Expression Levels Of Vimentin Research Articles

MiR-770-5p regulates EMT and invasion in TNBC cells by targeting DNMT3A

MicroRNAs (miRNAs) are shown to regulate various processes in cancer like motility and invasion that are key features of the metastatic triple negative breast cancer (TNBCs). Epithelial-mesenchymal transition (EMT) is one of the well-defined cellular transitioning processes characterized with reduced E-cadherin expression and increased mesenchymal molecules such as Vimentin or Snail thereby gives the cells mobility and invasive character. Aberrant DNA methylation by DNA methyltransferases (DNMTs) plays an important role in carcinogenesis. It is well known that DNMTs are required for transcriptional silencing of tumor-associated genes. DNMT3A-induced promoter hypermethylation of E-cadherin has also been known to improve cancer metastasis. Our results indicated that miR-770-5p could downregulate Vimentin and Snail expression levels, while increasing or restoring the expression of E-Cadherin hence, leading to inhibition of EMT phenotypes along with motility and invasion. Specifically, we showed that overexpression of miR-770-5p restored the expression of E-Cadherin in MDA-MB-231 cells via directly targeting DNMT3A. We also observed the change in the spindled shapes showing the loss of mesenchymal characteristics and gain of epithelial phenotype in miR-770-5p overexpressing cells. When considered together, our results show that miR-770-5p could effectively inhibit invasion potential driven by EMT.

Hypoxia enhances basal autophagy of epithelial-derived ameloblastoma cells.

Ameloblastoma is a locally aggressive odontogenic tumor. Etiopathogenesis and locally aggressive growth properties of ameloblastoma can be attributed to a hypoxic microenvironment conducive to tumor cell survival. Epithelial-derived follicular ameloblastoma cells (EP-AMCs) display enhanced basal autophagy, but the interplay of hypoxia and autophagy in EP-AMCs survival and ameloblastoma recurrence is unclear. We evaluated differential expression of autophagic markers in primary and recurrent ameloblastomas and hypothesized that hypoxia-induced autophagy supports EP-AMC survival. Primary and recurrent ameloblastomas were comparatively assessed for expression levels of pan-cytokeratin, Vimentin, and autophagic markers SQSTM1/p62, LC3, and pS6. EP-AMCs compared with human odontoma-derived cells (HODCs) were subjected to severe hypoxia to determine the interplay of hypoxia and autophagic process in posthypoxia survival. Pan-cytokeratin and SQSTM1/p62 were expressed by both primary and recurrent ameloblastoma epithelial cells while the ameloblastoma connective tissues displayed weak reactivity to vimentin. Under hypoxia, EP-AMC expression levels of hypoxia-inducible factor (HIF)-1α, p62, and LC3 were increased while pS6 was decreased posthypoxia. The combined decrease in pS6 and enhanced LC3 in EP-AMCs under hypoxia indicate that EP-AMCs re-establish basal autophagy under hypoxia. Taken together, these suggest a possible role of LC3-associated phagocytosis (LAP) in ameloblastoma cell survival.

Morphofunctional analysis of human pancreatic cancer cell lines in 2- and 3-dimensional cultures

Genetic, transcriptional, and morphological differences have been reported in pancreatic ductal adenocarcinoma (PDAC) cases. We recently found that epithelial or mesenchymal features were enhanced in three-dimensional (3D) cultures compared to two-dimensional (2D) cultures. In this study, we examined the differences in the morphological and functional characteristics of eight PDAC cell lines in 2D and 3D cultures. Most PDAC cells showed similar pleomorphic morphologies in 2D culture. Under 3D culture, PDAC cells with high E-cadherin and low vimentin expression levels (epithelial) formed small round spheres encircled with flat lining cells, whereas those with high vimentin and low E-cadherin expression levels (mesenchymal) formed large grape-like spheres without lining cells and were highly proliferative. In 3D culture, gemcitabine was more effective for the spheres formed by PDAC cells with epithelial features, while abraxane was more effective on those with mesenchymal features. The expression levels of drug transporters were highest PDAC cells with high vimentin expression levels. These findings indicate that PDAC cells possess various levels of epithelial and mesenchymal characteristics. The 3D-culture method is useful for investigating the diversity of PDAC cell lines and may play important roles in the development of personalized early diagnostic methods and anticancer drugs for PDAC.

