Epithelial-mesenchymal Transition Biomarkers Research Articles

Liver kinase B1 correlates with prognosis and epithelial-mesenchymal transition of resectable early stage non-small cell lung cancer.

BackgroundRecent evidences support that low expression of liver kinase B1 (LKB1) triggers epithelial-mesenchymal transition (EMT) through induction of Zinc finger E-box binding homeobox 1 (ZEB1) expression, which downregulates E-cadherin in human lung cancer cell lines. However, the clinicopathological significance of LKB1, EMT, salt-inducible kinase 1 (SIK1), ZEB1 and their relationship in early stage non-small cell lung cancer (ES-NSCLC) patients remain to be determined. In this study, the correlation among expression of LKB1, risk of distant metastasis, prognostic significance, and EMT in ES-NSCLC after surgery was investigated by immunohistochemistry.MethodsCase notes and pathology records of 103 patients with ES-NSCLC were retrospectively analyzed. Immunohistochemical staining was employed to detect LKB1, EMT biomarkers (E-cadherin and vimentin), SIK1 and ZEB1 expression in ES-NSCLC tissues.ResultsLKB1 expression is associated with distant metastasis after treatment (P=0.017). LKB1-high expression group has better overall survival (OS) (P=0.000) and disease-free survival (DFS) (P=0.000). LKB1 expression is correlated with E-cadherin (r=0.231, P=0.019), vimentin (r=−0.225, P=0.022), SIK1 (r=0.218, P=0.027) and ZEB1 (r=−0.242, P=0.014).ConclusionsOur findings suggest that LKB1-high expression is possibly associated with favourable prognosis and LKB1 expression is correlated with EMT and expression of SIK1 and ZEB1.

Gli promotes tumor progression through regulating epithelial-mesenchymal transition in non\u2013small-cell lung cancer

IntroductionLung cancer is the leading causes of cancer-related deaths globally. The most frequent histologic type of lung cancer is non–small-cell lung cancer (NSCLC). NSCLC often undergo epithelial-mesenchymal transition (EMT). The components that control this process are thus promising therapeutic targets.Materials and methodsGli/EMT protein expression levels were examined by western blot in paired NSCLC patient tissues and NSCLC cell lines. Functional analyses were performed to investigate SHH/Gli signaling and EMT in NSCLC cell lines. MTS cell viability, luciferase reporter, and western blot assays were performed to analyze pathway activity, while wound healing and transwell assays were executed to measure cell migration and invasion.ResultsHigher Gli1 expressions were detected in tumor samples than in paired normal tissues. Differential expression of EMT biomarkers and activation of p-AKT were observed in tumor tissues. N-Shh stimulation of cells significantly increased reporter activity in NSCLC cell lines, while Gli-i treatment of transfected cells showed less relative reporter activity. When subjected to both Gli-i and N-Shh treatment, NSCLC cell lines continued to demonstrate decreased Gli transcriptional activity. Gli inhibition is associated with decreased expression level of p-AKT, N-cadherin and Vimentin. Knockdown of both Gli1 and Gli2 showed decreased EMT, migrative and invasive ability. Cells stimulated by N-Shh demonstrated greater mobility. In addition, AKT-i treated cells also demonstrated inhibited EMT activity.ConclusionsThis study provides evidence for aberrant upregulation of the Gli signaling pathway and a strong association between expression of Gli versus AKT and EMT markers in NSCLC.

EphA5 knockdown enhances the invasion and migration ability of esophageal squamous cell carcinoma via epithelial-mesenchymal transition through activating Wnt/\u03b2-catenin pathway

BackgroundThe erythropoietin-producing hepatocellular (Eph) receptor A5 (EphA5) has been found to be overexpressed in some malignant tumors and is associated with disease prognosis. However, the role of EphA5 in esophageal squamous cell carcinoma (ESCC) is not clear.MethodsIn the present study, we measured the expression of EphA5 in ESCC tissues and cell lines including KYSE150 and KYSE450 cells. siRNA transfection was used to interfere with EphA5 expression in ESCC cell lines. Cell viability, colony formation, scratch and invasion assays were performed to explore the roles of EphA5 in ESCC cell lines. Flow cytometry analysis was performed to investigate whether EphA5 could affect the cell apoptosis and cycle. The biomarkers related to epithelial-mesenchymal transition (EMT) and molecules associated with Wnt/β‑catenin signaling were also measured by western blot and immunofluorescence.ResultsThe protein and mRNA expression of EphA5 were significantly higher in fresh ESCC tissues and cell lines compared with normal control groups and human normal esophageal epithelial cells (HEEC). The cell viability assay and colony formation assay revealed that EphA5 knockdown enhanced the proliferation of KYSE150 and KYSE450 cells in vitro. The invasion and migration of ESCC cells were accelerated after EphA5 knockdown. The expression of EMT biomarkers was altered in ESCC cells transfected with siRNA targeting EphA5. Moreover, EphA5 downregulation enhanced the protein levels of β‑catenin and p-GSK-3βSer9, which play a key role in the Wnt/β‑catenin pathway.ConclusionsEphA5 knockdown promotes the proliferation of esophageal squamous cell carcinoma,enhances invasion and migration ability via epithelial-mesenchymal transition through activating Wnt/β‑catenin pathway.

