Epithelial-mesenchymal Transformation Research Articles

Profiling of idiopathic macular hole vitreous proteome identifies the role of extracellular matrix remodelling, epithelial–mesenchymal transformation and unfolded protein-response pathways

Purpose: To analyze and describe the proteome of the vitreous humour in eyes with idiopathic macular holes. Methods: We performed mass spectrometry (MS)-based label-free quantitative analysis of the vitreous proteome of idiopathic macular hole (IMH) and control donor vitreous. Comparative quantification was performed using SCAFFOLD software which calculated fold changes of differential expression. Bioinformatics analysis was performed using DAVID and STRING software. Results: A total of 448 proteins were identified by LC-MS/MS in IMH and cadaveric eye vitreous samples, of which 199 proteins were common. IMH samples had 189 proteins that were unique and 60 proteins were present only in the control cadaveric vitreous. We found upregulation of several extracellular matrix (ECM) and cytoskeletal proteins, namely collagen alpha-1 (XVIII) chain, N-cadherin, EFEMP1/fibulin-3, basement membrane-specific heparan sulfate proteoglycan core protein, and target of Nesh-3. Several cytoskeleton proteins, namely tubulin, actin, and fibronectin levels, were significantly lower in IMH vitreous, probably reflecting increased ECM degradation. IMH vitreous also had a downregulation of unfolded protein response-mediated-mediated apoptosis proteins, possibly related to a state of increased cell survival and proliferation, along with a remodelling and aberrant production of ECM contents. Conclusion: The pathogenesis of macular holes may involve ECM remodelling, epithelial–mesenchymal transformation, downregulation of apoptosis, protein folding defects, and complement pathway. The vitreo-retinal milieu in macular holes contain molecules related to both ECM degradation and inhibition of the same, thereby maintaining a homeostasis.

Aristolocholic acid I promotes renal tubular epithelial fibrosis by upregulating matrix metalloproteinase-9 expression via activating the C3a/C3aR axis of macrophages

Aristolochic acid I (AAI) can cause nephrotoxicity and is characterized by interstitial fibrosis. The C3a/C3aR axis of macrophages and matrix metalloproteinase-9 (MMP-9) play important roles in fibrosis, but whether they are involved in AAI-induced renal interstitial fibrosis and are related remains to be elucidated. In this study, we investigated whether C3a/C3aR axis of macrophages promotes renal interstitial fibrosis by regulating MMP-9 in aristolochic acid nephropathy (AAN). Intraperitoneal injection of AAI for 28 days successfully induced AAN in C57bl/6 mice. The content of C3a in the kidney of AAN mice was increased, and there was a significant distribution of macrophages in the renal tubules. The same results were observed in the in vitro experiment. We also explored the role and mechanism of macrophages after AAI administration in the epithelial–mesenchymal transformation (EMT) of renal tubular epithelial cells (RTECs) and found that AAI could activate the C3a/C3aR axis of macrophages to upregulate p65 expression in macrophages. p65 upregulated MMP-9 expression in macrophages not only directly but also by promoting the secretion if interleukin-6 by macrophages and then activating STAT3 in RTECs. The upregulation of MMP-9 expression could promote the EMT of RTECs. Taken together, our study demonstrated that the AAI-activated the C3a/C3aR axis of macrophages, which induced MMP-9 production, was one of the causes of renal interstitial fibrosis. Therefore, targeting the C3a/C3aR axis of macrophages is an effective therapeutic strategy for the prevention and treatment of renal interstitial fibrosis in AAN.

Morphological features of epithelial-mesenchymal transformation and its effect on tumor progression of breast cancer