MiR-424-5p reduces 5-fluorouracil resistance possibly by inhibiting Src/focal adhesion kinase signalling-mediated epithelial-mesenchymal transition in colon cancer cells.

miR-424-5p negatively regulates various malignant biological behaviours in tumour cells. We explored the relationship between miR-424-5p and 5-fluorouracil resistance in colon cancer cells. We developed 5-fluorouracil-resistant HT-29 cells and detected miR-424-5p expression using real-time fluorescence quantitative PCR. Cell viability was assessed using Cell Counting Kit-8 (CCK-8) assay. Immunofluorescence and western blotting were performed to determine protein levels. Apoptosis was detected by Annexin V-FITC/PI staining. miR-424-5p was downregulated in 5-fluorouracil-resistant HT-29 cells. A miR-424-5p mimic enhanced the sensitivity of the resistant cells to 5-fluorouracil, whereas a miR-424-5p inhibitor promoted 5-fluorouracil resistance in HT-29 cells. Furthermore, the miR-424-5p mimic downregulated vimentin and upregulated E-cadherin in 5-fluorouracil-resistant HT-29 cells, whereas the miR-424-5p inhibitor exhibited opposite effects. The miR-424-5p inhibitor significantly inhibited 5-fluorouracil-induced HT-29 cell apoptosis and Src and focal adhesion kinase phosphorylation, whereas the miR-424-5p mimic showed opposite effects. Pretreatment with Src inhibitor 1 or focal adhesion kinase inhibitor 2 blocked the increase in Src and focal adhesion kinase phosphorylation and vimentin expression level and the decrease in E-cadherin expression level in miR-424-5p inhibitor-exposed HT-29 cells. miR-424-5p suppressed epithelial-mesenchymal transition by inhibiting the Src/focal adhesion kinase signalling pathway to reduce 5-fluorouracil resistance in colon cancer cells.

Decreased chromobox homologue 7 expression is associated with epithelial-mesenchymal transition and poor prognosis in cervical cancer.

The aim of this study was to evaluate the association of the chromobox homologue 7 (CBX7) expression with the epithelial–mesenchymal transition in cervical cancer (CC), as well as with the disease prognosis. CBX7, E-cadherin (E-cad), and vimentin (VIM) expression levels were detected with immunohistochemistry. The relationship between the expression of CBX7, E-cad, and VIM expression and conventional clinicopathological characteristics of CC were evaluated. The positive expression rates of CBX7 and E-cad in the CC tissues were lower than the adjacent non-tumorous cervical tissues. Moreover, the VIM expression level was higher. The CBX7 expression was positively correlated with the E-cad expression, whereas was negatively correlated with the VIM expression. Furthermore, CBX7 was associated with the disease clinical staging, histological differentiation, lymph node metastasis, and vascular invasion. Patients with negative CBX7 expression showed decreased overall survival rates compared with those with low or high CBX7 expression. Multivariate Cox regression analysis indicated that the decreased CBX7 expression was an independent predictor for the poor prognosis of CC. In conclusion, the absence of CBX7 is associated with the histologic differentiation, lymphatic metastasis, vascular invasion, and poor prognosis of CC. CBX7 may be an independent prognostic factor for the prognosis of CC patients.

Stachydrine inhibits TGF-β1-induced epithelial-mesenchymal transition in hepatocellular carcinoma cells through the TGF-β/Smad and PI3K/Akt/mTOR signaling pathways.

Stachydrine is a bioactive alkaloid that has been found to exert tumor-suppressive potential. However, the effect of stachydrine on hepatocellular carcinoma (HCC) has not been previously investigated. In the present study, we investigated the effect of transforming growth factor-β1 (TGF-β1)-induced epithelial-mesenchymal transition (EMT) in HepG2 cells. Our results showed that stachydrine significantly suppressed TGF-β1-induced HepG2 cell migration and invasion in a dose-dependent manner. Stachydrine prevented TGF-β1-induced EMT in HepG2 cells, as proved by the increased expression level of E-cadherin and decreased expression levels of N-cadherin and vimentin. In addition, stachydrine attenuated TGF-β1-induced upregulation of TGF-β receptor I (TβRI) in both protein and mRNA levels. Further mechanism investigations proved that stachydrine prevented TGF-β1-induced activation of Smad2/3 and phosphoinositol-3-kinase (PI3K)/Akt/mTOR signaling pathways in HepG2 cells. In conclusion, these findings demonstrated that stachydrine prevented TGF-β1-induced EMT in HCC cells through Smad2/3 and PI3K/Akt/mTOR signaling pathways. Thus, stachydrine might be a potential therapeutic agent for the treatment of HCC.

Long non-coding RNA OIP5-AS1 contributes to cisplatin resistance of oral squamous cell carcinoma through the miR-27b-3p/TRIM14 axis.