Prognostic value of epithelial-mesenchymal transition markers in clear cell renal cell carcinoma.

Epithelial-to-mesenchymal transition (EMT) is important in tumor invasiveness and metastasis. We aimed to determine prognostic value of six key EMT markers (CDH1, CDH2, SNAI1, SNAI2, VIM, TWIST1) in clear cell renal cell carcinoma (ccRCC). A total of 533 ccRCC patients with RNASeq data from The Cancer Genome Atlas (TCGA) cohort were included for analysis. Gene expression of these EMT markers was compared between tumor and normal tissues based on Oncomine database and TCGA cohort. Their correlations with progression-free survival (PFS) and overall survival (OS) were also examined in both TCGA cohort and FUSCC (Fudan University Shanghai Cancer Center) cohort. Cox proportional hazards regression model and Kaplan-Meier plot were used to assess the relative factors. Functional enrichment analyses were utilized to describe biologic function annotations and significantly involved hallmarks pathways of each gene. We found that Epithelial marker, CDH1 expression was lower, while mesenchymal markers (CDH2, SNAI1, VIM, TWIST1) expression was higher in ccRCC primary tumors. In the TCGA cohort, we found that patients with higher expression of VIM, TWIST1 or lower expression of CDH1 had worse prognosis. Further, in the FUSCC cohort, we confirmed the predictive ability of mesenchymal markers and epithelial marker expression in PFS and OS of ccRCC patients. After generating Cox regression models, EMT markers (CDH1, SNAI1, VIM, and TWIST1) were independent prognostic factors of both PFS and OS in ccRCC patients. Our preliminary EMT prediction model can facilitate further screening of EMT biomarkers and cast a better understanding of EMT gene function in ccRCC.

Insufficient radiofrequency ablation promotes epithelial-mesenchymal transition mediated by interleukin-6/signal transducer and activator of transcription 3/Snail pathway in the H22 cells.

Radiofrequency ablation (RFA), an established and minimally invasive therapy for hepatocellular carcinoma, has become an important treatment strategy. However, tumor aggressiveness remains a common problem. The epithelial-mesenchymal transition (EMT) is thought to play an important role in this process. Due to limited sample volumes harvested from patients, we established a heat-treated cell line and a mouse model to investigate the mechanisms of incomplete ablation in EMT. We heat-treated H22 and HepG2 cells using a water bath to determine a suitable temperature for incomplete RFA. Male BALB/c mice were orthotopically transplanted with H22 cells and then subjected to incomplete ablation. Changes in the EMT biomarkers were detected by real-time polymerase chain reaction, western blotting, and immunofluorescence. The experimental results are expressed as means ± standard deviations. Incomplete RFA promoted EMT, downregulated E-cadherin, upregulated vimentin and Snail, and enhanced the phosphorylation of signal transducer and activator of transcription 3 (STAT3) both in vivo and in vitro. Moreover, interleukin (IL)-6 secretion increased after heat treatment in the H22 cells. AG490, an IL-6 inhibitor, inhibited the occurrence of EMT. Insufficient ablation performed at low temperature successfully induces EMT and promotes tumor aggressiveness, which is mediated by the IL-6/STAT3/Snail pathway in both cell and mouse models.

Prediction of metastasis in oral squamous cell carcinoma through phenotypic evaluation and gene expression of E-cadherin, β-catenin, matrix metalloproteinase-2, and matrix metalloproteinase-9 biomarkers with clinical correlation.