Introduction. Breast cancer is in the first place in the structure of morbidity among all malignant neoplasms in women. The prognosis of the disease depends on the tumor degree, including the presence of epithelial-mesenchymal transformation (EMT), the degree of invasion, the proliferative index, the preservation or absence of estrogen, progesterone, and epidermal growth factor receptors.Aim: To study the immunohistochemical and morphological characteristics of the epithelial-mesenchymal transformation of breast cancer.Material and Methods. Immunohistochemical study with antibodies to AE1/AE3, HMW, CK18, Snail, HER2/neu, E-cadherin, Vimentin, α-SMA, CD34, Ki-67 and p63 was performed in 60 patients of different age with breast cancer. Native preparations were stained with picrofuchsin according to van Gieson Alcian blue. Inflammatory infiltrate cells were examined for antibodies CD45, CD3, CD4, CD8, CD20, CD68.Results. In ductal carcinoma, positive expression for estrogen and progesterone was found in 82.7% and 86.3%, respectively, the proliferation index ranged before 66,6%, and p-53 was positive in 97%. In lobular cancer, positive expression to estrogen and progesterone was observed in 83.4% and 66.6%, respectively, the index of proliferative activity at the level of 50%, and p-53 was positive in 66.6%. Positive moderate expression of HER-2/neu was determined in 47% of ductal and 50% of lobular cancers. Estrogen plays an important role in the development of invasive breast cancer, leads to tumor progression and contributes to EMT. EMT, in turn, leads to the expression of E-cadherin associated with a worse survival prognosis. EMT indirectly leads to the intensification of angiogenesis, and the presence of a large number of newly formed vessels increases the risk of metastasis. Histochemical methods were used to determine the growth of fibrous tissue around invasively growing cancer complexes. Cells located perifocally looked like fibrobla ts, immunohistochemically moderately expressed Vimentin and weakly expressed pancytokeratin, which proved the tumor nature of the cells and the acquisition of mesenchymal features by them. The inflammatory infiltrate along the periphery of the tumor growth consisted mainly of T- and B-lymphocytes, and around the cancer complexes - of B-lymphocytes and macrophages.Conclusion. The study of the immunohistochemical tumor phenotype will make it possible to prescribe adequate polychemotherapy and determine the prognosis of the course of the disease.

Protective effects of Qing-Re-Huo-Xue formula on bleomycin-induced pulmonary fibrosis through the p53/IGFBP3 pathway

BackgroundIdiopathic pulmonary fibrosis (IPF) is a chronic, progressive fibrosing lung disease with high mortality. Inflammation and epithelial mesenchymal transformation (EMT) may play an important role in the occurrence and development of IPF. Qing-Re-Huo-Xue formula (QRHXF) has been used clinically by our team for half a century and has obvious therapeutic effects on lung disease. Nevertheless, the role and mechanism of QRHXF in the treatment of IPF have never been studied.MethodsA mouse pulmonary fibrosis model was established by intratracheal injection of BLM. The effects of QRHXF on the treatment of pulmonary fibrosis were studied by pulmonary function testing, imaging examination, pathological staining, transmission electron microscopy (TEM) observation and mRNA expression. Tandem mass tag (TMT)-based quantitative proteomics was carried out to analyse the lung protein expression profiles between the control (CTL), bleomycin (BLM) and QRHXF (BLM + QRHXF) groups. Immunohistochemistry and qRT-PCR were used to verify the possible existence of drug target proteins and signalling pathways.ResultsThe results of pulmonary function, lung pathology and imaging examinations showed that QRHXF could significantly alleviate BLM-induced pulmonary fibrosis in vivo. Additionally, inflammatory cell infiltration and EMT were markedly reduced in BLM-induced PF mice administered QRHXF. Proteomics detected a total of 35 proteins, of which 17 were upregulated and 18 were downregulated. A total of 19 differentially expressed proteins (DEPs) overlapped between the BLM versus CTL groups and the BLM + QRHXF versus BLM groups. The expression of p53 and IGFBP3 was reversed in the QRHXF intervention group, which was verified by immunohistochemistry and qRT-PCR.ConclusionsQRHXF attenuated BLM-induced pulmonary fibrosis, and regulation of the p53/IGFBP3 pathway might be associated with its efficacy, which holds promise as a novel treatment strategy for pulmonary fibrosis patients.Graphical

Inhibitory Effects of 3-Cyclopropylmethoxy-4-(difluoromethoxy) Benzoic Acid on TGF-β1-Induced Epithelial-Mesenchymal Transformation of In Vitro and Bleomycin-Induced Pulmonary Fibrosis In Vivo.

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease characterized by lung inflammation and excessive deposition of extracellular matrix components. Transforming growth factor-β1 (TGF-β1) induced epithelial-mesenchymal transformation of type 2 lung epithelial cells leads to excessive extracellular matrix deposition, which plays an important role in fibrosis. Our objective was to evaluate the effects of 3-cyclopropylmethoxy-4-(difluoromethoxy) benzoic acid (DGM) on pulmonary fibrosis and aimed to determine whether EMT plays a key role in the pathogenesis of pulmonary fibrosis and whether EMT can be used as a therapeutic target for DGM therapy to reduce IPF. Firstly, stimulation of in vitro cultured A549 cells to construct EMTs with TGF-β1. DGM treatment inhibited the expression of proteins such as α-SMA, vimentin, and collagen Ⅰ and increased the expression of E-cadherin. Accordingly, Smad2/3 phosphorylation levels were significantly reduced by DGM treatment. Secondly, models of tracheal instillation of bleomycin and DGM were used to treat rats to demonstrate their therapeutic effects, such as improving lung function, reducing lung inflammation and fibrosis, reducing collagen deposition, and reducing the expression of E-cadherin. In conclusion, DGM attenuates TGF-β1-induced EMT in A549 cells and bleomycin-induced pulmonary fibrosis in rats.