Oral squamous cell carcinoma (OSCC) accounts for 90% of oral cavity cancer types, but the overall prognosis for patients with OSCC remains unfavorable. Cisplatin (DDP) is an effective drug in OSCC treatment, but DDP resistance weakens its therapeutic effect. Opa-interacting protein 5 antisense RNA 1 (OIP5-AS1) can trigger DDP resistance. The purpose of the current study was to explore the role and mechanism ofOIP5-AS1 in OSCC DDP resistance. In the present study, the expression levels of OIP5-AS1, microRNA (miR)-27b-3p and tripartite motif-containing 14 (TRIM14) were detected by reverse transcription-quantitative PCR. DDP resistance was measured using an MTT assay. Moreover, cell proliferation, migration and invasion were assessed by MTT, Transwell, and Matrigel assays. Protein expression levels of TRIM14, E-cadherin, N-cadherin and Vimentin were detected by western blot analysis. Putative binding sites between miR-27b-3p andOIP5-AS1 or TRIM14werepredicted with starBase and verified using a dual-luciferase reporter assay. The role of OIP5-AS1 in DDP resistance of OSCC in vivo was measured using a xenograft tumor model. It was observed that OIP5-AS1 was upregulated in DDP-resistant OSCC cells, and the knockdown of OIP5-AS1 improved DDP sensitivity in DDP-resistant OSCC cells. The present study identified that miR-27b-3p was a target of OIP5-AS1. Furthermore, miR-27b-3p silencing reversed the effect of OIP5-AS1 knockdown on DDP sensitivity in DDP-resistant OSCC cells. TRIM14was shown to be a direct target of miR-27b-3p, and TRIM14 overexpression abolished the effect of miR-27b-3p on DDP sensitivity in DDP-resistant OSCC cells. The results suggested that OIP5-AS1 increased TRIM14 expression by sponging miR-27b-3p. In addition, OIP5-AS1 knockdown enhanced DDP sensitivity of OSCC in vivo. Data from the present study indicated that OIP5-AS1 may improve DDP resistance through theupregulationTRIM14 mediated bymiR-27b-3p, providing a possible therapeutic strategy for OSCC treatment.

Effects of targeted Notch1 silencing on the biological processes of the T24 and 5637 cells in vitro.

The present study aimed to investigate the roles of Notch1 in the biological processes of bladder cancer cells (BCCs) in vitro. Short hairpin (sh)RNA targeting Notch1 was designed and constructed, and the T24 and 5637 BCCs were selected for transfection. The cells were classified into two groups: shRNA negative control (NC) and Notch1 shRNA. MTT and Transwell assays, and flow cytometry were performed to examine the changes in cell proliferation, invasiveness, and apoptosis, respectively. In addition, reverse transcription-quantitative PCR and western blot analysis was used to detect the mRNA and protein expression levels of apoptosis-related proteins (Bax, Bid and Bcl2) and epithelial-mesenchymal transition factors (vimentin and E- and N-cadherin). Compared with that in the shRNA NC group, the Notch1 shRNA group showed significantly decreased cell proliferation rate and invasiveness; increased apoptotic rate; elevated mRNA expression levels of Bad, Bid and E-cadherin; and reduced mRNA expression levels of Bcl2, N-cadherin and vimentin. The trends for protein expression levels were the same as those for mRNA levels. Notch1 silencing inhibited invasion and promoted apoptosis of BCCs.

Role of up-regulated DDX3 in the proliferation of human cervical cancer cells

Objective: To investigate the role of DDX3 up-regulation in the proliferation of human cervical cancer cells and its correlation with clinical prognosis. Methods: Expression levels of DDX3 in the 59 specimens of cervical cancer and adjacent non-neoplastic tissue collected at Henan Provincial People's Hospital from April 2012 to March 2013 were detected using immunohistochemistry. A lentivirus-mediated DDX3-over-expression cell line was constructed based on HeLa cells of cervical cancer. CCK-8 assay was used to evaluate cell survival rate. Boyden chamber was used to measure the cell migration and invasion. Real-time fluorescence quantitative PCR was used to detect DDX3 expression level and Western blot was used to detect the expression of EMT and PI3K/Akt signal pathway-related proteins. Results: DDX3 overexpression was associated with FIGO stage, depth of cervical invasion and lymph node metastasis (P<0.05). Kaplan-Meier analysis revealed that cervical cancer patients with high expression of DDX3 had a poor overall survival (P<0.05). Compared with the cells transfected with pLVX-Con vector, the expression of DDX3 protein and mRNA was significantly increased in the cells transfected with pLVX-DDX3 (all P<0.01). Cell proliferation was significantly increased following transfection with pLVX-DDX3 for 72 h in HeLa cells compared with that transfected with pLVX-Con (P<0.05). Compared with the controls, DDX3 overexpression significantly promoted the migration and invasion of HeLa cells (P<0.05), and increased the expression of N-Cadherin, vimentin and Snail in HeLa cells (P<0.05). In pLVX-DDX3 group, the expression levels of β-catenin, phosphorylated Akt, and pAkt's downstream target p-GSK3β were significantly higher than those of pLVX-Con group (P<0.05). The expression levels of p-Akt, p-GSK3β and β-catenin were decreased when the PI3K/Akt pathway was blocked using the PI3K inhibitor LY294002 (P<0.05), and the expression levels of N-Cadherin, vimentin and Snail were also significantly decreased (P<0.05). Conclusions: DDX3 overexpression promotes proliferation, migration and invasion of cervical cancer cells, and induces epithelial-mesenchymal transition (EMT). Its mechanism may be related to activation of the PI3K/Akt signaling pathway.