CONTEXT:Controversies prevail regarding the true predictive role of epithelial–mesenchymal transition (EMT) biomarkers in metastasis of oral squamous cell carcinoma (OSCC). There is also limited research carried on till date wherein the protein and gene expression of EMT biomarkers have been investigated simultaneously in the Indian population.AIM:The aim of this study was to assess the gene expression and quantitative protein expression of EMT biomarkers using conventional method and MATLAB software and to determine if there is any difference in the expression between metastatic and nonmetastatic OSCCs with clinicopathologic correlation.SETTINGS AND DESIGN:Twenty metastatic and nonmetastatic OSCC tissue sections each were obtained from department archives. Gene expression and quantified protein expression of EMT markers were done and correlated with clinical parameters.SUBJECTS AND METHODS:Sections immunostained for EMT biomarkers were evaluated using semi-quantitative and quantitative (MATLAB) methods. Gene expression using semi-quantitative reverse transcriptase–polymerase chain reaction was done. These findings were correlated with clinical parameters.STATISTICAL ANALYSIS USED:Pearson's Chi-square test, Student's t-test, and univariate logistic regression analysis were performed using SPSS software.RESULTS:The low immunoexpression of E-cadherin and β-catenin and the high expression of matrix metalloproteinase (MMP)-2 and MMP-9 correlate with Stages III and IV showing high metastatic risk. Furthermore, the upregulated MMP-2 and MMP-9 gene expressions in advanced clinical stages of OSCC have high metastatic potential.CONCLUSIONS:This study is the first of its kind to employ texture and color segmentation in MATLAB to objectively assess the protein expression of EMT biomarkers. This research is instrumental in studying the protein and gene expressions of EMT markers with clinical correlation.

Prognostic value of the immunohistochemical detection of epithelial-mesenchymal transition biomarkers in oral epithelial dysplasia: A systematic review.

Background Oral potentially malignant disorders (OPMDs) comprise a range of clinical-pathological alterations that are frequently characterized as architectural and cytological derangements upon histological analysis. Epithelial-mesenchymal transition (EMT) has been proposed as a critical mechanism for the acquisition of the malignant phenotype in neoplastic epithelial processes. This study aims to systematically review the current findings on the immunohistochemical expression of epithelial-mesenchymal transition markers in oral potentially malignant disorders and to evaluate their possible application as biomarkers associated with the progression of oral epithelial dysplasias. Material and Methods A systematic search was performed in the following databases: PubMed, EMBASE, Chinese BioMedical Literature Database, and Cochrane Library. Articles that evaluated the relationship between the expression of EMT markers and the degree of oral epithelial dysplasia were selected for the systematic review. The quality of each eligible study was evaluated by independent reviewers that used operationalized prognostic biomarker reporting guidelines (REMARK). Results Seventeen articles met all inclusion criteria and were selected. The EMT markers analyzed exhibited an important association with the prognosis of the cases evaluated. The results showed a progressive increase in the expression of nuclear transcription factors and markers of mesenchymal differentiation, as well as negative regulation of epithelial and cell adhesion markers, according to the stage of oral epithelial dysplasia. Conclusions The dysregulation of expression of important EMT components in oral dysplastic epithelium is a potential prognostic marker in OPMDs. Key words:Oral potentially malignant disorder, oral epithelial dysplasia, epithelial-mesenchymal transition, biomarker, prognosis.

Mir-21-5p inhibits the progression of human chondrosarcoma by regulating CCR7/STAT3/NF-κB pathway

ABSTRACT Purpose: MicroRNAs (miRNAs or miRs) play an important role in the initiation and development of chondrosarcoma (CS). However, the role of miR-21-5p in CS progression and its underlying molecular mechanisms remains unclear. Materials and Methods: miR-21-5p expression was measured by qRT-PCR. Cell proliferation, migration, and invasion were detected by CCK-8 and Transwell assay. Dual-luciferase reporter assay was used to validate the target of miR-21-5p. Western blot was applied to explore the expressions of CCR7 and EMT biomarkers. Then, the xenograft model was established to confirm the effects of miR-21-5p. Results: miR-21-5p was significantly downregulated in CS tissues and cells and negatively correlated with grade, recurrence, and 5-year overall survival. In vitro, miR-21-5p caused G0/G1 cell cycle arrest and induced apoptosis by decreasing cyclin D1 expression and Bcl-2/Bax ratio. miR-21-5p suppressed cell migration and invasion of CS cells by inhibiting epithelial–mesenchymal transition (EMT). In vivo, miR-21-5p inhibited xenograft tumor formation of SW1353 cells. Mechanistically, miR-21-5p targeted the 3’-UTR of C-C chemokine receptor 7 (CCR7) mRNA to inhibit its expression. Overexpression of CCR7 reversed the inhibitory effects of miR-21-5p on CS cell behaviors. However, the silencing of CCR7 enhanced the inhibitory effects of miR-21-5p on CS cells. Besides, the overexpression of miR-21-5p or silencing of CCR7 obviously reduced the expression levels of p-STAT3, p-p56, and p-IκBα. Conclusion: miR-21-5p targeted CCR7 expression to inhibit the STAT3 and NF-κB signaling, thereby suppressing cell proliferation, migration, invasion, and EMT in CS cells, which might be a novel mechanistic study for CS therapy. Abbreviations: 3’-UTR: 3’-untranslated region; CCR7: C-C chemokine receptor type 7; CS: chondrosarcoma; DMEM: dulbecco’s modified eagle’s medium; EMT: epithelial–mesenchymal transition; HEK-293T: human embryonic kidney-293T; miR-21-5p: microRNA-21-5p; miR-NC: negative control miRNA; SD: standard deviation; si-CCR7: CCR7 siRNAs

