therapeutic development.

The Role of Erythrocyte Membrane Protein Band 4.1-like 3 in Idiopathic Pulmonary Fibrosis.

Novel genetic and epigenetic factors involved in the development and prognosis of idiopathic pulmonary fibrosis (IPF) have been identified. We previously observed that erythrocyte membrane protein band 4.1-like 3 (EPB41L3) increased in the lung fibroblasts of IPF patients. Thus, we investigated the role of EPB41L3 in IPF by comparing the EPB41L3 mRNA and protein expression of lung fibroblast between patients with IPF and controls. We also investigated the regulation of epithelial-mesenchymal transition (EMT) in an epithelial cell line (A549) and fibroblast-to-myofibroblast transition (FMT) in a fibroblast cell line (MRC5) by overexpressing and silencing EPB41L3. EPB41L3 mRNA and protein levels, as measured using RT-PCR, real-time PCR, and Western blot, were significantly higher in fibroblasts derived from 14 IPF patients than in those from 10 controls. The mRNA and protein expression of EPB41L3 was upregulated during transforming growth factor-β-induced EMT and FMT. Overexpression of EPB41L3 in A549 cells using lenti-EPB41L3 transfection suppressed the mRNA and protein expression of N-cadherin and COL1A1. Treatment with EPB41L3 siRNA upregulated the mRNA and protein expression of N-cadherin. Overexpression of EPB41L3 in MRC5 cells using lenti-EPB41L3 transfection suppressed the mRNA and protein expression of fibronectin and α-SMA. Finally, treatment with EPB41L3 siRNA upregulated the mRNA and protein expression of FN1, COL1A1, and VIM. In conclusion, these data strongly support an inhibitory effect of EPB41L3 on the process of fibrosis and suggest the therapeutic potential of EPB41L3 as an anti-fibrotic mediator.

Abstract 1234: Tumor cell-intrinsic PD-L1 promotes epithelial-to-mesenchymal transition in non-small cell lung cancer in interferon-g-independent and dependent way

Abstract The immune checkpoint molecule programmed cell death-ligand 1 (PD-L1, CD274) suppresses immunity when the cell surface PD-L1 is engaged by programmed death-1 (PD-1) on antitumor immune cells. Although PD-L1 has been established and targeted in cancer immunotherapy, the tumor-intrinsic role of PD-L1 is less appreciated in tumor biology and therapeutic development. In this study, we showed that PD-L1 facilitates epithelial-to-mesenchymal transition (EMT) in non-small cell lung cancer (NSCLC) cells by upregulating EMT transcription factors and mesenchymal markers, and downregulating epithelial marker. RNA-sequencing was performed on paired exogenously PD-L1-overexpressed A549 cells and wild-type cells, and PD-L1-depleted H460 cells and wild-type cells, and transcriptomic features associated with PD-L1 status were identified using Gene Set Enrichment Analysis (GSEA). In both the paired samples, PD-L1 expression was associated with EMT-related pathways. Subsequent in vitro experiment showed that exogenous overexpression of PD-L1 in A549 and H522 cells increased both mRNA and protein expression of Twist, Zeb1, Snail, Slug, Vimentin, Fibronectin, and N-Cadherin. Correspondingly, the transcription factors and mesenchymal markers were decreased in PD-L1-depleted H460 and H596 cells. Recombinant PD-1 treatment also increased the expression of EMT transcription factors and markers on A549 and H460 cells. Moreover, interferon-γ stimulation on tumor cells increased expression of EMT transcription factors and markers, which was suppressed by PD-L1-knockdown, suggesting that interferon-γ-induced PD-L1 contributes to EMT. Our findings suggest that PD-L1 facilitates NSCLC progression by promoting the EMT in interferon-γ-independent and dependent way, and this intrinsic PD-L1 function could be targeted for better clinical management of PD-L1-overexpressing NSCLCs. Citation Format: Hyein Jeong, Hyun Kyung Ahn, Sehui Kim, Yoon Kyung Jeon. Tumor cell-intrinsic PD-L1 promotes epithelial-to-mesenchymal transition in non-small cell lung cancer in interferon-g-independent and dependent way [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1234.

Sortilin 1 Promotes Hepatocellular Carcinoma Cell Proliferation and Migration by Regulating Immune Cell Infiltration.