Attenuating role of withaferin A in the proliferation and migration of lung cancer cells via a p53-miR-27a/miR-10b pathway.

MicroRNAs (miRNAs/miRs) are known to play a key role in tumorigenesis and usually serve as therapeutic targets in cancer treatment. In the present study, the inhibitory effects and the targeting miRNAs of withaferin A (WA) were investigated in human lung cancer cells. Different lung cancer cell lines were administrated with different concentrations of WA for different time interval followed by western blot or reverse transcription-quantitative PCR analyses to determine the underlying signaling pathway. The results demonstrated that WA decreased the viability of lung cancer cells in a caspase-dependent manner. Further investigations indicated that treatment with WA induced the expression of proapoptotic molecules, p53 and Bax, and decreased Bcl-2 expression in A549 cells. Notably, the results demonstrated that WA also decreased the motility of lung cancer cells in a dose-dependent manner, at a relatively lower concentration. Western blot analysis revealed increased E-cadherin and decreased vimentin expression levels in lung cancer cells treated with WA. In addition, two oncomiRs, including miR-10b and miR-27a, which regulate the expression of E-cadherin and Bax, respectively, were downregulated in the presence of WA. The ectopic expression of miR-10b mimics was able to recover the WA-decreased motility of lung cancer cells, which was accompanied by a reduction in E-cadherin expression. Conversely, the ectopic expression of miR-27a mimics decreased the expression of Bax and recovered the viability of lung cancer cells attenuated by WA. In addition, the ectopic expression of p53-wild type decreased the expression levels of both miR-10b and miR-27a, whereas p53 knockdown induced their expression. Transient knockdown of p53 decreased the inhibitory effects of WA in the motility and viability of lung cancer cells, suggesting an association between WA-p53-miR-10b/27a and motility/viability. Further investigations demonstrated that p53 knockdown in lung cancer stable cell lines exhibited higher levels of both miR-10b and miR-27a, and higher motility and viability following treatment with WA. However, suppression of miR-10b and miR-27a effectively decreased motility and viability, respectively, following treatment with WA. Taken together, the results of the present study suggest that WA inhibits the functionality of lung cancer cells by decreasing the expression levels of both miR-10b and miR-27a in a p53-dependent manner.

Curcumin-enriched Gemini surfactant nanoparticles exhibited tumoricidal effects on human 3D spheroid HT-29 cells in vitro

BackgroundHere, we examined the tumoricidal effect of Gemini surfactant nanoparticles enriched with curcumin on 3D spheroid HT-29 cells. The delivery of curcumin and other phytocompounds to the tumor niche is an important challenge.MethodsSpheroid HT-29 cells were generated by using a conventional hanging drop method and exposed to different concentrations of Gemini-curcumin nanoparticles. The changes in spheroid integrity and cell viability were evaluated by measuring the spheroid diameter and LDH release, respectively. The uptake of Gemini-curcumin nanoparticles was detected by flow cytometry assay. Flow cytometric of Rhodamine 123 efflux was also performed. Migration capacity was analyzed using a Transwell insert assay. By using real-time PCR analysis and Western blotting, we studied the expression level of MMP-2, -9, Vimentin, and E-cadherin genes.ResultsGemini-curcumin nanoparticles had the potential to disintegrate spheroids and decrease central density compared to the control group (p < 0.05). These changes coincided with enhanced LDH release by the increase of nanoparticle concentration (p < 0.05). Data highlighted the ability of cells to uptake synthetic nanoparticles in a dose-dependent manner. We found reduced Rhodamine 123 efflux in treated HT-29 spheroid cells compared to the control (p < 0.05). Nanoparticles significantly decreased the metastasis and epithelial-mesenchymal transition (EMT) rate by the suppression of MMP-2 and MMP-9, Vimentin, and induction of E-cadherin (p < 0.05).ConclusionOur data confirmed that Gemini curcumin has the potential to suppress cell proliferation and inhibit metastasis in 3D spheroid HT-29 cells in vitro.