Blockade of MDM2 with inactive Cas9 prevents epithelial to mesenchymal transition in retinal pigment epithelial cells

Epithelial to mesenchymal transition (EMT) plays an important role in the pathogenesis of proliferative vitreoretinopathy (PVR). We aimed to demonstrate the role of mouse double minute 2 (MDM2) in transforming growth factor-beta 2 (TGF-β2)-induced EMT in human retinal pigment epithelial cells (RPEs). Immunofluorescence was used to assess MDM2 expression in epiretinal membranes (ERMs) from patients with PVR. A single guide (sg)RNA targeting the second promoter of MDM2 was cloned into a mutant lentiviral Clustered Regularly Interspaced Short Palindromic Repeats (lentiCRISPR) v2 (D10A and H840A) vector for expressing nuclease dead Cas9 (dCas9)/MDM2-sgRNA in RPEs. In addition, MDM2-sgRNA was also cloned into a pLV-sgRNA-dCas9-Kruppel associated box (KRAB) vector for expressing dCas9 fused with a transcriptional repressor KRAB/MDM2-sgRNA. TGF-β2-induced expression of MDM2 and EMT biomarkers were assessed by quantitative polymerase chain reaction (q-PCR), western blot, or immunofluorescence. Wound-healing and proliferation assays were used to evaluate the role of MDM2 in TGF-β2-induced responses in RPEs. As a result, we found that MDM2 was expressed obviously in ERMs, and that TGF-β2-induced expression of MDM2 and EMT biomarkers Fibronectin, N-cadherin and Vimentin in RPEs. Importantly, we discovered that the dCas9/MDM2-sgRNA blocked TGF-β2-induced expression of MDM2 and the EMT biomarkers without affecting their basal expression, whereas the dCas9-KRAB/MDM2-sgRNA suppressed basal MDM2 expression in RPEs. These cells could not be maintained continuously because their viability was greatly reduced. Next, we found that Nutlin-3, a small molecule blocking the interaction of MDM2 with p53, inhibited TGF-β2-induced expression of Fibronectin and N-cadherin but not Vimentin in RPEs, indicating that MDM2 functions in both p53-dependent and -independent pathways. Finally, our experimental data demonstrated that dCas9/MDM2-sgRNA suppressed TGF-β2-dependent cell proliferation and migration without disturbing the unstimulated basal activity. In conclusion, the CRISPR/dCas9 capability for blocking TGF-β2-induced expression of MDM2 and EMT biomarkers can be exploited for a therapeutic approach to PVR.

Notch1 and PI3K/Akt signaling blockers DAPT and LY294002 coordinately inhibit metastasis of gastric cancer through mutual enhancement.

Blockade of either Notch1 or PI3K/Akt pathway inhibits metastasis of gastric cancer. However, whether blockade of both pathways coordinately exerts such an effect remains unknown. In this study, we aimed to investigate the effects of combined treatment with Notch1 signaling blocker DAPT and PI3K/Akt signal blocker LY294002 on metastasis of gastric cancer. Notch intracellular domain (NICD) and phosphorylated Akt (p-Akt) levels in gastric cancer tissues and their adjacent normal tissue samples and gastric cancer SGC7901 and AGS cells and normal GES-1 cells were determined using immunohistochemistry and Western blotting. The effects of combined DAPT and LY294002 on metastasis of gastric cancer were evaluated by examining migration and invasion potential of SGC7901 cells using wound healing and transwell assays, determining changes in the levels of epithelial-mesenchymal transition biomarkers and MMP-9, Notch1, HES1, and phosphorylation of Akt in gastric cancer SGC7901 cells and/or AGS cells in vitro using Western blotting, and metastasis of gastric cancer to lungs in BALB/c nude mice after treatment. NICD and p-Akt levels were significantly higher in gastric cancer tissues and SGC7901 and AGS cells than those in the normal control and GES-1 cells. Migration and invasion potential of SGC7901 cells, EMT biomarkers and MMP-9 in SGC7901 cells, and metastasis of gastric cancer to lungs in mice were coordinately inhibited by DAPT and LY294002. In addition, DAPT and LY294002 coordinately inhibited the levels of Notch1, HES1, and p-Akt in gastric cancer cells. DAPT and LY294002 coordinately inhibited metastasis of gastric cancer through mutual enhancement.

