Vimentin Gene Expression Research Articles

195 Development of a fetal porcine model for congenital viral infections in the developing CNS

To develop a porcine model for analysis of the effects of congenital viral infections on CNS and spinal (CSF) derived fetal neural precursor (NPC) and neural progenitor cells. Isolation of NPCs from the subventricular zone (SVZ) of the porcine brain or CSF derived from premature (day 104) and post-term (day 135) Cesarean-delivered piglets were plated in 12-well plates. Nonadherent plates were used to generate neurospheres with self-renewal capacity and for long term passages. After a 2-day incubation for neurosphere formation and removal of apoptotic debris, cells were seeded in various conditions including incubation in (1) 10% FBS, (2) Rock Inhibitor (RI)-specific inhibitor of apoptosis, (3) FBS and RI, and (4) Neurobasal media alone (control). After purification and identification of NPCs, cells were then infected with low MOI of clinically isolated first passage ZIKV HN16, then fixed and stained with Flavivirus-specific antibody (MAb 4G2) followed by FITC labeled secondary antibody staining visualization under fluorescence microscopy. Gene expression studies were also performed from RNA from ZIKV infected and mock infected samples to determine effects on genes related to NPC self-renewal and survival. The brain derived cells cultured for 4 days showed three distinct populations including NSCs, NPCs, and a mixed NSC/NPC population. After 21 days of culture, cells shifted from an NSC population toward later within the NPC lineage. Both brain and CSF cells showed relatively equal permissivity to ZIKV viral infection (Fig.1). Moreover, there was no difference in Vimentin gene expression in ZIKV infected cells, while SOX-2 and Nestin gene expression following ZIKV infection was significantly lower (p<0.05; Fig.2). The findings that Nestin expression is downregulated when comparing brain and CSF cells, particularly after ZIKA infection, is of interest since Nestin is postulated to play a role in axonal growth. This study demonstrated the ability to utilize porcine fetal NPCs in a safe and effective manner for the study of NPC development and effects of viral infections

MicroRNA-193a-5p Suppresses the Migratory Ability of Human KATO III Gastric Cancer Cells through Inhibition of Vimentin and MMP-9.

Purpose: microRNA-193a-5p is one of the well-known tumor suppressor miRNAs in the body but in many cases, its expression became reduced in patients suffering from gastric cancer (GC). The main purpose of this study was to restore the function of this miRNA in human GC cells and investigating the effects of enhanced expression of miR-193a-5p on proliferation, apoptosis, and migration of GC cells upon in vitro transfection. Methods: The KATO III GC cells were treated with 100 nM of miR-193a-5p or negative control sequences. Following that, the MTT assay, flow cytometry assay, and wound-healing assay were applied to estimate the impacts of enhanced expression of this miRNA on the viability, apoptosis, and migration rate of the cells, respectively. Moreover, the total RNA was isolated and alterations in the mRNA expression ratio of migratory genes were measured by qRT-PCR techniques. Results: The findings designated that enhanced expression of miR-193a-5p suppressed the migratory ability of the cells, but had no significant effects on cell survival or apoptosis of the transfected cells. In addition, this inhibitory function of miR-193a-5p on the migration rate of the KATO III cell line occurs with concurrent suppression of vimentin and MMP-9 gene expression. Conclusion: It can be concluded that miR-193a-5p negatively influences the migratory ability of the cancerous cells and restoring its effects can be regarded as a promising target of future therapeutic interventions, especially for GC metastasis.