PDLIM2 prevents the malignant phenotype of hepatocellular carcinoma cells by negatively regulating β-catenin.

Hepatocellular carcinoma (HCC) is one of the most common malignancies and leading causes of cancer-related deaths globally. Despite significant advances in therapy, the molecular mechanisms underlying HCC development and progression remain unclear. Here, we aimed to explore the potential role of PDLIM2 in the development and epithelial-mesenchymal transition (EMT) of HCC via a possible modulation of β-catenin. We first confirmed that PDLIM2 was downregulated in HCC tissues and cells and found lower PDLIM2 expression was associated with worse prognosis in HCC patients. Loss- and gain- of function experiments were performed to evaluate the roles of PDLIM2 and β-catenin in HCC cell proliferation, migration, invasion, EMT, and colony formation. EMT was determined based on the levels of E-cadherin, zonula occludens-1, N-cadherin, and vimentin expression. In vivo, the roles of PDLIM2 and β-catenin in HCC were investigated by using a nude mouse xenograft model. It should be noted that PDLIM2 led to the inhibition of β-catenin activity and its downstream gene expression. Importantly, ectopic PDLIM2 expression inhibited the proliferation, migration, invasion, and EMT of HCC cells by reducing β-catenin expression both in vitro and in vivo, thereby suppressing the occurrence and progression of HCC. Taken together, our results demonstrated that overexpressed PDLIM2 exerts a tumor-suppressive role in HCC by regulating β-catenin. This study suggests that the PDLIM2 may be a promising target for the treatment of HCC.

Chrysin inhibited epithelial-mesenchymal transition of type Ⅱ alveolar epithelial cell by regulating NF-κB/Twist 1 signaling pathway

This study aimed to assess whether chrysin(ChR) can inhibit epithelial-mesenchymal transition(EMT) of type Ⅱ alveolar epithelial cell and produce anti-pulmonary fibrosis effect by regulating the NF-κB/Twist 1 signaling pathway. Sixty rats were randomly divided into the control group, the bleomycin(BLC) group, BLC+ChR(50 mg·kg~(-1)) group and BLC+ChR(100 mg·kg~(-1)) group, with 15 rats in each group. The pulmonary fibrosis model was induced by intratracheal injection of BLC(7 500 U·kg~(-1)). Rats were orally administered with different doses of ChR after BLC injection for 28 days. The cells were divided into control group, TGF-β1 group(5 ng·mL~(-1)), and TGF-β1+ChR(1, 10, 100 μmol·L~(-1)) groups. The type Ⅱ alveolar epithelial cells were treated with TGF-β1 for 24 h, and then treated with TGF-β1 for 48 h in the presence or absence of different doses of ChR(1, 10 and 100 μmol·L~(-1)). The morphological changes and collagen deposition in lung tissues were analyzed by HE staining, Masson staining and immunohistochemistry. The mRNA and protein expression levels of collagen Ⅰ, E-cadherin, zonula occludens-1(ZO-1), vimentin, alpha smooth muscle actin(α-SMA), inhibitor of nuclear factor kappa B alpha(IκBα), nuclear factor-kappa B p65(NF-κB p65), phospho-NF-κB p65(p-p65) and Twist 1 in lung tissues and cells were detected by qPCR and Western blot, respectively. The animal experiment results showed that as compared with the BLC group, after administration of ChR for 28 days, bleomycin-induced pulmonary fibrosis in rats was significantly relieved, collagen Ⅰ expression in lung tissues was significantly reduced(P&lt;0.05 or P&lt;0.01), and EMT of alveolar epithelial cells was obviously inhibited [the expression levels of E-cadherin and ZO-1 were increased and the expression levels of vimentin and α-SMA were decreased(P&lt;0.05 or P&lt;0.01)], concomitantly with significantly reduced IκBα and p65 phosphorylation level in cytoplasm and decreased NF-κB p65 and Twist 1 expression in nucleus(P&lt;0.05 or P&lt;0.01). The cell experiment results showed that different doses of ChR(1, 10 and 100 μmol·L~(-1)) significantly reduced TGF-β1-induced collagen Ⅰ expression(P&lt;0.05 or P&lt;0.01), significantly inhibited EMT of type Ⅱ alveolar epithelial cells[the expression levels of E-cadherin and ZO-1 were increased and the expression levels of vimentin and α-SMA were decreased(P&lt;0.05 or P&lt;0.01)], and inhibited IκBα and p65 phosphorylation in cytoplasm and down-regulated NF-κB p65 and Twist 1 expression in nucleus induced by TGF-β1(P&lt;0.05 or P&lt;0.01). The results suggest that ChR can reverse EMT of type Ⅱ alveolar epithelial cell and alleviate pulmonary fibrosis in rats, and its mechanism may be associated with reducing IκBα phosphorylation and inhibiting NF-κB p65 phosphorylation and nuclear transfer, thus down-regulating Twist 1 expression.