Objectives Recent evidence suggests that Sort1 promotes carcinogenesis and tumor progression in multiple types of cancers. This study investigates the role of Sort1 in hepatocellular carcinoma (HCC). Methods The differentially expressed gene was screened through GEO and TCGA databases. The Sort1 gene was identified and its expression was then verified by TCGA and HCCDB (a database of hepatocellular carcinoma expression atlas) databases. The Human Protein Atlas database was used to assess the gene expression in tissues. The TCGA and KM-plotter databases were used to study the relationship between Sort1 and HCC. The correlation between Sort1 and immune cells was evaluated through the TIMER database. GO and KEGG enrichment analysis was used to investigate the possible mechanism. The role of Sort1 in cell proliferation and invasion of HCC was further explored through in vitro experiments. Result The differentially expressed molecule obtained from database screening was Sort1. Its expression was higher in cancer tissues than in paracancerous ones, and it was mainly located in the cytoplasm. The TCGA, KM-plotter databases, and our study data showed that low expression of Sort1 in HCC patients had better overall survival (OS), progression-free survival (PFI), and disease-specific survival (DSS). Further analysis indicated a significant correlation between Sort1 expression and immune cell infiltration. The gene set enrichment analysis (GSEA) analysis showed that Sort1 affected the biological events of HCC by participating in the WNT, TGF-BETA, JAK, STAT, and CALCIUM signaling pathways. In vitro, cytological experiments demonstrated reduced expression of PCNA, Ki-67, Vimentin, N-cadherin, and MMP-9 mRNA after knocking down Sort1, although E-cadherin expression was promoted. Overall, these processes reduced the ability of proliferation and invasion of HCC cells. Conclusion Downregulation of Sort1 can prolong the OS, PFI, and DSS of HCC patients. Furthermore, due to its link with immune cell infiltration, the Sort1 gene represents a potentially novel predictive biomarker of HCC. The growth of HCC can be significantly inhibited by interfering with Sort1; therefore, these results provide a potential target for developing anticancer strategies for HCC.

Iodine-125 Seeds Inhibit Carcinogenesis of Hepatocellular Carcinoma Cells by Suppressing Epithelial-Mesenchymal Transition via TGF-β1/Smad Signaling.

To investigate the radioactive iodine-125 (I-125) seed on migrating and invading of hepatocellular carcinoma (HCC) cells and its mechanism, the irradiation of PLC and Huh7 cells was carried out with I-125 seeds in vitro. Cell counting kit 8 assay was employed to measure cell viability. Cell migration was evaluated by using wound-healing assay. Cell invasion was detected by Transwell assay; RT-PCR and Western blot were used for the detection of the mRNA and proteins of TGF-β1 signaling pathway-related genes. The viability of PLC and Huh7 cells declined in a dose-dependent manner with increasing irradiation from 0 Gy, 2 Gy, 4 Gy, and 6 Gy, to 8 Gy, respectively. The IC50 of PLC and Huh7 cells were 6.20 Gy and 5.39 Gy, respectively, after 24 h of irradiation. Migration and invasion abilities of I-125 group cells were greatly weakened (P < 0.05) comparing with the control group. According to the outcomes of RT-PCR and WB, I-125 seed irradiation significantly inhibited the mRNA and protein expression of N-cadherin, vimentin, TGF-β1, p-Smad2/3, and Snail. But the mRNA and protein expressions of E-cadherin were enhanced. Rescue experiment demonstrates that TGF-β1 activator could reverse the inhibitory effects of I-125 on invasion and migration of cells. The results of in vivo experiments further verified that the I-125 seeds can inhibit the proliferation and TGF-β1 of xenographed PLC cells. In conclusion, I-125 seeds restrain the invasion and migration of HCC cells by suppressing epithelial to mesenchymal transition, which may associate with the inhibition of the TGF-β1 signaling.