CTNNB1 AND VIMENTIN SWITCH EXPRESSION IN ORAL SQUAMOUS CELL CARCINOMA CELL LINES UNDER CHOLESTEROL DEPLETION

Objective: Evaluating the expression of epithelial marker β-catenin (CTNNB1) and the mesenchymal marker vimentin (VIM) in the oral squamous cell carcinoma (OSCC) cell line SCC-9) under caveolae disruption through different concentrations of Methyl-β-cyclodextrin (MβCD) substance. Study Design: The OSCC cell line SCC-9 was treated with MβCD at different concentrations (7.5 mM, 10 mM, and 15 mM) for 1 hour. After 24 hours of depletion, the gene expression of VIM and CTNNB1 was analyzed by quantitative reverse transcription polymerase chain reaction (RT-qPCR), comparing to untreated SCC-9 cells. Results: Negative expression of VIM was observed at the 10 mM and 15 mM concentrations (mean 0.89 and 0.67, respectively). Gene expression at 15 mM was significantly lower compared with 7.5 mM (mean, 1.39; P value, .029; 1-way analysis of variance [ANOVA]). On the other hand, CTNNB1 was positively expressed by SCC-9 cells after cholesterol depletion with MβCD at all concentrations of 7.5 mM, 10 mM, and 15 mM (mean 3.23, 2.66, and 1.78, respectively), showing a decrease in expression in a dose-dependent manner (P value, .277; 1-way ANOVA).Conclusion: The inversion of expression between the epithelial (CTNNB1) and mesenchymal (VIM) marker after cholesterol depletion by MβCD may reflect biological changes that correspond to the reversal of the epithelial- mesenchymal transition process in OSCC. Objective: Evaluating the expression of epithelial marker β-catenin (CTNNB1) and the mesenchymal marker vimentin (VIM) in the oral squamous cell carcinoma (OSCC) cell line SCC-9) under caveolae disruption through different concentrations of Methyl-β-cyclodextrin (MβCD) substance. Study Design: The OSCC cell line SCC-9 was treated with MβCD at different concentrations (7.5 mM, 10 mM, and 15 mM) for 1 hour. After 24 hours of depletion, the gene expression of VIM and CTNNB1 was analyzed by quantitative reverse transcription polymerase chain reaction (RT-qPCR), comparing to untreated SCC-9 cells. Results: Negative expression of VIM was observed at the 10 mM and 15 mM concentrations (mean 0.89 and 0.67, respectively). Gene expression at 15 mM was significantly lower compared with 7.5 mM (mean, 1.39; P value, .029; 1-way analysis of variance [ANOVA]). On the other hand, CTNNB1 was positively expressed by SCC-9 cells after cholesterol depletion with MβCD at all concentrations of 7.5 mM, 10 mM, and 15 mM (mean 3.23, 2.66, and 1.78, respectively), showing a decrease in expression in a dose-dependent manner (P value, .277; 1-way ANOVA).Conclusion: The inversion of expression between the epithelial (CTNNB1) and mesenchymal (VIM) marker after cholesterol depletion by MβCD may reflect biological changes that correspond to the reversal of the epithelial- mesenchymal transition process in OSCC.