Identification of key modules and candidate genes associated with endometriosis based on transcriptome data via bioinformatics analysis

BackgroundsEndometriosis is a common disease in women, but the signaling pathways and genes involved remain unclear. This study screened genes that were differentially expressed in ectopic endometrium (EC) and eutopic endometrium (EU) in endometriosis and provided clues for subsequent experimental verification. MethodsEndometriosis samples were harvested from inpatients that underwent surgery from 2017 to 2019 with pathological evidence of endometriosis. We assessed the mRNA expression profiles in endometriosis and further conducted gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, Gene Set Enrichment Analysis (GSEA) and weighted gene co-expression network analysis (WGCNA) to identify potential biomarkers in endometriosis. Finally, we further validated hub genes using public databases and immunohistochemistry assays. ResultsThe upregulated DEGs of ectopic endometrium from endometriosis patients were mainly involved in cell adhesion, MAPK signaling, PI3K-Akt signaling pathways, cytokine receptor interactions, and epithelial-mesenchymal transformation (EMT)-associated signaling pathways. The downregulated DEGs between ectopic endometrium and eutopic endometrium were related to decidualization-associated genes in endometriosis. The correlated gene modules in eutopic endometrial cells were mainly enriched in cell adhesion, embryo implantation and inflammation. The eutopic and ectopic endometrial lesions in endometriosis were involved in the EMT process. Furthermore, we identified 18 co-expression modules during WGCNA analysis. Hub genes in the pale turquoise module were FOSB, JUNB, ATF3, CXCL2, FOS, etc. Significantly enriched KEGG pathways included the TNF, MAPK, foxO, oxytocin, and p53 signaling pathways. Enrichment pathways were directly related to immune surveillance, stem cell self-renewal, and epithelial-mesenchymal transformation. Several pathways and modules of endometriosis are related to cancer-associated pathways, which substantiates the correlation between endometriosis and various gynecological tumors. ConclusionsEndometriosis was tightly correlated with EMT and fibrosis mediated by inflammatory immunity, cytokines, estrogen, kinases and protooncogene through transcriptomics. Overall, our findings lay the groundwork for understanding the pathogenesis of endometriosis and its relationship with malignant transformation.

Tcf21 Alleviates Pancreatic Fibrosis by Regulating the Epithelial-Mesenchymal Transformation of Pancreatic Stellate Cells.

The activation of pancreatic stellate cells (PSCs) plays a key role in the occurrence and development of chronic pancreatitis (CP) and pancreatic fibrosis, which is related to the process of epithelial-mesenchymal transition (EMT). This study was designed to investigate the effect and mechanism of Tcf21 (one of tumor suppressor genes) on pancreatic inflammation and fibrosis in vivo and in vitro. C57BL/6 male mice were intraperitoneally injected with caerulein for 6weeks to establish CP animal model. Fixed pancreatic tissue paraffin-embedded sections were used for immunohistochemistry staining of Tcf21, fibrosis-related markers (α-SMA), interstitial markers (Vimentin) and epithelial markers (E-cadherin). Western blotting and qRT-PCR assay were performed to analyze the change of expression of the above markers after stimulation of TGF-β1 or overexpressed Tcf21 lentivirus transfection in human pancreatic stellate cells (HPSCs). The pancreatic expression of α-SMA and Vimentin of CP mice significantly increased, while the expression of Tcf21 and E-cadherin significantly decreased. TGF-β1 could promote activation and EMT process of HPSCs, and inhibited the expression of Tcf21. Overexpression of Tcf21 could significantly down-regulate the expression of α-SMA, Fibronectin and Vimentin, and up-regulated the expression of ZO-1 of HPSCs. Cell Counting Kit-8 assay and scratch wound-healing assay results showed that overexpression of Tcf21 could significantly inhibit the cell migration and proliferation of HPSCs. Overexpression of Tcf21 could significantly alleviate the activation, proliferation, migration of PSCs by regulating the EMT process. Tcf21 had a potential prospect of a new target for CP therapy.

