Expression Levels Of Epithelial-mesenchymal Transition Markers Research Articles

Apelin-13 alleviates intrauterine adhesion by inhibiting epithelial-mesenchymal transition of endometrial epithelial cells and promoting angiogenesis.

Intrauterine adhesion (IUA) is a common complication of surgical manipulation of the uterine cavity such as abortion. The pathology of IUA is characterized by fibrosis, but the pathogenesis is not fully understood. The function of Apelin-13 in IUA and related mechanisms were investigated in this study. The IUA rat model was established. The pathological changes and fibrosis degree of rat uterine tissues were detected by HE and Masson staining after intraperitoneal injection of Apelin-13. Epithelial-mesenchymal transition (EMT) of endometrial epithelial cells and endothelial-mesenchymal transition (EnMT) of vein endothelial cells were induced by TGF-β1. Tube-forming assay using HUVEC was implemented to detect the effect of Apelin-13 upon angiogenesis. IHC staining, immunofluorescence staining, and Western blot were conducted to detect the expression levels of EMT markers, angiogenesis, and key proteins of the TGF-β1/Smad signaling. Apelin-13 significantly alleviated IUA and fibrosis, and increased endometrial thickness and gland number in IUA rats. In addition, Apelin-13 significantly reversed EMT and EnMT induced by IUA modeling and TGF-β1, promoted the tube-forming ability of HUVEC, and up-regulated the expression of angiogenesis-related proteins. Mechanistically, Apelin-13 significantly suppressed smad2/3 phosphorylation and inhibited the TGF-β1/Smad signaling via its receptor APJ. Apelin-13 might alleviate IUA via repressing the TGF-β1/Smad pathway and is expected to be a potent therapeutic option for the clinical treatment of IUA.

MiR-21-5p knockdown inhibits epithelial to mesenchymal transition in A549 lung adenocarcinoma cells by upregulating RhoB.

MiR-21-5p is a highly expressed microRNA that plays an important role in various cancer-promoting processes, including anchorage-independent growth, invasion, migration metastasis, and drug resistance in lung cancer. Studies indicate that miR-21-5p may contribute to these processes by promoting epithelial-mesenchymal transition (EMT). Ras homolog gene family member B (RhoB), a gene downregulated by miR-21-5p, has also been linked to EMT in lung cancer. However, the role of the miR-21-5p/RhoB axis in EMT regulation in lung adenocarcinoma remains unclear. In this study, we aimed to investigate the regulatory role of the miR-21-5p/RhoB axis in EMT and related in vitro functional characteristics such as migration, invasion, cisplatin resistance, and the formation of tumor spheroids. A549 cells were transfected with the miR-21-5p inhibitor, RhoB siRNA, and their corresponding negative controls. Wound healing, transwell invasion, Methyl thiazole tetrazolium (MTT), and sphere formation assays were also performed to evaluate the migration, invasion, cisplatin resistance, and anchorage-independent growth of A549 cells. RT-qPCR was used to determine the mRNA expression levels of EMT markers. MiR-21-5p knockdown inhibited migration, invasion, cisplatin resistance, and sphere formation while upregulating E-cadherin and downregulating Slug. Furthermore, RhoB silencing restored EMT and related in vitro functional characteristics in A549 cells. Knockdown of miR-21-5p inhibits EMT and related in vitro functional characteristics by upregulating RhoB, suggesting that miR-21-5p may promote EMT through downregulation of RhoB.

MFAP2 induces epithelial-mesenchymal transformation of osteosarcoma cells by activating the Notch1 pathway.

Osteosarcoma (OS) is a malignancy originating from mesenchymal tissue. Microfibril-associated protein 2 (MFAP2) plays a crucial role in cancer, notably promoting epithelial-mesenchymal transition (EMT). However, its involvement in OS remains unexplored. MFAP2 was silenced in U2OS cells using shRNA targeting MFAP2 (sh-MFAP2) and validated by quantitative real-time polymerase chain reaction (qRT-PCR). We extracted gene chip data of MFAP2 from multiple databases (GSE28424, GSE42572, and GSE126209). Correlation analyses between MFAP2 and the Notch1 pathway identified through the gene set variation analysis (GSVA) enrichment analysis were conducted using the Pearson correlation method. Cellular behaviors (viability, migration, and invasion) were assessed via the Cell Counting Kit-8 (CCK-8), wound healing, and Transwell assays. EMT markers (N-cadherin, vimentin, and β-catenin) and Notch1 levels were examined by western blotting and qRT-PCR. Cell morphology was observed microscopically to evaluate EMT. Finally, the role of MFAP2 in OS was validated through a xenograft tumor model. OS cell lines exhibited higher MFAP2 mRNA expression than normal osteoblasts. MFAP2 knockdown in U2OS cells significantly reduced viability, migration, and invasion, along with downregulation of N-cadherin and vimentin, as well as upregulation of β-catenin. MFAP2 significantly correlated with the Notch1 pathway in OS and its knockdown inhibited Notch1 protein expression. Furthermore, Notch1 activation reversed the inhibitory effects of MFAP2 knockdown on the malignant characteristic of U2OS cells. Additionally, MFAP2 knockdown inhibited tumor growth, expression levels of EMT markers, and Notch1 expression in OS tumor tissues. Our study revealed that MFAP2 was an upstream regulator of the Notch1 signaling pathway to promote EMT in OS. These findings suggested MFAP2 as a potential OS therapy target.