Effect of RBP2 gene silencing on proliferation, migration and invasion of ovarian epithelial cancer SKOV3/DDP cells and its mechanism

Objective: To explore the effect of down-regulation of retinol binding protein 2 (RBP2) expression on the biological characteristics of ovarian cancer cells and its mechanism. Methods: Knockdown of RBP2 and cisplatin (DDP)-resistant ovarian cancer cell line SKOV3/DDP-RBP2i was established, the negative control group and blank control group were also set. Cell counting kit 8 (CCK-8) was used to detect the cell proliferation ability, flow cytometry was used to detect cell apoptosis, scratch test and Transwell invasion test were used to detect cell migration and invasion ability, real-time fluorescent quantitative polymerase chain reaction (RT-qPCR) and western blot were used to detect the expressions of molecular markers related to epithelial-mesenchymal transition (EMT). The effect of RBP2 on the growth of ovarian cancer was verified through experiment of transplanted tumors in nude mice, and the relationships between RBP2 expression and tumor metastasis and patient prognosis were analyzed using the clinical data of ovarian cancer in TCGA database. Results: After down-regulating the expression of RBP2, the proliferation ability of SKOV3/DDP cell was significantly reduced. On the fifth day, the proliferation activities of SKOV3/DDP-RBP2i group, negative control group and blank control group were (56.67±4.16)%, (84.67±3.51) and (87.00±4.00)% respectively, with statistically significant difference (P<0.001). The apoptosis rate of SKOV3/DDP-RBP2i group was (14.19±1.50)%, higher than (8.77±0.75)% of the negative control group and (7.48±0.52)% of the blank control group (P<0.001). The number of invasive cells of SKOV3/DDP-RBP2i group was (55.20±2.39), lower than (82.60±5.18) and (80.80±7.26) of the negative control group and the blank control group, respectively (P<0.001). The scratch healing rate of SKOV3/DDP-RBP2i group was (28.47±2.72)%, lower than (50.58±4.06)% and (48.92±4.63)% of the negative control group and the blank control group, respectively (P<0.001). The mRNA and protein expressions of E-cadherin in the SKOV3/DDP-RBP2i group were higher than those in the negative control group (P=0.015, P<0.001) and the blank control group (P=0.006, P<0.001). The mRNA and protein expression of N-cadherin in SKOV3/DDP-RBP2i group were lower than those in the negative control group (P=0.012, P<0.001) and the blank control group (P=0.005, P<0.001). The mRNA and protein expressions of vimentin in SKOV3/DDP-RBP2i group were also lower than those in the negative control group (P=0.016, P=0.001) and the blank control group (P=0.011, P=0.001). Five weeks after the cells inoculated into the nude mice, the tumor volume of SKOV3/DDP-RBP2i group, negative control group and blank control group were statistically significant different. The tumor volume of SKOV3/DDP-RBP2i group was smaller than those of negative control group and blank control group (P=0.001). Bioinformatics analysis showed that the expression of RBP2 in patients with metastatic ovarian cancer was higher than that without metastasis (P=0.043), and the median overall survival of ovarian cancer patients with high RBP2 expression was 41 months, shorter than 69 months of low RBP2 expression patients (P<0.001). Conclusion: Downregulation of the expression of RBP2 in SKOV3/DDP cells can inhibit cell migration and invasion, and the mechanism may be related to the inhibition of EMT.

Correlation between Lactobacillus and expression of E-cadherin, β-catenin, N-cadherin, and Vimentin in postmenopausal cervical lesions.

To detect the correlation between Lactobacillus vaginalis and the expression of epithelial-mesenchymal transition (EMT)-related factors, E-cadherin, β-catenin, N-cadherin, and Vimentin, in postmenopausal cervical squamous intraepithelial lesions (SILs) and cervical squamous cell carcinoma (SCC), and to explore the possible mechanism. From January 2016 to January 2020, 30 postmenopausal patients with low-grade SIL (LSIL), 18 patients with high-grade SIL (HSIL), and 30 patients with SCC who underwent colposcopy biopsy in the Outpatient Department of the First Affiliated Hospital of Inner Mongolia Medical University were selected as the experimental group, and 30 postmenopausal normal women were selected as the control group. The expression of 16SrRNA of Lactobacillus vaginalis in each group was determined by the 16S third-generation full-length amplification sequencing technique. The mRNA expression levels of E-cadherin, β-catenin, N-cadherin, and Vimentin were detected by quantitative real-time polymerase chain reaction (qPCR). The correlation between the 16SrRNA expression level of Lactobacillus vaginalis and the mRNA expression level of the EMT-related proteins was compared among all groups. (I) The progression of postmenopausal cervical SILs to cervical SCC was significantly positively correlated with age, number of pregnancies, smoking, pH value, positive rate of HPV16, and negatively correlated with total Lactobacillus 16SrRNA expression (P<0.0001). (II) The level of vaginal microbiota in postmenopausal women showed that Lactobacillus iners was dominant. With the progression of the disease, the expression levels of 16SrRNA in Lactobacillus iners and Lactobacillus total vagina decreased gradually, and the differences were statistically significant (P<0.05). (III) With the disease progresses. The expression of total Lactobacillus 16SrRNA was positively correlated with the mRNA expression of β-catenin and E-cadherin (r>0; P<0.05), and negatively correlated with the mRNA expression of Vimentin and N-cadherin (r<0; P<0.05). In postmenopausal women, Lactobacillus vaginalis interacts with HPV and is associated with the occurrence of EMT, promoting the development of cervical lesions.