KCNH2 regulates the growth and metastasis of pancreatic cancer

Objective: Due to the characteristics of insidious onset and early metastasis of pancreatic cancer (PC), patients are often diagnosed at an advanced stage and often delayed in completing surgical resection timely, resulting in poor prognosis. Therefore, this study aims to explore the expression of potassium voltage-gated channel subfamily H member 2 (KCNH2) in PC and its relationship with clinicopathological parameters and the related mechanisms. Methods: GEPIA database and immunohistochemical staining were used to analyze the difference in KCNH2 expression between PC and adjacent tissue in RNA and protein levels. Chi-squared test was used to evaluate the relationship between KCNH2 expression and clinicopathological features. The Cox regression model was used for multivariate analysis and univariate analysis. Histological diagnosis was performed according to World Health Organization (WHO) criteria to evaluate the relationship between KCNH2 expression and clinicopathological features. Results: KCNH2 expression was upregulated in PC compared with normal pancreatic tissue. In addition, the knockdown of KCNH2 inhibits PC cell proliferation, migration, invasion, and epithelial-mesenchymal transformation and promotes their apoptosis. In addition, clinical data showed that the abnormal expression of KCNH2 in PC was related to the tumor stage. Patients with high expression of KCNH2 had a poor prognosis. Conclusions: KCNH2 is expected to be a novel targeted molecule in treating PC.

Using Nicotine in A Silica-Exposed Mouse Model to Promote Lung Epithelial-Mesenchymal Transition.

Smoking and exposure to silica are common among occupational workers, and silica is more likely to injure the lungs of smokers than non-smokers. The role of nicotine, the primary addictive ingredient in cigarettes, in silicosis development is unclear. The mouse model employed in this study was simple and easily controlled, and it effectively simulated the effects of chronic nicotine ingestion and repeated exposure to silica on lung fibrosis through epithelial-mesenchymal transition in human beings. In addition, this model can help in the direct study of the effects of nicotine on silicosis while avoiding the effects of other components in cigarette smoke. After environmental adaptation, mice were injected subcutaneously with 0.25 mg/kg nicotine solution into the loose skin over the neck every morning and evening at 12 h intervals over 40 days. Additionally, crystalline silica powder (1-5 µm) was suspended in normal saline, diluted to a suspension of 20 mg/mL, and dispersed evenly using an ultrasonic water bath. The isoflurane-anesthetized mice inhaled 50 µL of this silica dust suspension through the nose and were awoken via chest massage. Silica exposure was administrated daily on days 5-19. The double-exposed mouse model was exposed to nicotine and then silica, which matches the exposure history of workers who are exposed to both harmful factors. In addition, nicotine promoted pulmonary fibrosis through epithelial-mesenchymal transformation (EMT) in mice. This animal model can be used to study the effects of multiple factors on the development of silicosis.

Multiomics analysis revealed the mechanisms related to the enhancement of proliferation, metastasis and EGFR-TKI resistance in EGFR-mutant LUAD with ARID1A deficiency

IntroductionDysregulated ARID1A expression is frequently detected in lung adenocarcinoma (LUAD) and mediates significant changes in cancer behaviors and a poor prognosis. ARID1A deficiency in LUAD enhances proliferation and metastasis, which could be induced by activation of the Akt signaling pathway. However, no further exploration of the mechanisms has been performed.MethodsLentivirus was used for the establishment of the ARID1A knockdown (ARID1A-KD) cell line. MTS and migration/invasion assays were used to examine changes in cell behaviors. RNA-seq and proteomics methods were applied. ARID1A expression in tissue samples was determined by IHC. R software was used to construct a nomogram.ResultsARID1A KD significantly promoted the cell cycle and accelerated cell division. In addition, ARID1A KD increased the phosphorylation level of a series of oncogenic proteins, such as EGFR, ErbB2 and RAF1, activated the corresponding pathways and resulted in disease progression. In addition, the bypass activation of the ErbB pathway, the activation of the VEGF pathway and the expression level changes in epithelial–mesenchymal transformation biomarkers induced by ARID1A KD contributed to the insensitivity to EGFR-TKIs. The relationship between ARID1A and the sensitivity to EGFR-TKIs was also determined using tissue samples from LUAD patients.ConclusionLoss of ARID1A expression influences the cell cycle, accelerates cell division, and promotes metastasis. EGFR-mutant LUAD patients with low ARID1A expression had poor overall survival. In addition, low ARID1A expression was associated with a poor prognosis in EGFR-mutant LUAD patients who received first-generation EGFR-TKI treatment.7Ri7oJWPiw-vggUB99u973Video abstract

LncRNA MBNL1-AS1 Suppresses Cell Proliferation and Metastasis of Pancreatic Adenocarcinoma through Targeting Carcinogenic miR-301b-3p.