High expression of long noncoding RNA UCA1 promotes invasion, migration and epithelial-mesenchymal transition of trophoblasts in vitro

To investigate the role of urothelial carcinoma antigen 1 (UCA1) in regulation of invasion, migration and epithelial-mesenchymal transition (EMT) of trophoblast HTR-8/SVneo cells and its association with tubal pregnancy. Cultured HTR- 8/SVneo cells stimulated with interleukin-6 (IL-6) were examined for changes in UCA1 expression and cell migration ability using qRT-PCR and scratch assay, respectively. A HTR-8/SVneo cell model with UCA1 silencing was constructed by transient transfection, and the migration and invasion abilities of the cells were assessed using Scratch assay and Transwell assay; qRT-PCR and Western blotting were performed to detect the mRNA and protein expression levels of EMT markers. HTR-8/SVneo cells stimulated with IL-6 exhibited significantly increased migration ability and up-regulated expression of UCA1 (P < 0.01). UCA1 silencing obviously suppressed migration and invasion abilities of HTR-8/SVneo cells (P < 0.01), significantly up-regulated the mRNA and protein expressions of EMT epithelial marker E-cadherin (P < 0.01), and down-regulated the expressions of the mesenchymal markers integrin β3, vimentin and N-cadherin (P < 0.05). UCA1 may be a key gene that promotes the occurrence of tubal pregnancy and thus provides a new therapeutic target for tubal pregnancy.

Abstract P2-19-02: Androgen mediated signaling induces epithelial to mesenchymal transition phenotype in MDA-MB-453 breast cancer cells and human breast tumors

Abstract Background: Epithelial-mesenchymal transition (EMT) phenotype is a complex process and plays a central role in tumor progression, aggression, invasion, metastasis, and resistance to therapy. Role of androgen receptor (AR) and androgens in inducing EMT has been well established in experimental systems of prostate cancer. AR is widely expressed in all subtypes of breast cancer (BC) and is considered to have context dependent role based on the steroid hormone microenvironment. Previous studies have shown AR as the only sex steroid receptor detectable in BC metastases and in metastatic lesion. We investigated the role of androgens in driving the EMT phenotype and association of AR induced genes with EMT in BC tumors using public dataset. Methods: We treated AR positive breast cancer cell line MDA-MB-453 with the androgen, dihydrotestosterone (DHT) followed by AR antagonist bicalutamide (Bic) and examined the expression levels of AR induced genes by quantitative real time PCR (qPCR) and AR induced protein GCDFP-15 by western blot. AR driven genes (SEC14L2, C1orf116, FKBP5, UGT2B11, UGT2B28, KCNMA1, PIP and ABCC11) were identified by bioinformatic pipeline using a systematic bioinformatic approach from public microarray data sets derived from AR positive breast cancer cell lines (S Rajarajan et al, SABCS 2021). Mesenchymal phenotype of the cell lines under DHT/Bic was evaluated morphologically and expression levels of EMT markers (ZEB1, Slug, Vimentin) and E-cadherin was estimated by qPCR, western blot and immunofluorescence. Migratory potential of the cell line under treatment was evaluated by wound healing assay and proliferation was examined by the MTT assay. Association of AR induced genes and levels of enzymes involved in intracrinal testosterone metabolism (SRD5A1 &amp; SRD5A3) with EMT in breast tumors was evaluated using transcript data from METABRIC database. EMT scores were derived from previous publications (Byers et al, 2013, Mak et al, 2016) and correlation was tested with AR induced genes. Results: The MDAMB-453 cell line was treated with 10nM DHT for 72hrs and this increased the expression of AR downstream protein GCDFP-15 by 31.47% (p=0.022) when compared to the control. Further, a significant increase in the expression levels of AR driven genes with a fold change &amp;gt;2 was observed in the DHT treated cells (p&amp;lt; 0.05). The expression of GCDP15 protein (p=0.028) and the AR downstream genes (p&amp;lt; 0.05) were significantly repressed upon treatment with bicalutamide. DHT treatment also showed a significant higher expression of EMT master regulator, SLUG (fold change&amp;gt;3, p=0.045) and a wound healing assay showed a 22% increase in migration (p=0.027). The expression of SLUG was significantly repressed (p=0.003) and the migratory ability of the DHT treated cells reduced upon treatment with bicalutamide (p=0.145). We also observed a significant loss of E-cadherin protein (by 23.65%; p=0.024) with DHT treatment and this was completely reversed upon treatment with bicalutamide (p=0.047). No significant change in proliferation was observed among the different treatment conditions. A significant positive correlation was observed between AR induced genes and the EMT scores (p&amp;lt; 0.05). Expression level of SRD5A3 was also positively correlated with EMT score (p=0.043) and AR induced genes (p&amp;lt; 0.05), except SEC14L2. Conclusion: Androgen receptor plays an important role in the biology of breast cancer and is considered a useful marker for prognosis. However, antiandrogen therapies haven’t seen the expected success in clinical trial settings. Our results support the controversial role played by AR in higher androgenic environment within breast cancer and warrant the consideration of the intratumoral levels of sex steroid hormones. Citation Format: Savitha Rajarajan, Snijesh VP, Madhumathy G Nair, Apoorva D, Chandrakala M, Vidya P Nimbalkar, Maalavika Pillai, Mohit Kumar Jolly, Jyothi S Prabhu. Androgen mediated signaling induces epithelial to mesenchymal transition phenotype in MDA-MB-453 breast cancer cells and human breast tumors [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-19-02.