Silencing Of MAGI1 Promotes The Proliferation And Inhibits Apoptosis Of Glioma Cells Via The Wnt/β-Catenin And PTEN/AKT Signaling Pathways.

BackgroundMembrane-associated guanylate kinase (MAGUK) with inverted orientation protein 1 (MAGI1) is a novel member of the MAGUK family with a vital role in tumor progression related to invasion and metastasis. However, the function of MAGI1 in glioma is currently unknown. We therefore analyzed the expression of MAGI1 protein in human glioma samples, glioma cell lines and glioma stem cells (GSCs), and explored its effects on glioma cell proliferation and apoptosis.MethodsMAGI1 expression in glioma tissues was examined by Western blotting and real-time polymerase chain reaction and its relationships with clinical pathological features were analyzed. The effects of MAGI1 knockdown on the proliferation of glioma cell lines and GSCs were detected by CCK8 and colony-formation assays, and apoptosis was assessed by flow cytometry. We also investigated the effects of MAGI1 silencing on protein expression levels of epithelial-mesenchymal transition biomarkers, as well as β-catenin, cyclin D1, PTEN and phospho-Akt by Western blotting.ResultsMAGI1 was significantly downregulated in glioma tissues and its expression was related to cancer progression. Silencing of MAGI1 in both glioma cell lines and GSCs enhanced proliferation and inhibited apoptosis. MAGI1 knockdown also significantly increased the expression levels of N-cadherin, vimentin, β-catenin, cyclin D1 and phospho-Akt and reduced the expression of E-cadherin and PTEN.ConclusionsOur results indicated that MAGI1 might play a vital role in glioma progression and may represent a potential therapeutic target for the treatment of glioma.

Lipopolysaccharide induces epithelial-mesenchymal transition of alveolar epithelial cells cocultured with macrophages possibly via the JAK2/STAT3 signaling pathway.

Epithelial-mesenchymal transition (EMT) plays a key role in the process of pulmonary fibrosis (PF). Increasing evidences have shown that exaggerated EMT in recurrent pulmonary injury mediates the early pathogenesis of PF. This study aimed to evaluate EMT of human alveolar epithelial cells (A549) when cocultured with human macrophages Tohoku hospital pediatrics-1 (THP-1) induced by lipopolysaccharide (LPS) and investigate the role of Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling pathway. Firstly, we detected the inflammatory and EMT biomarkers in A549 cells monoculture and A549/THP-1 cells coculture in the presence or absence of LPS. Then, the activation of JAK2/STAT3 signaling pathway was determined in coculture. Interestingly, inflammatory markers, such as interleukin (IL)-6, matrix metalloproteinase (MMP)-9, transforming growth factor (TGF)-β, and collagen type 1 (COL-1), were enhanced in LPS treated coculture. Besides, the expression of E-cadherin decreased but α-smooth muscle actin expression increased, indicating the presence of EMT in A549 cells when cocultured with THP-1 macrophages. However, these phenotypes could not be observed in LPS-treated A549 cells monoculture. Meanwhile, JAK2/STAT3 signaling pathway was activated, and the STAT3 DNA-binding and inflammatory markers were inhibited by Stattic. Together, these findings demonstrate the key role of JAK2/STAT3 signaling pathway in LPS promoted EMT of A549 in the presence of THP-1 macrophages as an in vitro PF model.

36P - Expression of IL-17RA promotes cancer stem-like properties of colorectal cancer cells by Stat3 activation

BackgroundColorectal cancer stem cells (CSCs) serve crucial functions in tumor relapse, metastasis and therapy failure. Interleukin 17 receptor A (IL-17RA) is a potent mediator in the pathogenesis and progression of colorectal cancer. Our previous study showed that IL-17RA could promote angiogenesis, tumor growth and metastasis. In addition, IL-17RA was correlated with CRC recurrence. Recent study showed IL-17RA play a potential role in self-renewal in glioma stem cells. This study aims to evaluate the functional role and mechanism of IL-17RA in colorectal CSCs. MethodsIn this study, IL-17RA stable overexpression cells were used to examine the change of sphere formation, proliferation, CSC markers and epithelial-mesenchymal transition biomarkers and the regulation mechanism of signaling pathway. The specific IL-17RA signaling inhibitors were used to evaluate the function of the IL-17RA signaling affecting the CSC markers, epithelial-mesenchymal transition gene expression and sphere formation. ResultsWe observed high IL-17RA expression can significantly promote the self-renewal by increasing stem cell markers CD133, ALDH1, Lgr5 and Sox2 expression and the ability to form tumorsphere in SW480 and SW620 cells. In additional, IL-17RA overexpression markedly increased chemoresistant ability and mesenchymal markers Vimentin, Slug, Snail, Zeb1expression. The STAT3 inhibitor remarkably decreased the CD133 stem cell gene, drug resistance and tumorsphere ability. ConclusionsIL-17RA can enhance the self-renewal ability via activating the STAT3 pathway. Therefore, we expect that IL-17RA could serves as a prognosis marker and a potential therapeutic target in CRC. Legal entity responsible for the studyChih-Yung Yang. FundingHas not received any funding. DisclosureAll authors have declared no conflicts of interest.