Pancreatic adenocarcinoma (PAAD) has been a huge challenge to public health due to its increasing incidence, frequent early metastasis, and poor outcome. The molecular basis of tumorigenesis and metastasis in PAAD is largely unclear. Here, we identified a novel tumor-suppressor long noncoding RNA (lncRNA) MBNL1-AS1, in PAAD and revealed its downstream mechanism. Quantitative real-time PCR (qRT-PCR) data showed that MBNL1-AS1 expression was significantly downregulated in PAAD tissues and cells, which was closely associated with metastasis and poor prognosis. Cell counting kit-8 (CCK-8) assay, transwell assay, and western blot verified that overexpression of MBNL1-AS1 suppressed cell proliferation, migration, and epithelial mesenchymal transformation (EMT) behavior in PAAD cells. By using a dual luciferase reporter gene system, we confirmed that miR-301b-3p was a direct target of MBNL1-AS1. Further mechanismic study revealed that upregulation of miR-301b-3p abolished the inhibitory effect of MBNL1-AS1 overexpression on cell proliferation, tumorigenesis, migration and EMT. Our results demonstrate that MBNL1-AS1 plays a tumor-suppressive role in PAAD mainly by downregulating miR-301b-3p, providing a novel therapeutic target for PAAD.

Effect of long non-coding RNA 114227 on gastric cancer cell proliferation and migration.

Gastric cancer is a common cancer of the digestive system. Long non-coding RNA (lncRNA) plays an important role in the formation and development of gastric cancer. This study aims to investigate the effect of long non-coding lncRNA 114227 on biologic behaviors in gastric cancer cells. The experiment was divided into 4 groups: a negative control (NC) group, a lncRNA 114227 small interference (si-lncRNA 114227) group, an empty vector (Vector) group, and an overexpression vector (OE-lncRNA 114227) group. The expressions of lncRNA 114227 in gastric mucosa and gastric cancer tissues, gastric mucosal epithelial cells and different gastric cancer strains were determined by real-time reverse transcription PCR (real-time RT-PCR).The proliferation were detected by CCK-8 assay in gastric cancer cells. The epithelial-mesenchymal transformation (EMT) was utilized by Transwell assay, scratch healing assay, and Western blotting in gastric cancer cells. The effect of lncRNA 114227 on proliferation of gastric cancer cells was detected by tumor bearing experiment in nude mice in vivo. The expression level of lncRNA 114227 in the gastric cancer tissues was significantly lower than that in the gastric mucosa tissues, and in 4 kinds of gastric cancer strains was all significantly lower than that in gastric mucosal epithelial cells (all P<0.01). In vitro, the proliferation and migration abilities of gastric cells were significantly reduced after overexpressing lncRNA 114227, and cell proliferation and migration were enhanced after silencing lncRNA 114227 (all P<0.05). The results of in vivo subcutaneous tumorigenesis in nude mice showed that the tumorigenic volume of the tumor-bearing mice in the OE-lncRNA 114227 group was significantly smaller than that of the Vector group, and the tumorigenic quality was lower than that of the Vector group (P<0.05), indicating that lncRNA 114227 inhibited tumorigenesis. The expression of lncRNA 114227 is downregulated in gastric cancer gastric cancer tissues and cell lines. LncRNA 114227 may inhibit the proliferation and migration of gastric cancer cells through EMT process.

Effect of electroacupuncture on transforming growth factor-β1 and renal tubular epithelial mesenchymal transformation in renal tissue of spontaneously hypertensive rats