Implications of Axis Inhibition Protein 2 in Breast Cancer Progression.

Although axis inhibition protein 2 (Axin2) has been reported to act as a tumour suppressor, recent findings suggest that it exhibits oncogenic effects by mediating Snail1-induced epithelial-mesenchymal transition (EMT) in breast cancer cells. EMT is a crucial biological process involved in the initiation of metastasis in cancer progression. This study elucidated the biological significance and mechanism of Axin2 in breast cancer using transcriptomic and molecular techniques. The expression of Axin2 and Snail1 in MDA-MB-231 breast cancer cells was determined by western blotting analysis, and the role of Axin2 in breast cancer tumorigenesis was investigated in xenograft mouse models constructed using pLKO-Tet-shAxin2-transfected triple negative (TN) breast cancer cells. Additionally, the expression levels of EMT markers were determined using qRT-PCR, and clinical data were analysed using Kaplan-Meier (KM) plotter and The Cancer Genome Atlas (TCGA). Axin2 knockdown significantly decreased (p<0.001) the proliferation of MDA-MB-231 cells in vitro and attenuated (p<0.05) the tumorigenic potential of the cells in vivo. Moreover, Axin2 knockdown significantly increased the relative mRNA levels of epithelial markers but decreased the expression of mesenchymal markers in MDA-MB-231 cells. Axin2 may be involved in the progression of breast cancer, particularly triple-negative breast cancer, through the regulation of Snail1-induced EMT, making it a potential therapeutic target.

CircPCSK5 is highly expressed in gastric cancer and promotes cancer cell proliferation, invasion and epithelial-mesenchymal transition

To investigate the expression of circPCSK5 in gastric cancer (GC) and its role in regulation of the proliferation, invasion and epithelial-mesenchymal transition (EMT) of GC cells. High-throughput sequencing was performed in 3 pairs of GC and adjacent gastric mucosa tissues to obtain the differential expression profile of circRNA. The expression of circPCSK5 was detected in 62 patients undergoing radical surgery for GC using RT-qPCR, and the correlation between circPCSK5 expression level and clinicopathological data of the patients was analyzed. The overall survival and disease-free survival of the patients were assessed with Kaplan-Meier survival analysis, and the independent risk factors affecting the patients' prognosis were analyzed using Cox proportional hazards regression model. The stability and subcellular localization of circPCSK5 were assessed using RNase R and actinomycin D assays, fluorescence in situ hybridization and nucleocytoplasmic separation assay. CCK-8 assay, EdU assay and Transwell assay were employed to examine the changes in proliferation, migration and invasion of GC cells with circPCSK5 knockdown or overexpression; Western blotting and RT-qPCR assays were used to detect the expression levels of EMT markers in the transfected cells. The expression of circPCSK5 was significantly upregulated in GC tissues and cells (P < 0.001, P < 0.01). The expression level of circPCSK5 was positively correlated with tumor size, vascular invasion, lymph node metastasis and AJCC stage of GC (P < 0.05). The overall survival and disease-free survival were significantly lower in GC patients with high circPCSK5 expression than in those with low circPCSK5 expression (P < 0.001). High circPCSK5 expression was an independent risk factor for a poor prognosis of GC patients (P < 0.05). Knockdown of circPCSK5 significantly inhibited the proliferation, migration and invasion of HGC27 cells (P < 0.01), increased the expressions of E-cadherin, and decreased the expression of N-cadherin and vimentin (P < 0.01). CircPCSK5 overexpression promoted the proliferation, migration and invasion of MKN45 cells (P < 0.01), reduced E-cadherin expression and increased N-cadherin and vimentin expressions (P < 0.01). CircPCSK5 is highly expressed in GC and promotes the proliferation, invasion and EMT of GC cells, suggesting its potential as a prognostic biomarker and therapeutic target for GC.

Lysophosphatidic Acid Promotes Epithelial-Mesenchymal Transition in Kidney Epithelial Cells via the LPAR1/MAPK-AKT/KLF5 Signaling Pathway in Diabetic Nephropathy.