P1142DICHLOROACETATE AND L-CARNITINE REDUCED TGF-BETA-INDUCED MESOTHELIAL-TO-MESENCHYMAL TRANSITION

Abstract Background and Aims Mesothelial to Mesenchymal Transition (MMT) plays a main role in the induction of peritoneal fibrosis in long term peritoneal dialysis. Mesothelial cells exposed to high glucose produce great amount of TGF-beta that plays a pivotal role in MMT. Recent findings suggest that TGF-beta controls mesothelial fibrosis via the activation of hyperglycolysis, thus we suppose that coupling glycolysis with Krebs cycle may improve mesothelial cells tolerance to TGF-beta. Method To test this hypothesis, we assessed the effects of Dichloroacetate (DCA), a pyruvate dehydrogenase kinase inhibitor, and L-carnitine (which sustains the production of Acetyl-CoA in multiple ways) on mesothelial cells treated with TGF-beta. Recent findings, in fact, highlight the potential anti-fibrotic effects of DCA and L-carnitine (through energy metabolic balancing) in different organs. Particularly, human peritoneal mesothelial cells (HMrSV5) were cultured in Dulbecco's Modified Eagle Medium (DMEM; Gibco) containing 10% fetal bovine serum. Then, cells, cultured to confluence, were pre-treated for one hour with or without 10mM DCA, 1 or 5 mM L-Carnitine and then with 10ng/ml TGF-beta for 4 hours. Gene expression of alpha-SMA and vimentin (markers of MMT) and IL1-beta and IL-6 (inflammatory markers) was evaluated by real-time PCR. Results Our results revealed that TGF-beta significantly increased the gene expression level of alpha-SMA (2.04±0.3, p&amp;lt;0.05 versus control) and vimentin (1.61±0.23, p&amp;lt;0.05 versus control) as well as the expression level of the pro-inflammatory cytokines IL1-beta (3.44±0.87, p&amp;lt;0.05 versus control) and IL-6 (2.56±0.51, p&amp;lt;0.05 versus control). Pre-treatment with DCA and L-carnitine (at 1 or 5 mM) significantly reduced these effects. In detail, pre-treatment with DCA reduced alpha-SMA expression (0.97±0.24, p&amp;lt;0.05), vimentin (1.03±0.1, p&amp;lt;0.05), IL1-beta (1.47±0.16, p&amp;lt;0.05) and IL-6 (0.88±0.28, p&amp;lt;0.05) respect to TGF-beta treated cells. Pre-treatment with 1 and 5 mM L-carnitine reduced respectively alpha-SMA expression at 0.96±0.09 (p&amp;lt;0.05) and 0.98±0.06 (p&amp;lt;0.05), vimentin 1.01±0.12 (p&amp;lt;0.05) and 1.02±0.1 (p&amp;lt;0.34), IL1-beta 1.02±0.15 (p&amp;lt;0.05) and 1.68±0.33 (p&amp;lt;0.05), and IL-6 0.72±0.04 (p&amp;lt;0.05) and 0.85±0.01 (p&amp;lt;0.05) respect to TGF-beta treated cells. Conclusion These preliminary results suggest, for the first time, that the use of both Dichloroacetate and L-carnitine, coupling glycolysis with Krebs cycle and oxidative phosphorylation, could restore physiological energy metabolism in mesothelial cells and control fibrosis.

Myostatin in the Arterial Wall of Patients with End-Stage Renal Disease.

Aim: Myostatin (Mstn) has been described as a trigger for the progression of atherosclerosis. In this study, we evaluated the role of Mstn in arterial remodeling in patients with end-stage renal disease (ESRD).Methods: Vascular specimens were collected from 16 ESRD patients (56.4 ± 7.9 years) undergoing renal transplant (recipients) and 15 deceased kidney non-uremic donors (55.4 ± 12.1 years). We studied gene and protein expression of Mstn, ubiquitin ligases, Atrogin-1, and muscle ring finger protein-1 (MuRF-1), inflammatory marker CCL2, cytoskeleton components, and Klotho by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. Moreover, we assessed vascular calcification and collagen deposition. Finally, we studied the effects of recombinant Mstn on rat vascular smooth muscle cells (VSMCs, A7r5) and evaluated the effects of uremic serum (US) on primary human VSMCs.Results: Myostatin mRNA was upregulated in the arterial vascular wall of recipients compared with donors (∼15-folds, p < 0.05). This response was accompanied by the upregulation of gene expression of Atrogin-1 and MuRF-1 (+2.5- and +10-fold) and CCL2 (+3-fold). Conversely, we found downregulation of protein expression of Smoothelin, α-smooth muscle actin (α-SMA), vimentin, and Klotho (−85%, −50%, −70%, and −80%, respectively; p < 0.05) and gene expression of vimentin and Klotho. Exposition of A7r5 to Mstn induced a time-dependent SMAD 2/SMAD 3 phosphorylation and expression of collagen-1 and transforming growth factor β (TGFβ) mRNA, while US induced overexpression of Mstn and Atrogin-1 and downregulation of Smoothelin and Klotho.Conclusions: Our data suggest that uremia might induce vascular Mstn gene expression together with a complex pathway of molecular and structural changes in the vascular wall. Myostatin, in turn, can translate the metabolic alterations of uremia into profibrotic and stiffness inducing signals.