To observe the effects of electroacupuncture (EA) on renal fibrosis, the expression of transforming growth factor-β1 (TGF-β1) and epithelial mesenchymal transition (EMT) marker proteins in renal tissue of spontaneously hypertensive rats (SHR), so as to explore the underlying mechanism on EA alleviating hypertensive renal impairment. Twenty-four male SHR were randomly divided into model group, losartan group and EA group, with 8 rats in each group, and eight male Wistar-Kyoto rats were taken as the normal group. Rats in the losartan group received gavage of losartan potassium solution (3 mg/mL, 30 mg·kg-1·d-1),once every other day for 12 weeks. Rats in the EA group received EA stimulation at bilateral "Shenshu" (BL23) and "Geshu" (BL17) (2 Hz/15 Hz, 1.0 mA), 15 min each time, once every other day for 12 weeks. The systolic blood pressure of caudal artery was measured before, and 4, 8 and 12 weeks after the intervention. The 24-hour urinary protein was measured before, and 6 and 12 weeks after the intervention. Histopathological changes of the left renal tissue were observed under light mircoscope after H.E. stain. Extracellular matrix (ECM) in renal tissues was observed after periodate Schiff staining. Basement membrane and collagen fibers were observed after Masson staining with collagen volume fraction (CVF) evaluated. The expression of TGF-β1 mRNA in the right renal was detected by real-time fluorescence quantitative PCR. The expression of TGF-β1 and EMT marker E-cadherin, α-SMA and fibronectin (FN) proteins in the left renal tissue was measured by immunohistochemistry. In the model group, irregular arrangement of nephrocytes, renal tubule atrophy, lumen stenosis, ECM hyperplasia and deposition, scar and sclerosis were observed, which were relatively milder in the EA and losartan groups. Compared with the normal group, tubulointerstitium CVF, systolic blood pressure of caudal artery before, and at 4, 8 and 12 weeks after the intervention, 24-hour urinary protein before, and at 6 and 12 weeks after the intervention, the expression of TGF-β1 mRNA, area of TGF-β1, α-SMA and FN positive staining in renal tissues were significantly increased (P<0.01), while the area of E-cadherin positive staining was significantly decreased (P<0.01) in the model group. Compared with the model group, tubulointerstitium CVF, systolic blood pressure of caudal artery at 4, 8 and 12 weeks after the intervention, 24-hour urinary protein at 6 and 12 weeks after the intervention, the expression of TGF-β1 mRNA, area of TGF-β1, α-SMA and FN positive staining in renal tissues were significantly decreased (P<0.01，P<0.05), while area of E-cadherin positive staining was significantly increased (P<0.01) in the losartan and EA groups. Compared with the losartan group, the area of E-cadherin was conside-rately increased (P<0.01), while the area of α-SMA protein decreased (P<0.01) in the EA group. EA could effectively alleviate hypertension and renal interstitial fibrosis in SHR, the mechanism of which may be related to its function in reducing the expression of TGF-β1 and inhibiting EMT in renal tissue.

Blocking xCT and PI3K/Akt pathway synergized with DNA damage of Riluzole-Pt(IV) prodrugs for cancer treatment

Cancer treatment requires the participation of multiple targets/pathways, and single approach is hard to effectively curb the proliferation and metastasis of carcinoma cells. In this work, we conjugated FDA-approved riluzole and platinum(II) drugs into a series of unreported riluzole-Pt(IV) compounds, which were designed to simultaneously target DNA, the solute carrier family 7 member 11 (SLC7A11, xCT), and the human ether a go-go related gene 1 (hERG1), to exert synergistic anticancer effect. Among them, c,c,t-[PtCl2(NH3)2(OH)(glutarylriluzole)] (compound 2) displayed excellent antiproliferative activity with IC50 value of 300-times lower than that of cisplatin in HCT-116, and optimal selectivity index between carcinoma and human normal liver cells (LO2). Mechanism studies indicated that compound 2 released riluzole and active Pt(II) species after entering cells to exhibit a prodrug behavior against cancer, which obviously increased DNA-damage and cell apoptosis, as well as suppressed metastasis in HCT-116. Compound 2 persisted in the xCT-target of riluzole and blocked the biosynthesis of glutathione (GSH) to trigger oxidative stress, which could boost the killing to cancer cells and reduce Pt-drug resistance. Meanwhile, compound 2 significantly inhibited invasion and metastasis of HCT-116 cells by targeting hERG1 to interrupt the phosphorylation of phosphatidylinositide 3-kinases/proteinserine-threonine kinase (PI3K/Akt), and reverse epithelial-mesenchymal transformation (EMT). Based on our results, the riluzole-Pt(IV) prodrugs studied in this work could be regarded as a new class of very promising candidates for cancer treatment compared to traditional platinum drugs.

Cytoskeleton regulator RNA expression on cancer-associated fibroblasts is associated with prognosis and immunotherapy response in bladder cancer