The epithelial–mesenchymal transition (EMT) is a differentiation process associated with fibrogenesis in diabetic nephropathy (DN). Lysophosphatidic acid (LPA) is a small, naturally occurring glycerophospholipid implicated in the pathogenesis of DN. In this study, we investigated the role of LPA/LPAR1 signaling in the EMT of tubular cells as well as the underlying mechanisms. We observed a decrease in E-cadherin and an increase in vimentin expression levels in the kidney tubules of diabetic db/db mice, and treatment with ki16425 (LPAR1/3 inhibitor) inhibited the expression of these EMT markers. Ki16425 treatment also decreased the expression levels of the fibrotic factors fibronectin and alpha-smooth muscle actin (α-SMA) in db/db mice. Similarly, we found that LPA decreased E-cadherin expression and increased vimentin expression in HK-2 cells, which was reversed by treatment with ki16425 or AM095 (LPAR1 inhibitor). In addition, the expression levels of fibronectin and α-SMA were increased by LPA, and this effect was reversed by treatment with ki16425 and AM095 or by LPAR1 knockdown. Moreover, LPA induced the expression of the transcription factor, Krüppel-like factor 5 (KLF5), which was decreased by AM095 treatment or LPAR1 knockdown. The expression levels of EMT markers and fibrotic factors induced by LPA were decreased upon KLF5 knockdown in HK-2 cells. Inhibition of the extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and serine-threonine kinase (AKT) pathways decreased LPA-induced expression of KLF5 and EMT markers. In conclusion, these data suggest that LPA contributes to the pathogenesis of diabetic nephropathy by inducing EMT and renal tubular fibrosis via regulation of KLF5 through the LPAR1.

3D Disease Modelling of Hard and Soft Cancer Using PHA-Based Scaffolds.

Simple SummaryTumour progression in vivo was able to be well mimicked in 3D culture by utilizing biodegradable 10 mm × 10 mm × 8 mm P(3HO-co-3HD) and P(3HB)-based 3D scaffolds with a pore size of 30 to 300 µm. Both hard (MCF7 and MDA-MB-231) and soft (HCT116) tumour-related cells were successfully grown on the scaffolds, and their growth patterns were studied for 5 days. MDA-MB-231 tend to grow in clusters, and MCF7 cells form an evenly dispersed layer, which covered most of the 3D PHA scaffolds, while HCT116 formed large colonies within the pockets of the 3D PHA scaffold. Epithelial mesenchymal transition (EMT) marker genes, including Wnt-11, E-cadherin, Vim and Snail expression profiles, were like those seen in real tumour samples, which confirmed that the cancer models were exhibiting real tumour-like characteristics with high fidelity. These models are important in mimicking hypoxic tumours and in studying gene expression, cellular signalling, angiogenesis and drug response for translational research.Tumour cells are shown to change shape and lose polarity when they are cultured in 3D, a feature typically associated with tumour progression in vivo, thus making it significant to study cancer cells in an environment that mimics the in vivo milieu. In this study we established hard (MCF7 and MDA-MB-231, breast cancer) and soft (HCT116, colon cancer) 3D cancer tumour models utilizing a blend of P(3HO-co-3HD) and P(3HB). P(3HO-co-3HD) and P(3HB) belong to a group of natural biodegradable polyesters, PHAs, that are synthesised by microorganisms. The 3D PHA scaffolds produced, with a pore size of 30 to 300 µm, allow for nutrients to diffuse within the scaffold and provide the cells with the flexibility to distribute evenly within the scaffold and grow within the pores. Interestingly, by Day 5, MDA-MB-231 showed dispersed growth in clusters, and MCF7 cells formed an evenly dispersed dense layer, while HCT116 formed large colonies within the pockets of the 3D PHA scaffolds. Our results show Epithelial Mesenchymal Transition (EMT) marker gene expression profiles in the hard tumour cancer models. In the 3D-based PHA scaffolds, MDA-MB-231 cells expressed higher levels of Wnt-11 and mesenchymal markers, such as Snail and its downstream gene Vim mRNAs, while MCF7 cells exhibited no change in their expression. On the other hand, MCF7 cells exhibited a significantly increased E-Cadherin expression as compared to MDA-MB-231 cells. The expression levels of EMT markers were comparative to their expression reported in the tumour samples, making them good representative of cancer models. In future these models will be helpful in mimicking hypoxic tumours, in studying gene expression, cellular signalling, angiogenesis and drug response more accurately than 2D and perhaps other 3D models.

Citromycin Isolated from the Antarctic Marine-Derived Fungi, Sporothrix sp., Inhibits Ovarian Cancer Cell Invasion via Suppression of ERK Signaling.