House dust mite induces Sonic hedgehog signaling that mediates epithelial‑mesenchymal transition in human bronchial epithelial cells.

Epithelial-mesenchymal transition (EMT) provides a valuable source of fibroblasts that produce extracellular matrix in airway walls. The Sonic hedgehog (SHH) signaling pathway plays an essential role in regulating tissue turnover and homeostasis. SHH is strikingly upregulated in the bronchial epithelia during asthma. Snail1 is a major target of SHH signaling, which regulates EMT and fibroblast motility. The present study was designed to ascertain whether the combination of house dust mite (HDM) and transforming growth factor β1 (TGF-β1) could induce EMT via the SHH signaling pathway in human bronchial epithelial cells (HBECs). HBEC cultures were treated with HDM/TGF-β1 for different periods of time. The involvement of SHH signaling and EMT biomarkers was evaluated by quantitative real-time PCR, western blotting and immunofluorescence staining. Small-interfering RNA (siRNA) for glioma-associated antigen-1 (Gli1) or cyclopamine was used to inhibit SHH signaling in HBECs. HBECs stimulated by HDM/TGF-β1 exhibited morphological features of EMT. E-cadherin (an epithelial marker) was decreased after a 72-h exposure to HDM/TGF-β1 compared to that in the control cells, and the expression of type I collagen and FSP1 (mesenchymal markers) was increased. HDM/TGF-β1 activated the SHH signaling pathway in HBECs, which led to Gli1 nuclear translocation and the transcriptional activation of Snail1 expression. Moreover, gene silencing or the pharmacological inhibition of Gli1 ameliorated EMT. In summary, these findings suggest that HDM/TGF-β1 may induce EMT in HBECs via an SHH signaling mechanism. Inhibition of SHH signaling may be a novel therapeutic method for preventing airway remodeling in asthma.

Low Vitamin D Status Is Associated with Epithelial-Mesenchymal Transition in Patients with Chronic Obstructive Pulmonary Disease.

Vitamin D deficiency is correlated with the increased morbidity of chronic obstructive pulmonary disease (COPD). However, the mechanisms underlying these effects have largely remained elusive. This study analyzed the correlations among COPD, vitamin D concentration, and epithelial-mesenchymal transition (EMT). Ninety-five patients with newly diagnosed COPD and 190 age- and sex-matched healthy subjects were recruited for this research. Serum 25(OH)D levels were detected, and pulmonary EMT biomarkers and TGF-β/Smad signaling were evaluated. Serum 25(OH)D level was remarkably decreased in COPD patients compared with that in control subjects. Furthermore, serum 25(OH)D concentration gradually decreased in COPD patients ranging from grade 1-2 to 4. However, reduced expression of the epithelial biomarker E-cadherin and increased expression of the mesenchymal biomarkers vimentin and α-SMA were found in COPD patients. Mechanistic analysis showed that pulmonary nuclear vitamin D receptor (VDR) was decreased in patients with COPD. In contrast, TGF-β/Smad signaling was obviously activated in COPD patients. Furthermore, the level of serum TGF-β in COPD patients increased in parallel with COPD severity. Serum 25(OH)D concentration was inversely associated with TGF-β levels in COPD patients. In vitro experiments showed that active vitamin D3 inhibits TGF-β-induced Smad2/3 phosphorylation in MRC-5 cells. Furthermore, vitamin D concentration was inversely correlated with TGF-β/Smad signaling and EMT in COPD patients, suggesting EMT as a vital mediator of COPD development in patients with low vitamin D concentrations.

Curcumin decreases epithelial‑mesenchymal transition by a Pirin‑dependent mechanism in cervical cancer cells.