Resveratrol affects Proliferation, Migration, Invasion and Apoptosis of Oral Squamous Cell Carcinoma Cells by Inhibiting the WNT Pathway

Objective: To explore the mechanism by which resveratrol (RES) affects the proliferation, migration, invasion and apoptosis of oral squamous cell carcinoma (OSCC) cells through inhibiting the Wnt pathway. Methods: Ki-67, matrix metalloproteinase-9 (MMP-9) and B-cell lymphoma-2 (Bcl-2) protein expression levels were detected by immunohistochemical staining. Tca8113 cells were treated with different concentrations of RES (0, 50, 100 and 200 μmol/L) and divided into control, low-dose RES, medium-dose RES and high-dose RES groups. EdU staining,Transwell migration and invasion assays and Annexin V-FITC/PI double-staining were employed to determine cell proliferation, migration, invasion and apoptosis, respectively. E-cadherin and N-cadherin mRNA expression levels in cells were analyzed by RT-qPCR. Wnt1,β-catenin and glycogen synthase kinase-3β (GSK-3β) protein expression levels were detected by Western blotting. Results: Ki-67, MMP-9 and Bcl-2 were positively expressed in OSCC tissues, with higher rates than those in normal oral mucosal tissues. RES significantly inhibited the proliferation, migration and invasion ability of Tca8113 cells, down-regulated the expression levels of N-cadherin mRNA and Wntl, β-catenin and GSK-3β proteins, promoted apoptosis, and up-regulated E-cadherin mRNA expression level dose-dependently (P&lt;0.05). Conclusion: RES can inhibit the proliferation, migration and invasion of OSCC cells and promote apoptosis possibly by inhibiting the Wnt signaling pathway, providing a potential target for targeted therapy of OSCC.

Identification of the Hub Genes Associated with the Prognosis of Ovarian Cancer Patients via Integrated Bioinformatics Analysis and Experimental Validation.

BackgroundThis study aimed to identify the hub genes associated with prognosis of patients with ovarian cancer by using integrated bioinformatics analysis and experimental validation.MethodsFour microarray datasets (GSE12470, GSE14407, GSE18521 and GSE46169) were analyzed by the GEO2R tool to screen common differentially expressed genes (DEGs). Gene Ontology, the Kyoto Encyclopedia of Genes and Genomes, the (KEGG) pathway and Reactome pathway enrichment analysis, protein–protein interaction (PPI) construction, and the identification of hub genes were performed. Furthermore, we performed the survival and expression analysis of the hub genes. In vitro functional assays were performed to assess the effects of hub genes on ovarian cancer cell proliferation, caspase-3/7 activity and invasion.ResultsA total of 89 common DEGs were identified among these four datasets. The KEGG and Reactome pathway results showed that the DEGs were mainly associated with cell cycle, mitotic and p53 signaling pathway. A total of 20 hub genes were identified from the PPI network by using sub-module analysis. The survival analysis revealed that high expression of six hub genes (AURKA, BUB1B, CENPF, KIF11, KIF23 and TOP2A) were significantly correlated with shorter overall survival and progression-free survival of patients with ovarian cancer. Furthermore, the expression of the six hub genes were validated by the GEPIA database and Human Protein Atlas, and functional studies revealed that knockdown of KIF11 and KIF23 suppressed the SKOV3 cell proliferation, increased caspase-3/7 activity and attenuated invasive potentials of SKOV3 cells. In addition, knockdown of KIF11 and KIF23 up-regulated E-cadherin mRNA expression but down-regulated N-cadherin and vimentin mRNA expression in SKOV3 cells.ConclusionOur results showed that six hub genes were up-regulated in ovarian cancer tissues and may predict poor prognosis of patients with ovarian cancer. KIF11 and KIF23 may play oncogenic roles in ovarian cancer cell progression via promoting ovarian cancer cell proliferation and invasion.