Recently, microorganisms and their metabolites in the Antarctic marine environment have attracted attention as useful sources for novel therapeutics, including anticancer drugs. Here, we investigated the effects of citromycin, isolated from the Antarctic marine-derived fungus, Sporothrix sp., on human ovarian cancer cells. Citromycin inhibited the migration and invasion of human ovarian cancer SKOV3 and A2780 cells, but had no cytotoxic activity against them. Additionally, it inhibited the expression of epithelial–mesenchymal transition (EMT) markers and the activation of matrix metalloproteinase (MMP)-2 and MMP9. Moreover, extracellular signal-regulated kinase (ERK)-1/2 signaling was inhibited after citromycin treatment, and the ectopic expression of ERK negated the anti-invasive activity of citromycin. Our findings suggest that citromycin inhibits the migration and invasion of human ovarian cancer cells by downregulating the expression levels of EMT markers and MMP-2/9 via inhibition of the ERK1/2 pathway.

Factor Xa inhibitor, edoxaban ameliorates renal injury after subtotal nephrectomy by reducing epithelial-mesenchymal transition and inflammatory response.

Chronic kidney disease (CKD) is an increasing and life‐threatening disease worldwide. Recent evidence indicates that blood coagulation factors promote renal dysfunction in CKD patients. Activated factor X (FXa) inhibitors are safe and first‐line drugs for the prevention of thrombosis in patients with atrial fibrillation. Here, we investigated the therapeutic effects of edoxaban on CKD using the mouse 5/6 nephrectomy model. Eight‐week‐old wild‐type mice were subjected to 5/6 nephrectomy surgery and randomly assigned to two groups, edoxaban or vehicle admixture diet. Edoxaban treatment led to reduction of urinary albumin excretion and plasma UN levels compared with vehicle group, which was accompanied with reduced glomerular cross‐sectional area and cell number. Edoxaban treatment also attenuated fibrinogen positive area in the remnant kidneys after subtotal nephrectomy. Moreover, edoxaban treatment resulted in attenuated tubulointerstitial fibrosis after 5/6 nephrectomy, which was accompanied by reduced expression levels of epithelial‐mesenchymal transition (EMT) markers, inflammatory mediators, and oxidative stress markers in the remnant kidneys. Treatment of cultured proximal tubular cells, HK‐2 cells, with FXa protein led to increased expression levels of EMT markers, inflammatory mediators, and oxidative stress markers, which were abolished by pretreatment with edoxaban. Treatment of HK‐2 cells with edoxaban attenuated FXa‐stimulated phosphorylation levels of extracellular signal‐regulated kinase (ERK) and NF‐κB. Our findings indicate that edoxaban can improve renal injury after subtotal nephrectomy by reducing EMT and inflammatory response, suggesting that FXa inhibition could be a novel therapeutic target for CKD patients with atrial fibrillation.

Evaluation of hydroalcoholic extract effects of Ferula assa-foetida on expression change of EMT and CD44-related genes in gastric cancer stem cell

BackgroundGastric cancer (GC) with high incidence is a life-threatening malignant tumor in humans. Cancer stem cells (CSCs) due to tumor-initiating capacity, ability of invasion, metastasis, recurrence, and chemoresistance may be utilized for cancer treatment. Ferula assa-foetida as a herbal remedy in ancient Iranian medicine was shown to have various biological activities, including anti-cancer. Epithelial-mesenchymal transition (EMT) markers play an important role in migration, invasion, and metastatic dissemination of CSCs. The gastric CSCs-surface glycoprotein CD44 also responsible for high tumorigenicity, self-renewal, and metastasis through EMT. In this study, we investigated the hydroalcoholic extract effects of F. assa-foetida on the expression of EMT-related genes, anti-apoptotic factor Bcl-2 and CD44 cell-surface molecule in CSCs. MethodsCSCs were cultured in serum-free medium containing EGF and B-27 and exposed to different concentrations of F. assa-foetida hydroalcoholic extract and for different time durations. Cytotoxic effects of the extract compounds were evaluated using MTT assay. After CSCs treatment with IC50 dose of the extract, the expression level of EMT markers vimentin, Snail1, and Zeb1 and anti-apoptotic marker Bcl-2 was determined using relative quantitative real-time PCR as well as marker CD44 and CD54 were determined by flow cytometry. ResultsOur MTT assay results demonstrated that cytotoxicity effect of extract compounds was significantly dependent on time and concentration. After exposing CSCs to IC50 doses of extract in different times, mRNA expression levels of EMT markers (vimentin, Snail1, Zeb1) and anti-apoptotic marker Bcl-2, as well as protein expression of stemness marker CD44 and CD54 significantly decreased in treated CSCs compared with untreated (P < 0.05). ConclusionOur data demonstrated that extract compounds of F. assa-foetida were able to decrease EMT markers, Bcl-2 anti-apoptotic factor, surface marker of CD44 and CD54, leading to inhibition of CSCs proliferation as well as killing of them. Therefore, isolation and purification of F. assa-foetida hydroalcoholic extract could be used as a potential agent for cancer treatment.

Rosmarinic acid ameliorates renal interstitial fibrosis by inhibiting the phosphorylated-AKT mediated epithelial-mesenchymal transition in vitro and in vivo.