Curcumin is a natural antioxidant polyphenol, which decreases epithelial‑mesenchymal transition(EMT) and cell migration in cervical cancer cells. However, the mechanism by which such a decrease occurs is unclear. It is well established that cervical cancer can be caused by high‑risk human papillomavirus(HPV), which overexpresses E6andE7oncoproteins. Recent findings have suggested that viral oncoproteins regulate the expression of Pirin, which is an oxidative stress sensor involved in EMT and cell migration. Molecular markers associated with EMT, pirin and HPV were evaluated using reverse transcription‑reverse quantitative PCR and western blotting. In addition, the migratory ability of cells was evaluated using a Transwell assay. In order to evaluate the role of Pirin in curcumin‑mediated inhibition of EMT, SiHa cervical carcinoma cells, which contain two integrated copies of HPV16, were exposed to curcumin. Cell migration, and the expression levels of EMT biomarkers and the pirin protein, which is a product of the PIR gene, were subsequently evaluated. The results demonstrated a significant decrease in EMT following exposure to 20µM curcumin for 72h. This finding was supported by a decrease in the protein expression levels of N‑cadherin, Vimentin and Slug. Furthermore, it was observed that PIR expression and Pirin protein levels were significantly decreased when SiHacells were exposed to curcumin. Subsequently, to analyze the effects of Pirin on EMT, SiHacells were transfected with a small interfering RNA (siRNA) to knockdown PIR. A significant increase in E‑cadherin mRNA expression and a decrease in N‑cadherin protein expression were observed. In addition, a similar decrease was observed when SiHacells were exposed to both PIR siRNA and curcumin. Finally, a significant decrease in SiHacell migration was observed in the presence of 20µM curcumin compared with in the control group. These findings suggested that curcumin may decrease EMT, at least in part by a Pirin‑dependent mechanism. Therefore, Pirin protein may be an important pharmacological target for cervical cancer treatment.

Epithelial‑mesenchymal transition in chronic rhinosinusitis (CRS) and the prognostic value of α‑SMA in postoperative outcomes of patients with CRS

Tissue remodeling is the pathological basis of the symptoms encountered in chronic rhinosinusitis (CRS). Epithelial‑mesenchymal transition (EMT) may participate in this process. The present study was designed to investigate the involvement of EMT in CRS. In addition, the prognostic value of the EMT biomarker α‑smooth muscle actin (α‑SMA) was assessed in patients with CRS who underwent endoscopic sinus surgery (ESS). A total of 13 patients with CRS without nasal polyps (CRSsNP), 13 patients with CRS with nasal polyps (CRSwNP) and 13 control subjects were enrolled. The expression of EMT markers was determined in sinonasal specimens by qPCR, western blot and immunofluorescence assays. EMT features were evaluated in primary nasal epithelial cells (NECs) with transforming growth factor (TGF)‑β1 stimulation. The associations were assessed between α‑SMA expression and the clinical features of CRS. Epithelial and mesenchymal markers were overexpressed in the sinonasal specimens of both CRSsNP and CRSwNP patients. Alterations in the expression pattern were more apparent in the CRSsNP patients. Following incubation of primary NECs with TGF‑β1, a mesenchymal shape was acquired. In addition, NECs that co‑expressed α‑SMA and cytokeratin were readily detected and the protein levels of α‑SMA were elevated. In contrast to α‑SMA, the levels of E‑cadherin were decreased. The protein levels of α‑SMA were negatively correlated with endoscopic scores and several postoperative symptoms. In conclusion, partial EMT occurred in patients with CRS, notably in CRSsNP patients. Moreover, primary NECs could undergo EMT following TGF‑β1 treatment invitro. In addition, α‑SMA could be considered an efficient predictor for postoperative endoscopic and symptomatic outcomes in patients with CRS treated with ESS.

Inhibitor of growth 4 (ING4) inhibits hypoxia-induced EMT by decreasing HIF-1α and snail in HK2 cells