TGF-β1 induces amoeboid-to-mesenchymal transition of CD44high oral squamous cell carcinoma cells via miR-422a downregulation through ERK activation and Cofilin-1 phosphorylation.

The objective of this study was to clarify the molecular mechanism of amoeboid-to-mesenchymal transition (AMT) of CD44high oral squamous cell carcinoma (OSCC) cells. Morphology and expression of mesenchymal genes were investigated in CD44high OSCC cells (CD44high OM-1 cells) cultured on laminin-coated soft silicone gel. Additionally, microarray analysis was performed to investigate microRNA (miRNA) expression inhibited by transforming growth factor-β1 (TGF-β1) in CD44high OM-1 cells. When CD44high OM-1 cells were cultured on 2.0-kPa laminin-coated silicone gel, the cells exhibited an amoeboid-like round morphology. Cofilin-1 expression was found in the nucleus and cytoplasm of amoeboid-like CD44high OM-1 cells. The invasive capacity was significantly reduced after Cofilin-1 knockdown. Additionally, Cofilin-1 knockdown cells had an irregularly extended shape. Phosphorylated Cofilin-1 was significantly upregulated by TGF-β1. Additionally, TGF-β1 enhanced N-cadherin and Snail mRNA expression and induced a spindle-shaped morphology. ERK1/2 phosphorylation was induced by TGF-β1. Microarray analysis revealed that miR-422a exhibited the greatest downregulation (fold change: 0.22) in the presence of TGF-β1. Importantly, TGF-β1-inhibited miR-422a expression was recovered by the ERK inhibitor or ERK1/2 knockdown. Additionally, miR-422a inhibitor-transfected CD44high OM-1 cells exhibited high N-cadherin and Snail mRNA expression. Furthermore, Cofilin-1 knockdown and miR-422a inhibition induced a spindle cell morphology. Cofilin-1 is involved in the invasive ability of CD44high OSCC cells. TGF-β1 contributes to AMT by downregulation of miR-422a via ERK activation and Cofilin-1 phosphorylation. Our findings suggest that miR-422a and Cofilin-1 play major roles in the maintenance of amoeboid-like CD44high cells.

Aberrant Expression of Androgen Receptor Associated with High Cancer Risk and Extrathyroidal Extension in Papillary Thyroid Carcinoma

Male gender is a risk factor for mortality in patients with papillary thyroid carcinoma (PTC). This study investigated the impact of androgen receptor (AR) gene expression on the clinical features and progression of PTC. The levels of AR mRNA and protein in frozen, formalin-fixed, paraffin-embedded tissue samples from PTC and adjacent normal thyroid tissue were assessed by quantitative real-time polymerase chain reaction and immunohistochemical staining, respectively, and the relationships between AR expression and clinical features were analyzed. The thyroid cancer cell lines, BCPAP and TPC-1, were used to evaluate the effects of AR on the regulation of cell migration, and key epithelial–mesenchymal transition (EMT) markers. AR mRNA expression was significantly higher in normal thyroid tissue from men than women. The sex difference in AR mRNA expression diminished during PTC tumorigenesis, as AR mRNA expression levels were lower in PTC than normal thyroid tissues from both men and women. AR mRNA expression was significantly decreased in PTC patients with higher risk and in those with extrathyroidal extension. Overexpression of AR in BCPAP cells decreased cell migration and repressed the EMT process by down-regulating mRNA expression of N-cadherin, Snail1, Snail2, Vimentin, and TWIST1 and up-regulating E-cadherin gene expression. These results suggest that suppression of the androgen–AR axis may lead to aggressive tumor behavior in patients with PTC.

Benzo[a]pyrene at human blood equivalent level induces human lung epithelial cell invasion and migration via aryl hydrocarbon receptor signaling.