IMMUNOHISTOCHEMICAL AND MORPHOMETRIC CHARACTERISTICS OF CHORIONIC TROPHOBLAST AND DECIDUAL CELLS OF FETAL MEMBRANES IN CASE OF ANTE-INTRANATAL FETAL DEATH

The aim: To identify immunohistochemical and morphometric features of chorionic trophoblast cells and fetal membranes derived decidual cells, which were obtained from stillbirths associated with pre-eclampsia, iron deficiency anemia, and acute chorioamnionitis. Materials and methods: The study included 58 fetal membranes of fetuses, who died in the ante-intranatal period. The membranes were divided into 6 obstetric history-based groups: premature (n = 8) and full-term (n = 8) stillbirths complicated by preeclampsia; premature (n = 8) and full-term ( n = 8) stillbirths complicated by iron deficiency anemia, premature (n = 10) and full-term (n = 16) stillbirths complicated by chorioamnionitis. A control group consisted of 8 membranes obtained from physiological pregnancies followed by the birth of a live full-term baby. Samples (rupture site) were probed with cytokeratin to identify the fetal trophoblast layer of the chorion and with vimentin for further identification of the decidual cells. The thickness of the trophoblastic layer, expression levels of cytokeratin and vimentin were determined. Results: A decrease of the cytokeratin expression by the chorionic trophoblasts and a thinning of the chorionic trophoblast cell layer due to an increasing gestational age were shown in case of the mentioned pathological conditions. In comparison with the control group, the level of vimentin expression by decidual cells was increased in case of full-term pregnancy complicated by preeclampsia, decreased in pregnancies complicated by chorioamnionitis, and remained unchanged in case of accompanying anemia. Conclusions: The changes in the studied immunohistochemical parameters are more pronounced in case of chorioamnionitis, which indicates more severe morphological and functional changes.

Not Only Hypoxia- but Radiation-Induced Epithelial-Mesenchymal Transition Is Modulated by Hypoxia-Inducible Factor 1 in A549 Lung Cancer Cells

Hypoxia leads to post-treatment metastasis and recurrences of cancer via the epithelial-mesenchymal transition (EMT). Radiotherapy itself may also contribute to the acquisition of EMT phenotypes. Despite extensive studies on the EMT driven by either hypoxia or radiation stimuli, the molecular mechanisms characterizing these EMT events remain unclear. Thus, we aimed to evaluate the differences in the molecular pathways between hypoxia-induced EMT (Hypo-EMT) and radiation-induced EMT (R-EMT). Further, we investigated the therapeutic effects of HIF-1α inhibitor (LW6) on Hypo-EMT and R-EMT cells. A549 cells, lung adenocarcinoma cell line, acquired enhanced wound-healing activity under both hypoxia and irradiation. Localization of E-cadherin was altered from the cell membrane to the cytoplasm in both hypoxia and irradiated conditions. Of note, the expression levels of vimentin, one of the major EMT markers, was enhanced in irradiated cells, while it decreased under hypoxia condition. Importantly, LW6 significantly blocked EMTrelated malignant phenotypes in both Hypo-EMT cells and R-EMT cells with concomitant re-location of E-cadherin onto the cell membrane. Moreover, LW6 deflected stress responsive signalling, JNK, activated sustainably under hypoxic condition, and the blockage of JNK impaired EMT phenotypes. Together, this work demonstrated the molecular events underlying Hypo-EMT and R-EMT, and highlighted HIF-1α as a therapeutic target not only in Hypo-EMT, but also in R-EMT.

Thymocytes induce renal tubular epithelial cells to undergo the epithelial-to-mesenchymal transition.