Distribution and Expression of Vimentin and Desmin in Broiler Pectoralis major Affected by the Growth-Related Muscular Abnormalities.

Desmin (DES) and Vimentin (VIM) exert an essential role in maintaining muscle cytoarchitecture and since are considered reliable markers for muscle regeneration, their expression has been extensively investigated in dystrophic muscles. Thus, exhibiting features similar to those of human dystrophic muscles, the present study aimed at assessing the distribution of VIM and DES proteins and the expression of the corresponding genes in Pectoralis major muscles affected by white striping (WS), wooden breast (WB), and spaghetti meat (SM) abnormalities as well as in those having macroscopically normal appearance (NORM). For this purpose, 20 Pectoralis major muscles (5/group) were collected from the same flock of fast-growing broilers to perform immunohistochemistry, immunoblotting and gene expression. Immunohistochemical analyses showed an increased number of fibers immunoreactive to both VIM and DES in WS and WB, while only a few immunoreactive fibers were observed in NORM. Concerning the protein level, if compared with NORM, a 55% increase in VIM content was found in WB affected cases (P < 0.05) thus suggesting the development of intense regenerative processes in an early-stage within these muscles. The significantly higher amount of DES (+53%) found in WS might be attributed to a progression of the regenerative processes that require its synthesis to preserve the structural organization of the developing fibers. On the other hand, significantly lower VIM and DES contents were found in SM. About gene expression, VIM mRNA levels gradually increased from the NORM to the SM group, with significantly higher gene expressions in WB and SM samples compared to the NORM group (P = 0.009 for WB vs. NORM and P = 0.004 for SM vs. NORM). Similarly, the expression of DES gene showed an increase from the NORM to WB group (P = 0.05). Overall, the findings of the present study suggest that intense regenerative processes take place in both WB and WS muscles although a different progression of regeneration might be hypothesized. On the other hand, the lack of correspondence between VIM gene expression and its protein product observed in SM suggests that VIM may also exert a role in the development of the SM phenotype.

Efficacy of erythropoietin-pretreated mesenchymal stem cells in murine burn wound healing: possible in vivo transdifferentiation into keratinocytes.

Stem cells have shown promising potential to treat burn wounds. Erythropoietin was capable of promoting in vitro transdifferentiation of mesenchymal stem cells (MSCs). The aim of the study was to investigate possible role of erythropoietin-pretreated mesenchymal stem cells (EPOa/MSCs) in burn wounds healing and to evaluate its in vivo differentiation into keratinocytes. Forty rats were utilised in this study divided into four groups (n = 10 for each). Control group (I), burn group (II), burn + MSCs, group (III), burn + EPOa/MSCs. 1 × 10⁶ cells were injected locally for each 1 cm² of burn areas. Burn areas were followed-up morphologically. After 21 days of the experiment, the rats were euthanised, skin specimens were assessed biochemically, histologically and immunohistochemically. EPOa/MSCs enhanced significantly (p < 0.05) burn wound vimentin gene expression and level of interleukin (IL)-10 while decreased IL-1 and COX2 as compared to the burn group. Histologically, EPOa/MSCs improved epithelialisation despite stem cells' differentiation into keratinocytes was rarely detected by PKH26 red fluorescence. EPOa/MSCs promoted angiogenesis as detected by significant increase in VEGF and PDGF immunoexpression as compared to burn group. EPOa/MSCs may improve burn wound healing, probably through anti-inflammatory, immunomodulatory and angiogenic action. However, in vivo transdifferentiation into keratinocytes was rarely detected.

Long Noncoding RNA VIM Antisense RNA 1 (VIM-AS1) Plays an Important Role in Development of Preeclampsia by Regulation of Epithelial Mesenchymal Transition.