Benzo[a]pyrene (B[a]P), a typical carcinogenic polycyclic aromatic hydrocarbon, exists worldwide in vehicle exhaust, cigarette smoke and other polluted environments. Recent studies have demonstrated a strong association between B[a]P and lung cancer. However, whether B[a]P at human blood equivalent level can promote epithelial-mesenchymal transition (EMT), a crucial molecular event during cell malignant transformation, remains unclear. Besides, whether B[a]P facilitates this progress via aryl hydrocarbon receptor (AhR) signaling pathway also lacks scientific evidence. In our study, the transwell assay showed that 5 μg/L of B[a]P promoted BEAS-2B cell invasion and migration. In addition, the mRNA and protein expression levels of AhR and its target genes involved in B[a]P metabolism, such as AhR nuclear translocator, heat shock protein 90 and CYP1A1, were significantly increased by B[a]P exposure. Moreover, the mRNA expression levels of downstream regulatory factors related to both AhR signaling pathway and EMT, such as NRF2, K-RAS and hypoxia-inducible factor 1-alpha, were significantly increased. Furthermore, the expression level of the epithelial marker E-cadherin was significantly downregulated, while the mRNA expression of mesenchymal phenotype markers, N-cadherin, fibronectin and vimentin, were significantly upregulated. Notably, the above changes induced by B[a]P were significantly attenuated or even stopped by resveratrol (RSV), a natural phenol, also an AhR inhibitor, when the AhR signaling pathway was inhibited by RSV, demonstrating the regulatory role of AhR signaling pathway in B[a]P-induced EMT. In conclusion, B[a]P at the human blood equivalent level induces BEAS-2B cell invasion and migration through the AhR signaling pathway.

Overexpression of ALDH1 and EMT marker profile are linked with unfavorable outcome in head and neck cancer.

Background The aim of this research was to assess the expression of aldehyde dehydrogenase 1 (ALDH1) and epithelial-mesenchymal transition (EMT) markers in head and neck squamous cell carcinoma (HNSCC), and to correlate them with the clinical and histopathological parameters of a patient cohort with follow-up over an 8-year period. Material and Methods For this, seventeen HNSCC and non-neoplastic adjacent epithelium (AE) samples were subjected to laser microdissection and real-time PCR to evaluate the mRNA expression of ALDH1, E-cadherin (E-CAD), N-cadherin (N-CAD), and vimentin (VIM). Also, immunohistochemistry was performed for ALDH1, E-CAD, N-CAD, and VIM in the tumor center (TC), invasion front (IF), and AE of the seventeen samples. Mann-Whitney, Kruskal-Wallis and Chi-square tests were used to correlate the mRNA and immunohistochemical expression with different variables, considering p<0.05. Kaplan-Meier curves were produced for local recurrence, regional metastasis and treatment. Results A mRNA overexpression of ALDH1 in primary tumors was associated with regional metastasis and a high ALDH1 immunostaining was related to metastasis and a worse patient outcome. Additionally, a favorable outcome was associated with the transition phase and an unfavorable outcome was associated with EMT event. An overall 26.9 months was observed with longer survival associated with surgery and radiotherapy. Conclusions However, due to the intense variability inherent to the indicator proteins in the EMT process, the complete profile markers related to this biological process should be continuous investigated. Key words:Aldehyde dehydrogenase 1, epithelial mesenchymal transition, head and neck cancer, squamous cell carcinoma, follow-up study.

Knockdown of mediator complex subunit 19 suppresses the migration and invasion and epithelial-mesenchymal transition in prostate cancer PC3 cells

Objective: To studythe effect of lentivirus-mediated inhibition of Med19 on cell migration andinvasion in the PC3 cells, and explore the mechanism of epithelial-mesenchymal transition transformation. Methods: The lentivirus vectors containing small interferingRNA(siRNA) targeting Medl9 gene were constructed and transfected to PC3 cells.Quantitative real-time PCR(qRT-PCR) and Western blot analysis were used to detect the Medl9 expression in the siRNA group(PC3-Med 19-siRNA cells)and the NC group(PC3-Med 19-sc cells) at 72h after the transfection.The cell mobility,migration and invasion ability of PC3 cells were respectively measured by Boyden migration and woun-healing assay. The expression of E-Cadherin, N-Cadherin, Vimentin, ZEB2, Snail-1, and Snail-2 mRNA were detected by using qRT-PCR. Results: The expression of Medl9 mRNA in PC3-Med 19-siRNA cells was lower than that in PC3-Med 19-scRNA cells(P<0.01). The number of migrated cells and invaded cells were significantly decreased in PC3-Med 19-siRNA cells(P<0.01). The expression of N-Cadherin, Vimentin, ZEB2, Snail-1, and Snail-2 mRNA were remarkablylower and E-Cadherin was higher in PC3-Med 19-siRNA cells. Conclusion: Med 19 inhibitioncouldreduce migration abilityof prostate cancer PC3 cells by epithelial-mesenchymal transition.