Renal tubulointerstitial fibrosis is known to occur as a result of epithelial cell transformation into myofibroblasts via the epithelial-to-mesenchymal transition (EMT) process. It has been reported that macrophages, regulatory T (Treg) cells, and gamma delta T (γδ T) cells can promote fibrosis via EMT in vivo. Our study intended to detect whether thymocytes can induce renal tubular cells to undergo the EMT. Rat thymocytes were activated by phytohemagglutinin and concanavalin A. The rat renal tubular epithelial cells (NRK-52E) were incubated in a conditioned medium harvested from activated thymocytes or co-cultured with freshly isolated thymocytes for 48 hours. Real-time reverse transcription-polymerase chain reaction, immunofluorescence, and western blotting analysis were used to test the expression of the epithelial and mesenchymal markers in NRK-52E cells. Scratch assay was designed to test the cell migration abilities of NRK-52E cells. Student's t test and oneway analysis of variance test were used for statistical analysis. The combined stimulation with phytohemagglutinin and concanavalin A activated the primary isolated rat thymocytes. After treatment with conditioned medium or freshly isolated thymocytes, the expression levels of cytokeratin 19 and E-cadherin were downregulated in NRK-52E cells, while the mRNA and protein expression levels of alpha-smooth muscle actin, desmin, and vimentin were upregulated (P < 0.05). We found that the cell migration abilities of the induced NRK-52E cells were significantly improved. Both activated rat thymocytes (more percentage of CD8+ T cells) and freshly isolated thymocytes have promoting effects on the EMT of NRK-52E cells.

Down-regulation of PRR11 affects the proliferation, migration and invasion of osteosarcoma by inhibiting the Wnt/β-catenin pathway.

Purpose: This study aim to explore the effect of down-regulation of PRR11 (proline-rich protein 11) on the proliferation, invasion, migration, Wnt/β-catenin signaling pathway and EMT of osteosarcoma cells.Methods: Immunohistochemical staining, fluorescent quantitative PCR and western blotting were used to detect the expression level of PRR11 in osteosarcoma tissues and osteosarcoma cells. After SiRNA down-regulated the expression level of PRR11, CCK8 was used to detect cell proliferation ability, Transwell chamber to detect cell invasion ability, scratch test to detect cell migration ability, and flow cytometry to detect cell apoptosis. Western blotting was used to detect the expression levels of wnt/β-catenin pathway related proteins and key epithelial-mesenchymal transition proteins.Results: PRR11 is highly expressed in osteosarcoma tissues, and its expression level is related to tumor size, Enneking stage of tumor, lymph node metastasis and patient prognosis. The low expression of PRR11 can inhibit the proliferation, migration and invasion of osteosarcoma cells, and promote apoptosis. Down-regulating the expression of PRR11 will inhibit the expression of Wnt pathway related proteins β-catenin and p-GSK-3β, enhance the expression of p-β-catenin, GSK-3β, and increase the expression of downstream genes CyclinD1 and c-Myc in the Wnt pathway. At the same time, the expression of PRR11 was down-regulated, the epithelial marker E-cadherin was significantly increased, and the expression levels of mesenchymal markers Vimentin and Fibronectin were significantly reduced.Conclusion: Down-regulation of PRR11 can inhibit the proliferation, migration and invasion of osteosarcoma cells, and its mechanism may be related to down-regulation of PRR11 to inhibit the Wnt/β-catenin signaling pathway and thus prevent the EMT process. Therefore, PRR11 may be used as an oncogene to promote the occurrence and development of osteosarcoma, and is a potential prognostic indicator and therapeutic target in osteosarcoma.

YKL-40 promotes invasion and metastasis of bladder cancer by regulating epithelial mesenchymal transition

Objective This study aims to explore the relevance between YKL-40 and recurrence and progression of bladder cancer, and determine whether YKL-40 can be used as a potential target in patients with bladder cancer. Methods We analyzed the invasion and metastasis ability of BIU-87, UMUC3, J82, T24, 5637 and immortalized human bladder epithelial cells SVHUC1 by Transwell method. The YKL-40 expression levels in cell lines were analyzed by Western blot and qPCR. Results The increase of YKL-40 level, especially in tumour group, was related to tumour pathological stage and tumour invasion and metastasis. The cell lines with YKL-40 high expression had stronger invasion and metastasis ability. Overexpression of YKL-40 in SVHUC1 with the lowest YKL-40 expression can enhance the invasion and migration of cells. In T24 cells with YKL-40 high expression, transfection of shRNA plasmid targeting YKL-40 can down regulate the invasion and migration. The expression levels of N-cadherin and Vimentin in YKL-40 overexpressed SVHUC1 cells were increased, the E-cadherin expression was decreased, the Twist, Snail and Slug expression levels were increased, but they were opposite in T24 cells with down-regulation of YKL-40 expression. Conclusions YKL-40 promoted the migration and invasion of bladder cancer cells by up regulating the EMT gene expression. The YKL-40 expression is closely related to the invasion and migration of bladder cancer.