BackgroundLong noncoding RNAs play important roles in the development of various diseases. This study aimed to evaluate the effects and mechanism of VIM antisense RNA 1 (VIM-AS1) in the development of preeclampsia.Material/MethodsHTR-8/SVneo cells were divided into normal control (NC), Model, Blank, and VIM-AS1 groups. These groups were analyzed for their VIM-AS1 gene expressions by RT-PCR, HTR-8/SVneo cell invasion was assessed by transwell and migration by wound healing, cell morphology was assessed by microscopy examination, and E-cadherin, Snail, and Vimentin genes expressions were assessed by RT-PCR and WB assay.ResultsVIM-AS1 gene expression was significantly different among normal placenta tissue, mild preeclampsia tissues, and severe preeclampsia tissues (P<0.001 or P<0.01). VIM-AS1 gene expressions, cell invasions, and wound healing rates in the Model and Blank groups were significantly suppressed compared with that of NC group (P<0.001, all). With VIM-AS1 supplementation, VIM-AS1 gene expression, cell invasion, and wound healing rate in the VIM-AS1 group were significantly increased compared with that in the Model group (P<0.001). RT-PCR and WB assay showed that E-cadherin gene and protein expressions in Model and Blank groups were significantly upregulated compared with the NC group (P<0.001); Snail and Vimentin gene and protein expressions in the Model and Blank groups were significantly downregulated compared with the NC group (P<0.001). With VIM-AS1 supplementation, E-cadherin, Snail, and Vimentin gene and proteins expression levels in the VIM-AS1 group were significantly different compared with that in the Model group (P<0.001).ConclusionsVIM-AS1 promotes preeclampsia via inducing epithelial-to-mesenchymal transition (EMT).

Vimentin as a potential therapeutic target in sorafenib resistant HepG2, a HCC model cell line.

Background/AimsHepatocellular carcinoma (HCC) is the most common liver cancer with high mortality rate in patients suffering from liver diseases. The drug of choice used in advanced-stage of HCC is sorafenib. However, adaptive resistance has been observed in HCC patients undergoing long-term sorafenib treatment, lowering its effectiveness. Hence, it is important to overcome drug resistance to improve overall management of HCC. Here, we have identified a candidate biomarker for sorafenib resistance in a HCC model cell line, HepG2. MethodsInitially, comparative proteomic profiling of parental HepG2 [HepG2 (P)] and sorafenib-resistant HepG2 [HepG2 (R)] cells was performed via MALDI (matrix-assisted laser desorption/ionization) which revealed the deregulation of vimentin in HepG2 (R) cells. Gene and protein level expression of vimentin was also observed through quantitative real-time polymerase chain reaction (qRT PCR) and fluorescence-activated cell sorting (FACS), respectively. Furthermore, withaferin A was used to study regulation of vimentin expression and its significance in sorafenib resistance. ResultsBoth gene and protein level of vimentin expression was found to be downregulated in HepG2 (R) in comparison to HepG2 (P). Interestingly, the study demonstrated that withaferin A further lowered the expression of vimentin in HepG2 (R) cells in a dose-dependent manner. Also, inhibition of vimentin lowered ABCG2 expression and decreased cell viability in parental as well as sorafenib resistant HepG2 cells. ConclusionsHence, our study for the first time highlighted the probable therapeutic potential of vimentin in sorafenib resistant HepG2, a HCC model cell line.

Novel Role for miR-1290 in Host Species Specificity of Influenza A Virus

The role of microRNA (miRNA) in influenza A virus (IAV) host species specificity is not well understood as yet. Here, we show that a host miRNA, miR-1290, is induced through the extracellular signal-regulated kinase (ERK) pathway upon IAV infection and is associated with increased viral titers in human cells and ferret animal models. miR-1290 was observed to target and reduce expression of the host vimentin gene. Vimentin binds with the PB2 subunit of influenza A virus ribonucleoprotein (vRNP), and knockdown of vimentin expression significantly increased vRNP nuclear retention and viral polymerase activity. Interestingly, miR-1290 was not detected in either chicken cells or mouse animal models, and the 3′ UTR of the chicken vimentin gene contains no binding site for miR-1290. These findings point to a host species-specific mechanism by which IAV upregulates miR-1290 to disrupt vimentin expression and retain vRNP in the nucleus, thereby enhancing viral polymerase activity and viral replication.