BackgroundDysregulation of long noncoding RNAs (lncRNAs) has been reported to be associated with multiple tumors where they act as tumor suppressors or accelerators. The lncRNA CYTOR was identified as an oncogene involved in many cancers, such as gastric cancer, colorectal cancer, hepatocellular carcinoma, and renal cell carcinoma. However, the role of CYTOR in bladder cancer (BCa) has rarely been reported. MethodsUsing cancer datasets from The Cancer Genome Atlas (TCGA) program, we analyzed the association between CYTOR expression and prognostic value, oncogenic pathways, antitumor immunity and immunotherapy response in BCa. The influence of CYTOR on the immune infiltration pattern in the urothelial carcinoma microenvironment was further verified in our dataset. Single-cell analysis revealed the role of CYTOR in the tumor microenvironment (TME) of BCa. Finally, we evaluated the expression of CYTOR in BCa in the Peking University First Hospital (PKU–BCa) dataset and its correlation with the malignant phenotype of BCa in vitro and in vivo. ResultsThe results indicated that CYTOR was highly expressed in multiple cancer samples, including BCa, and increased CYTOR expression contributed to poor overall survival (OS). Additionally, elevated CYTOR expression was significantly correlated with clinicopathological features of BCa, such as female sex, advanced TNM stage, high histological grade and non-papillary subtype. Functional characterization revealed that CYTOR may be involved in immune-related pathways and the epithelial mesenchymal transformation (EMT) process. Moreover, CYTOR had a significant association with infiltrating immune cells, including M2 macrophages and regulatory T cells (Tregs). CYTOR facilitates the crosstalk between cancer-associated fibroblasts (CAFs) and macrophages, and mediates M2 polarization of macrophages. Correlation analysis revealed a positive correlation between CYTOR expression and programmed cell death-1 (PD-1)/programmed death ligand 1 (PD-L1)/expression and other targets for specific immunotherapy in BCa, which are recognized to predict the efficacy of immunotherapy. ConclusionsThese results suggest that CYTOR serves as a potential biomarker for predicting survival outcome, TME cell infiltration characteristics and immunotherapy response in BCa.

METTL3 depletion contributes to tumour progression and drug resistance via N6 methyladenosine-dependent mechanism in HR+HER2—breast cancer

BackgroundChemotherapy is an important strategy for the treatment of hormone receptor-positive/human epidermal growth factor receptor 2-negative (HR+HER2−) breast cancer (BC), but this subtype has a low response rate to chemotherapy. Growing evidence indicates that N6-methyladenosine (m6A) is the most common RNA modification in eukaryotic cells and that methyltransferase-like 3 (METTL3) participates in tumour progression in several cancer types. Therefore, exploring the function of METTL3 in HR+HER2− BC initiation and development is still important.MethodsmRNA and protein expression levels were analysed by quantitative real-time polymerase chain reaction and western blotting, respectively. Cell proliferation was detected by CCK-8 and colony formation assays. Cell cycle progression was assessed by flow cytometry. Cell migration and invasion were analysed by wound healing assays and transwell assays, respectively, and apoptosis was analysed by TUNEL assays. Finally, m6A modification was analysed by methylated RNA immunoprecipitation.ResultsChemotherapy-induced downregulation of the m6A modification is regulated by METTL3 depletion in HR+HER2− BC. METTL3 knockdown in MCF-7/T47D cells decreased the drug sensitivity of HR+HER2− BC cells by promoting tumour proliferation and migration and inhibiting apoptosis. Mechanistically, CDKN1A is a downstream target of METTL3 that activates the AKT pathway and promotes epithelial-mesenchymal transformation (EMT). Moreover, a decrease in BAX expression was observed when m6A modification was inhibited with METTL3 knockdown, and apoptosis was inhibited by the reduction of caspase-3/-9/-8.ConclusionMETTL3 depletion promotes the proliferation and migration and decreases the drug sensitivity of HR+HER2− BC via regulation of the CDKN1A/EMT and m6A-BAX/caspase-9/-3/-8 signalling pathways, which suggests METTL3 played a tumour-suppressor role and it could be a potential biomarker for predicting the prognosis of patients with HR+HER2− BC.

SCARA5 inhibits oral squamous cell carcinoma via inactivating the STAT3 and PI3K/AKT signaling pathways.

Oral squamous cell carcinoma (OSCC) is a common tumor in the world. Despite the rapid development of medical care, OSCC is also accompanied by high incidence and mortality every year. Therefore, it is still necessary to continuously develop new methods or find new targets to treat OSCC. Previous research showed that scavenger receptor class A member 5 (SCARA5) was one of the potential biomarkers of OSCC, and its expression is significantly low in OSCC. This study aimed to explore the role and related molecular mechanisms of SCARA5 in OSCC. In this study, we found that the SCARA5 expression was lower in CAL-27 and SCC-9 cells than that in human normal oral epithelial keratinocytes. SCARA5 overexpression significantly inhibited cell proliferation and induced apoptosis of CAL-27 and SCC-9 cells. In addition, SCARA5 repressed OSCC cell epithelial-mesenchymal transformation (EMT), evidenced by increased E-cadherin expression and reduced N-cadherin expression. Finally, we found that SCARA5 could suppress STAT3, PI3K, and AKT phosphorylation. Therefore, SCARA5 was related to STAT3 and PI3K/AKT signaling pathways in OSCC. In conclusion, SCARA5 inhibited the proliferation and EMT and induced the apoptosis of OSCC cells through the inhibition of STAT3 and PI3K/AKT signaling pathways, thereby exerting a tumor suppressor effect.