Indoxyl sulfate (IS), a uremic toxin, causes chronic kidney disease (CKD) progression via renal fibrosis. Epithelial-mesenchymal transition (EMT) is a crucial feature of renal fibrosis. Rosmarinic acid (RA) is an ester of caffeic acid and 3,4-dihydroxyphenylacetic acid with a wide range of desirable biological activities. In this study, we investigated whether RA exerted anti-renal fibrosis effects and its related mechanisms in a unilateral ureteral obstruction (UUO) mouse model. C57BL/6 mice were orally administered RA (10 and 20 mg kg-1 d-1) for 7 consecutive days before and after UUO surgery. The mice were then sacrificed to collect the blood and kidneys. Hematoxylin and eosin (H&E) and Masson's trichrome staining were used to evaluate the renal injury and function. Immunohistochemical analysis, reverse transcription-polymerase chain reaction (RT-PCR), and western blotting were used to detect the expression levels of EMT markers. In vitro studies were performed using the IS-stimulated NRK-52E cell line. Here, the pathological changes, collagen deposition, and mRNA and protein expression levels of profibrotic factors and fibrotic markers were found to be significantly elevated in the kidneys of UUO mice. We found that RA administration significantly ameliorated UUO-induced kidney damage by reversing abnormal serum creatinine and blood urea nitrogen levels. It was found that RA treatment decreased the expression levels of alpha-smooth muscle actin (α-SMA), collagen I, fibronectin, transforming growth factor (TGF)-β1, vimentin and phosphorylated AKT (p-AKT) while increasing the E-cadherin expression in both UUO kidneys and IS-treated NRK-52E cells. Our results demonstrate that RA may be a promising therapeutic agent for renal interstitial fibrosis.

Role of NELF-B in supporting epithelial-mesenchymal transition and cell proliferation during hepatocellular carcinoma progression.

Negative elongation factor-B (NELF-B), also known as cofactor of BRCA1 (COBRA1), is one of the four subunits of the NELF complex. It interacts with BRCA1, in addition to other transcription complexes in various tissues. The NELF complex represses the transcription of several genes by stalling RNA polymerase II during the early phase of transcription elongation. The role of NELF-B in liver cancer and hepatocellular carcinoma (HCC), the most prevalent type of liver cancer, remains to be elucidated. It has been previously demonstrated that silencing of NELF-B inhibits the proliferation and migration of HepG2 cells. The present study aimed to investigate the consequences of ectopic expression and silencing of NELF-B in liver cancer HepG2 and SNU449 cell lines. Functional assays were performed to examine the effects on gene and protein expression, viability, migration and invasion of cells. Overexpression of NELF-B did not alter the proliferation and migration of HepG2 cells, or the expression of tested genes, indicating that overexpression alone may not be sufficient for altering these features in HepG2 cells. By contrast, knockdown of NELF-B in SNU449 cells resulted in decreased cell proliferation, together with induction of apoptosis and decreased expression levels of Ki-67 and survivin, which are markers of proliferation and inhibition of apoptosis, respectively. Additionally, silencing of NELF-B resulted in a significant decrease in the hallmarks of epithelial-mesenchymal transition (EMT), including cell migration and invasion, and decreased the expression levels of EMT markers, such as N-cadherin, vimentin and β-catenin. Decreased expression levels of forkhead box F2 transcription factor and increased mRNA levels of trefoil factor 1, a putative tumor suppressor, were also detected following the silencing of NELF-B. The current results demonstrated that NELF-B enhanced the manifestation of most hallmarks of cancer, including cell proliferation, migration, invasion and inhibition of apoptosis, indicating its critical role in the progression of HCC.

Triptolide reverses epithelial-mesenchymal transition in glioma cells via inducing autophagy.

BackgroundTo observe the effects of triptolide (TP) on the proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) of glioma cells, and to explore the possible mechanisms of phenotypic changes in EMT.MethodsThe U87 and U251 glioma cell lines were treated TP. The Cell Counting Kit-8 (CCK-8) method was used to detect the half-maximal inhibitory concentration (IC50) of TP in these two cell lines and the inhibition of cell proliferation at the IC50 concentration. The wound-healing experiment and Transwell invasion assay were used to detect the cells’ migration and invasion abilities, respectively. Using western blot protocol, the expression levels of the EMT markers were analyzed, and the levels of the autophagy markers were also detected. The pEGFP-C2-LC3B plasmid was transfected into glioma cells, and the effect of TP on autophagy was detected by immunofluorescence. A subcutaneous tumor model in nude mice was established to observe the effect of TP on cell proliferation in vivo, and immunohistochemistry (IHC) was used to detect the expression levels of EMT markers in mouse tumor tissues.ResultsTP significantly inhibited the proliferation of U87 and U251 cells in a dose- and time-dependent manner. TP had a significant inhibitory effect on the migration and invasion of U87 and U251 cells. Western blot showed that TP reversed the process of EMT in glioma cells, which was evidenced by the upregulated expression of the epithelial marker E-cadherin, and the downregulated expression of the mesenchymal markers N-cadherin, Vimentin, ZEB1, Snail, and Slug. TP increased autophagy in glioma cells, increased the LC3B II/I ratio, and upregulated Beclin-1 and Atg-7 expression. Immunofluorescence showed that the number of autophagosomes increased significantly after TP was applied to cells. In the nude mouse subcutaneous tumor model, experiments revealed an inhibitory effect of TP on glioma cell proliferation in vivo. IHC confirmed that the expression of E-cadherin was upregulated in mouse tumor tissues, while the expression levels of N-Cadherin and Vimentin were downregulated.ConclusionsTP can inhibit glioma cell proliferation, migration, and invasion, and reverse EMT progression. The possible mechanism of EMT reversal in glioma cells is that TP induces autophagy.