Twist1 Correlates With Epithelial-Mesenchymal Transition Markers Fibronectin and Vimentin in Adrenocortical Tumors.

Although the knowledge regarding adrenocortical carcinomas (ACC) tumorigenesis has significantly improved during the last decade, it still remains to be completely determined. Epithelial-mesenchymal transition (EMT) is a well described transcription factor induced process, postulated as an essential step toward cancer progression and metastasis development. In this context, Twist1 has been described as the EMT master-regulator. The aim of this study was to assess the association among Twist1, fibronectin, vimentin and E-cadherin gene expression in adrenocortical tumor samples. Twist1, fibronectin, vimentin and E-cadherin gene expression in 18 adrenal adenomas, 18 ACC, and 24 childhood onset adrenocortical tumors were assessed in formalin-fixed paraffin-embedded tissues. The fold expression was calculated according to the 2ΔCt method. A significant correlation between mRNA levels of Twist1, fibronectin and vimentin was evident. Although their expression was inversely proportional, no association was observed between Twist1 and E-cadherin expression. The expression of Twist1, the major regulator of EMT, is directly correlated to the expression of mesenchymal markers fibronectin and vimentin in ACC samples.

An Optically Induced Dielectrophoresis (ODEP)-Based Microfluidic System for the Isolation of High-Purity CD45neg/EpCAMneg Cells from the Blood Samples of Cancer Patients-Demonstration and Initial Exploration of the Clinical Significance of These Cells.

Circulating tumour cells (CTCs) in blood circulation play an important role in cancer metastasis. CTCs are generally defined as the cells in circulating blood expressing the surface antigen EpCAM (epithelial cell adhesion molecule). Nevertheless, CTCs with a highly metastatic nature might undergo an epithelial-to-mesenchymal transition (EMT), after which their EpCAM expression is downregulated. In current CTC-related studies, however, these clinically important CTCs with high relevance to cancer metastasis could be missed due to the use of the conventional CTC isolation methodologies. To precisely explore the clinical significance of these cells (i.e., CD45neg/EpCAMneg cells), the high-purity isolation of these cells from blood samples is required. To achieve this isolation, the integration of fluorescence microscopic imaging and optically induced dielectrophoresis (ODEP)-based cell manipulation in a microfluidic system was proposed. In this study, an ODEP microfluidic system was developed. The optimal ODEP operating conditions and the performance of live CD45neg/EpCAMneg cell isolation were evaluated. The results demonstrated that the proposed system was capable of isolating live CD45neg/EpCAMneg cells with a purity as high as 100%, which is greater than the purity attainable using the existing techniques for similar tasks. As a demonstration case, the cancer-related gene expression of CD45neg/EpCAMneg cells isolated from the blood samples of healthy donors and cancer patients was successfully compared. The initial results indicate that the CD45neg/EpCAMneg nucleated cell population in the blood samples of cancer patients might contain cancer-related cells, particularly EMT-transformed CTCs, as suggested by the high detection rate of vimentin gene expression. Overall, this study presents an ODEP microfluidic system capable of simply and effectively isolating a specific, rare cell species from a cell mixture.