SLFN5 promotes reversible epithelial and mesenchymal transformation in ovarian cancer

Ovarian cancer is a disease with increasing incidence worldwide, and there is an urgent need for chemotherapy and biological targeted therapy. Epithelial-mesenchymal transformation (EMT) is an important initiation stage for tumor cells to acquire the ability to invade and metastasize. A growing number of findings suggest that human Schlafen family member 5(SLFN5) plays a key role in malignancy. However, the role of SLFN5 in ovarian cancer cells has not been fully elucidated. Samples were collected from patients with ovarian cancer diagnosed in Hangzhou First People's Hospital, and the expression of SLFN5 was detected by fluorescence quantitative PCR. The relationship between SLFN5 expression and the progression and malignancy of ovarian cancer was analyzed by using the expression profile data from the Cancer Genome Atlas (TCGA) database. The mRNA expression levels of SLFN5 related upstream and downstream signaling pathways were studied by fluorescence quantitative PCR. Silencing SLFN5 was performed by siRNA transfection. The expression of SLFN5 and transfer-related proteins was examined by Western blot. Transwell and wound healing experiments investigated the migration and invasion ability of ovarian cancer cells. TCGA database analysis results showed that in the population with high SLFN5 expression, compared with the group with low SLFN5 expression, OS was worse (P = 0.011). SLFN5 silencing had a significant inhibitory effect on EMT and invasion movement of ovarian cancer cells. RT-PCR method was used to detect the mRNA changes of SLFN5 in ovarian cancer tissue and adjacent tissue. It was found that the expression of SLFN5 in ovarian cancer tissue was increased, with a significant difference (P < 0.05). Together, these results suggest that SLFN5 may play a synergistic role in tumorigenesis and development of ovarian cancer cells, providing a potential target for future drug development for the treatment of ovarian cancer.

Імуногістохімічні критерії прогнозу епітеліально-мезенхімальної трансформації та стовбурового фенотипу дрібноклітинних раків легень

Small cell lung cancer (SCLC) is one of the most aggressive tumor of the human body and has morphological features of cancers with epithelial-mesenchymal transformation (EMT). The aim of our study to improve morphological features for SCLC course prediction based on researching of EMT and stem cells phenotype at the molecular-biological level. The prognostic significance of pancytokeratin, vimentin, E-cadherin and N-cadherin expression, EMT stage and expression of CD44 for overall survival and their influence on the spread of the tumor were established. It was proved that the presence of EMT is associated with extensive forms of SCLC (p &lt; 0.01), the overall survival of patients with SCLC depended on the presence of EMT (p &lt; 0.01) and its stage (p &lt; 0.01).

Down-regulation of FBP1 in lung adenocarcinoma cells promotes proliferation and invasion through SLUG mediated epithelial mesenchymal transformation.

Metabolic reprogramming and epithelial-mesenchymal transformation (EMT) play an important role in lung cancer. In recent studies, metabolic enzymes such as Fructose-1,6-bisphosphatase 1 (FBP1) have shown potential functions beyond regulating metabolism. Western blot assay was performed to detect glycolysis-related and EMT-related protein expression levels. The glucose uptake kit and adenosine triphosphate (ATP) detection kit were used to detect glucose uptake rate and ATP content. Transwell assay was used to determine the invasiveness of lung adenocarcinoma cells. Wound healing assay was used to determine the metastatic ability of lung adenocarcinoma cells. Methyl thiazolyl tetrazolium (MTT) assay and EdU staining were performed to investigate the effect of FBP1 overexpression on lung adenocarcinoma proliferation. Overexpression of FBP1 down-regulated glycolysis-related protein levels and inhibited glucose uptake and ATP production, while knockdown of FBP1 had the opposite effect. Overexpression of FBP1 reversed EMT and inhibited Slug expression. Meanwhile, overexpression of FBP1 impaired the invasion, metastasis and proliferation ability of lung adenocarcinoma cells. In contrast, FBP1 knockdown promoted the EMT process, up-regulated Slug expression and enhanced the invasion, metastasis and proliferation of lung adenocarcinoma cells. Therefore, FBP1 can be used as one of the potential clinical targets through inhibiting glycolysis, cell invasion and proliferation by inhibiting Slug mediated EMT processes.