Targeting the EMT transcription factor Snail overcomes resistance to osimertinib in EGFR-mutant non-small cell lung cancer.

BackgroundThe resistance mechanism of the third generation of epidermal growth factor (EGFR) tyrosine kinase inhibitor (TKI) osimertinib is complex. Epithelial mesenchymal transition (EMT) is a common mechanism of EGFR‐TKI acquired resistance. Snail is an important transcription factor related to EMT. Whether targeting Snail can reverse the resistance of osimertinib by downregulating Snail is unknown.MethodsThe presence of EMT in H1975/OR (osimertinib resistance) cells was confirmed by transwell assay. To explore the EMT role in resistance, the expression levels of EMT markers were detected in both parental cells H1975 and resistant cells H1975OR. We used RNA interference technology to knockdown the key regulator Snail in resistant cells. After the interference efficiency was confirmed, changes in EMT‐related molecules of Snail were explicitly downregulated, and changes in sensitivity and migration and invasion ability were also examined. We used CDK4/6 inhibitor to test the ability of reversing drug resistance by downregulating Snail.ResultsCompared with the H1975 cell line, the H1975/OR resistant cell line showed increased invasiveness, upregulated expression of vimentin and downregulation of E‐cadherin. EMT occurred in the H1975/OR resistant cell line. The expression of Snail was upregulated in the osimertinib‐resistant cell line H1975/OR. Knockdown of Snail increased the sensitivity of H1975/OR cells to osimertinib. CDK4/6 inhibitor palbociclib could downregulate the expression of Snail. CDK 4/6 inhibitor palbociclib combined with osimertinib could reverse the resistance of osimertinib in H1975/OR.ConclusionsSnail plays an important role in the third generation of EGFR‐TKI osimertinib resistance, which may be reversed by downregulating Snail.

Osteopontin‑c isoform inhibition modulates ovarian cancer cell cisplatin resistance, viability and plasticity.

Osteopontin (OPN) is upregulated in several types of tumor and has been associated with chemoresistance. However, the contribution of OPN splicing isoforms (OPN-SIs) to chemoresistance requires further investigation. The present study aimed to evaluate the expression patterns of each tested OPN-SI in cisplatin (CDDP)-resistant ovarian carcinoma cell lines, focusing on the role of the OPN-c isoform (OPNc) in drug resistance. ACRP ovarian cancer cells resistant to CDDP, as well as their parental cell line A2780, were used. Analyses of the transcriptional expression of OPN-SIs, epithelial-mesenchymal transition (EMT) markers and EMT-related cytokines were performed using reverse transcription-quantitative PCR. OPNc was silenced in ACRP cells using anti-OPNc DNA oligomers and stably overexpressed by transfecting A2780 cells with a mammalian expression vector containing the full length OPNc cDNA. Functional assays were performed to determine cell proliferation, viability and colony formation. The results demonstrated that among the three tested OPN-SIs, OPNc was the most upregulated transcript in the ACRP cells compared with the parental A2780 cells. In addition, the expression levels of P-glycoprotein multidrug transporter were upregulated in CDDP-resistant ACRP cells compared with those in A2780 cells. OPNc knockdown sensitized ACRP cells to CDDP treatment and downregulated P-gp expression levels compared with those in the negative control group. Additionally, silencing of OPNc impaired cell proliferative and colony formation abilities, as well as reversed the expression levels of EMT markers and EMT-related cytokines compared with those in the negative control cells. Notably, although stable OPNc overexpression resulted in increased A2780 cell proliferation, it notably increased CDDP sensitivity compared with that in the cells transfected with a control vector. These results suggested that OPNc silencing may represent a putative approach to sensitize resistant ovarian cancer cells to chemotherapeutic agents.

LncRNA-POIR promotes epithelial-mesenchymal transition and suppresses sorafenib sensitivity simultaneously in hepatocellular carcinoma by sponging miR-182-5p.