Renal fibrosis is a common mechanism that leads to all kidney diseases and Epithelial-mesenchymal transition (EMT) is considered as one of the potential mechanisms of renal fibrosis. Inhibitor of growth 4 (ING4) was reported to involve in several diseases; especially it was negatively correlated with lung fibrogenesis parameters. However, the role of ING4 and underlying mechanisms in EMT are still unknown. In this study, we used a UUO rat model to mimic renal fibrosis, which was examined by Masson and HE staining analysis. To explore the effects of ING4 on hypoxia-induced EMT, HK2 cells were treated with hypoxia to induce EMT and ING4 was over-expressed in hypoxia-treated HK2 cells by transfection of pEGFP-N1-ING4. MTT assay was used to describe the cell viability of HK2 cells under the hypoxic condition. The expression levels of ING4, hypoxia-inducible factor-1α (HIF-1α), and EMT markers (E-cadherin, N-cadherin and vimentin) were examined in vivo and in vitro by western blot, qRT-PCR, immunohistochemical staining or Immunofluorescence. Our results showed that, in a UUO rat model, ING4 was decreased and EMT was developed with reduction in E-cadherin and increase in N-cadherin and vimentin, suggesting a significant association between ING4 expression and EMT. Under hypoxia, E-cadherin was down-regulated and N-cadherin and vimentin were up-regulated, indicating that hypoxia induced EMT in HK2 cells. Nonetheless, changes in the expression of EMT biomarkers were inhibited by over-expression of ING4. Moreover, over-expressing ING4 decreased the expression of HIF-1α and snail in HK2 cells. These findings suggest that ING4 may inhibit hypoxia-induced EMT via decreasing HIF-1α and snail in HK2 cells, indicating the potential of ING4 as a therapeutic target for renal fibrosis.

MiR-124a enhances therapeutic effects of bone marrow stromal cells transplant on diabetic nephropathy-related epithelial-to-mesenchymal transition and fibrosis.

Epithelial-to-mesenchymal transition (EMT) has beengradually considered as one of the major pathways that causes the production of interstitial myofibroblasts in diseased kidneys. The study was done to investigate the effect of a bone marrow stromal cell(BMSCs) transplant on rat podocytes and diabetic nephropathy (DN) rats in high-glucose concentration, and to explore the effect of miR-124a on BMSCtherapy. High glucose-injured podocytes and streptozotocin-induced DN rats have beenrespectively used as injury models in in vitro and in vivo studies. Podocyte viability was measured using the Cell Counting Kit-8 assay. Renal pathological examination was observed by HE staining and Masson staining. The messenger RNA and protein levels were determined via real-time polymerase chain reaction and Western blotting, respectively. By mediating the activation of caveolin-1 (cav-1) and β-catenin and affecting the expression levels of EMT biomarkers including p-cadherin, synaptopodin, fibroblast-specific protein-1, α-smooth muscle actin and snail, our in vitro study confirmed that miR-124a played a significant role in the treatment of high glucose-induced podocyte injury by BMSCs. The therapeutic effects of the BMSCtransplant on DN rats were also proved to be further enhanced by miR-124a overexpression in BMSCs, and such a phenomenon was accompanied by the improvement of renal fibrosis and mitigation of DN-related kidney impairment. Regulation of fibronectin, collagen1, and EMT-related proteins was closely implicated with the mechanism, and the activation of cav-1 and β-catenin was also possibly involved. The study demonstrated thepivotal effect of miR-124a on BMSCtherapy for DN rats via mitigating EMT and fibrosis. Our results provide a novel insight into how therapeutic effects of BMSCs can be improved at theposttranscriptional level.

Bortezomib attenuates renal interstitial fibrosis in kidney transplantation via regulating the EMT induced by TNF-α-Smurf1-Akt-mTOR-P70S6K pathway.

Allograft interstitial fibrosis was characterized by massive extracellular matrix deposition caused by activated fibroblasts and myofibroblasts. Epithelial‐mesenchymal transition (EMT) is recognized as an important source of myofibroblasts contributing to the pathogenesis of allograft interstitial fibrosis. Smad ubiquitination regulatory factor 1 (Smurf1) has been recently reported to be involved in the progression of EMT. Our study was to detect the effect of Bortezomib and Smurf1 in the EMT and allograft interstitial fibrosis. Biomarkers of EMT, as well as Smurf1, were examined in human proximal tubular epithelial cells (HK‐2) treated with tumour necrosis factor‐alpha (TNF‐α) in various doses or at various time points by Western Blotting or qRT‐PCR. We knockdown or overexpressed Smurf1 in HK‐2 cells. Furthermore, rat renal transplant model was established and intervened by Bortezomib. Allograft tissues from human and rats were also collected and prepared for HE, Masson's trichrome, immunohistochemical staining and western blotting assays. As a result, we found that TNF‐α significantly promoted the development of EMT in a time‐dependent and dose‐dependent manner through Smurf1/Akt/mTOR/P70S6K signalling pathway. More importantly, Bortezomib alleviated the progression of EMT and allograft interstitial fibrosis in vivo and in vitro by inhibiting the production of TNF‐α and expression of Smurf1. In conclusion, Smurf1 plays a critical role in the development of EMT induced by TNF‐α. Bortezomib can attenuate the Sumrf1‐mediated progression of EMT and renal allograft interstitial fibrosis, which could be suggested as a novel choice for the prevention and treatment of renal allograft interstitial fibrosis.