LINC00662 Promotes Oral Squamous Cell Carcinoma Cell Growth and Metastasis through miR-144-3p/EZH2 Axis.

PurposeLong non-coding RNA (lncRNA) is identified as an important regulator involved in oral squamous cell carcinoma (OSCC) tumorigenesis. This study aimed to investigate the functional role and underlying mechanism of LINC00662 in OSCC.Materials and MethodsThe expression levels of LINC00662, miR-144-3p, and enhancer of zeste homolog 2 (EZH2) mRNA were quantified with quantitative real-time polymerase chain reaction in OSCC tissues and cell lines. Western blot analysis was used to assay the expression levels of E-cadherin, Vimentin, and EZH2. Cell proliferation, migration, and invasion were monitored by cell counting kit-8 and Transwell assays. Dual-luciferase reporter and RNA immunoprecipitation assays were employed to verify the regulatory relationship between LINC00662 and miR-144-3p.ResultsThe expression of LINC00662, positively associated with the increased TNM stage and lymph node metastasis of the patients, was up-regulated in OSCC tissues and cells. The overexpression of LINC00662 facilitated the proliferation, migration, and invasion of OSCC cells. MiR-144-3p could bind to LINC00662, and the promoting effect of LINC00662 overexpression was counteracted by miR-144-3p mimic. Moreover, EZH2 expression was negatively regulated by miR-144-3p and positively regulated by LINC00662. The silencing of EZH2 attenuated the promoting effects of overexpression of LINC00662 on cell proliferation, migration, invasion, and epithelial-mesenchymal transition.ConclusionLINC00662, as an oncogenic lncRNA of OSCC, accelerates OSCC progression by repressing miR-144-3p expression and increasing EZH2 expression.

VSP‑17 suppresses the migration and invasion of triple‑negative breast cancer cells through inhibition of the EMT process via the PPARγ/AMPK signaling pathway

VSP-17, a novel peroxisome proliferator-activated receptor γ (PPARγ) agonist, has been previously demonstrated to suppress the metastasis of triple-negative breast cancer (TNBC) by upregulating the expression levels of E-cadherin, which is a key marker of epithelial-mesenchymal transition (EMT). However, the mechanism of action of VSP-17, in particular whether it may be associated with the EMT process, remains unknown. The present study investigated the ability of VSP-17 to inhibit the invasiveness and migratory ability of TNBC cell lines (MDA-MB-231 and MDA-MB-453) performed in in vitro experiments. including cell migration assay, cell invasion assay, cell transfection, RT-qPCR, western blot (WB) analysis and immunofluorescence. The present study aimed to ascertain whether and how the PPARγ/AMP-activated protein kinase (AMPK) signaling pathway serves a role in the inhibitory effects of VSP-17 on cell migration and invasion. The results revealed that both treatment with compound C (an AMPK inhibitor) and transfection with small interfering RNA (si)AMPK notably diminished the inhibitory effect of VSP-17 treatment on the migration and invasion of MDA-MB-231 and MDA-MB-453 cells, indicating that VSP-17 may, at least partly, exert its effects via AMPK. Furthermore, both compound C and siAMPK markedly diminished the VSP-17-induced downregulation of vimentin expression levels and upregulation of E-cadherin expression levels, further indicating that the VSP-17-induced inhibition of the EMT process may be dependent on AMPK. The combination of GW9662 (a PPARγ antagonist) or siPPARγ diminished the inhibitory effect of VSP-17 treatment on the migration and invasion of the TNBC cells, indicating that PPARγ may serve an important role in the VSP-17-induced inhibition of the migration and invasion of TNBC cells. In addition, both GW9662 and siPPARγ significantly reversed the VSP-17-induced downregulation of vimentin expression levels and upregulation of E-cadherin expression levels, implying that the VSP-17-induced inhibition of the EMT process may be dependent on PPARγ. VSP-17 treatment also upregulated the expression levels of p-AMPK, which could be reversed by either GW9662 or siPPARγ, indicating that the VSP-17-induced activation of the AMPK signaling pathway was PPARγ-dependent. In conclusion, the findings of the present study indicated that VSP-17 treatment may inhibit the migration and invasion of TNBC cells by suppressing the EMT process via the PPARγ/AMPK signaling pathway.