Sorafenib (SOR) resistance remains a major obstacle in the effective treatment of hepatocellular carcinoma (HCC). A number of long noncoding RNAs (lncRNAs) are responsible for this chemoresistance. This study aimed to reveal the essential function of a recently defined lncRNA, lncRNA-POIR, in the epithelial-mesenchymal transition (EMT) and SOR sensitivity of HCC cells. SOR-induced cytotoxicity was analyzed via cell counting kit-8 and ethynyl-2'-deoxyuridine incorporation assays, whereas immunoblotting and confocal immunofluorescence were used to determine the expression levels of EMT markers. Furthermore, loss- or gain-of-function approaches were used to demonstrate the role of lncRNA-POIR/miR-182-5p on EMT and SOR sensitivity in HCC. The direct interaction between lncRNA-POIR and miR-182-5p was verified using a luciferase reporter assay. We found that knockdown of lncRNA-POIR sensitized HCC cells to SOR and simultaneously reversed EMT. As expected, miR-182-5p was confirmed as the downstream target of lncRNA-POIR. Moreover, miR-182-5p overexpression clearly reversed EMT and promoted SOR-induced cytotoxicity in representative HCC cells, whereas miR-182-5p downregulation played a contrasting role; miR-182-5p knockdown abolished the modulatory effects of lncRNA-POIR siRNA on EMT and SOR sensitivity. Together, these pieces of data suggest that lncRNA-POIR promotes EMT progression and suppresses SOR sensitivity simultaneously by sponging miR-182-5p. Thus, we proposed a compelling rationale for the use of lncRNA-POIR as a promising predictor of SOR response and as a potential therapeutic target for HCC treatment in the future.

Pristimerin exerts antitumor activity against MDA-MB-231 triple-negative breast cancer cells by reversing of epithelial-mesenchymal transition via downregulation of integrin β3

BackgroundPristimerin, a natural flavonoid compound, has potential anti-tumor activities. These activities have been illustrated in various cancer cell lines, including MDA-MB-231 cells. MDA-MB-231 cells are a representative mesenchymal subtype of triple negative breast cancer (MES-TNBC) cell line. Currently, the main treatment for patients with advanced MES-TNBC is cytotoxic chemotherapy. We tried to examine the role and effect of pristimerin on epithelial–mesenchymal transition (EMT) in MDA-MB-231 cells. MethodsThe effects of pristimerin on the proliferation of MDA-MB-231 cells were investigated by cloning formation growth assay. In vitro transwell and adhesion assays were performed for cell invasion and adhesion. The expression levels of EMT markers in E-cadherin and N-cadherin were examined by western blotting. We also established overexpressed- and silenced-integrin β3 cell lines to evaluate the role of integrin β3 in mediating the EMT reversion events in MDA-MB-231 cells. ResultsPristimerin inhibited cell proliferation, and its inhibitory effect was dose-dependent. We demonstrated that pristimerin reserved EMT by upregulating E-cadherin and downregulating N-cadherin expression. Meanwhile, we revealed that pristimerin inhibited mRNA and protein expression of integrin β3, which is a key heterodimeric transmembrane receptor associated with EMT. These inhibitory effects and reversion of EMT were enhanced when integrin β3 was knockdown in MDA-MB-231 cells, while the overexpression of integrin β3 attenuated these effects. In vivo studies using xenograft mouse model demonstrated that pristimerin inhibited tumor growth. ConclusionsOur findings provide important insights into the effects of pristimerin on inhibiting cancer progression and EMT reversion by suppression of integrin β3.

NRP1 regulates radiation-induced EMT via TGF-β/Smad signaling in lung adenocarcinoma cells

Purpose Radiation has been shown to promote the epithelial–mesenchymal transition (EMT) in tumor cells, and TGF-β/Smad and PI3K-Akt signaling pathways play an important role in the EMT. In this study, we investigated the effects of neuropilin-1 (NRP1) on radiation-induced TGF-β/Smad and non-classical Smad signaling pathways in lung cancer cells, as well as the effects of NRP1 on invasion and migration. Materials and methods Changes in the expression levels of EMT markers (β-catenin, N-cadherin, and vimentin) and related transcription factors (Twist and ZEB1) in stably transfected cells were detected by Western blotting and qPCR, and changes were assessed by TGF-β/Smad and non-classical Smad signaling. Immunofluorescence was used to detect the expression of the cytoskeletal protein F-actin. Expression of TGF-β1 and CXCL-12 was detected by ELISA. Transwell and scratch assays were used to detect the invasive ability and migration of lung cancer cells, respectively. Results Our results showed that ionizing radiation could induce the EMT as well as morphological changes in lung adenocarcinoma cells (A549); however, the effects were not significant in lung squamous carcinoma cells (SK-MES-1). Moreover, we showed that NRP1 promotes the EMT induced by ionizing radiation in A549 cells, which may be related to the increased expression of EMT-related transcription factors. NRP1 may promote the radiation-induced EMT of A549 cells mainly through TGF-β1/Smad2/3 signaling. NRP1 also enhanced radiation-induced invasion, migration, and CXCL-12 expression in A549 cells. Conclusions We conclude that NRP1 promotes radiation-induced EMT in lung adenocarcinoma cells via TGF-β1/Smad signaling and not non-classical Smad signaling, and enhances the invasion and migration of lung adenocarcinoma cells.