Pomegranate peel extract inhibits expression of β-catenin, epithelial mesenchymal transition, and metastasis in triple negative breast cancer cells

The standard treatment for triple-negative breast cancer (TNBC) is chemotherapy, which is highly toxic to patients; thereby, there is a need to identify safer and more effective therapeutic approaches. Medicinal plants constitute a common alternative for cancer treatment. Pomegranate is a well-known fruit in this context, but its antimetastatic property has not been extensively studied. As breast cancer-related deaths from TNBC are mainly due to metastasis, the present study was designed to investigate the antimigratory effect of pomegranate peel extract (PPE) on TNBC cells. For this purpose, the MDA-MB-231 cells were treated with different concentrations of PPE for 24, 48 and 72 hr. The effects of PPE on cell migration and invasion were determined by wound healing and transwell assays. To address the possible molecular mechanisms underlying the antimetastatic effect of PPE, real-time quantitative PCR analysis of selected epithelial mesenchymal transition (EMT) markers were performed. Moreover, the expression of β-catenin as a critical factor in promoting cancer metastasis was examined. PPE markedly inhibited the migration and invasion of cells at concentrations of 25, 50, 100, 250, 500, and 1000µg/ml. At relatively high concentrations (500, 1000µg/ml), PPE induced apoptosis. Moreover, PPE decreased the gene expression of vimentin, ZEB1, and β-catenin and also increased the expression of E-cadherin in TNBC cells. The protein level of β-catenin, as measured using western analysis, revealed a time-dependent decrease at the concentration of 1000µg/ml PPE. Downregulation of EMT markers and β-catenin showed accordance with the inhibition of migration and invasion. The present data show that PPE could be a promising drug candidate to reduce metastasis in TNBC cells.

Zinc increases nestin but not vimentin gene expression in mouse mesenchymal stem cells

Zinc increases nestin but not vimentin gene expression in mouse mesenchymal stem cells

Inhibition of Histone Deacetylases Reverses Epithelial-Mesenchymal Transition in Triple-Negative Breast Cancer Cells through a Slug Mediated Mechanism

High metastatic ability and poor clinical outcome are the most known clinical features of the triple-negative breast tumors. Given that the tumor cells undergoing epithelial-mesenchymal transition (EMT) often gain malignant and invasive features, we have investigated the possibility of EMT reversal in triple-negative breast cancer cells by targeting the epigenetic-modifying enzymatic complexes named histone deacetylases (HDACs) and examined the possible mechanism underlying the HDACs-based inversion in model MDA-MB-231 cells. Cells were treated with a maximal tolerable 200 nM concentrations of classical HDACs inhibitor Trichostatin A (TSA) for 48 h and afterwards the invasiveness and immigration of the cells were evaluated in TransWell Invasion Scratch Wound Healing assays. Then, in treated and control cells, quantitative real time-PCR reacions were performed for assessing the gene expression of EMT biomarkers E-cadherin, Vimentin and transcriptional factor Slug. After TSA treatment, the invasion and migration properties MDA-MB-231 cells significantly decreased, gene expression of E-cadherin was significantly up-regulated, while the levels of Slug and Vimentin encoding mRNAs were suppressed. We conclude that inhibition of HDACs in triple-negative breast cancer cells may lead to inversion of EMT and the decrease of invasiveness by down-regulating the gene expression of Slug. Since EMT is known as a pre-metastatic process, triple-negative breast tumors, the EMT reversal effects of HDACs inhibition may reduce tumor cell metastasis.

Development and characterization of 2-dimensional culture for buffalo intestinal cells.

Small intestinal epithelial cells (IEC) play a major role in the absorption of nutrients and toxins. Due to the similarity of genome-wide single copy protein orthologues between cattle and human, establishment of ruminant's primary small IEC culture could be a valuable tool for toxicity studies. Therefore, the current study focused on the development and characterization of buffalo IEC culture, as cattle slaughter is banned in India. The buffalo jejunum fragments were washed consecutively several times in saline, warm phosphate buffered saline (PBS), PBS with 5mM dithiothreitol, digesting solution and 2% sorbitol in PBS. The cells were cultured on 17µg/cm2 collagen coated plates and transwell plates with serum (2% Fetal bovine serum (FBS) and 10% FBS) and serum-free culture conditions. The cells were differentiated into typical epithelial cobblestone morphology from day 5 onwards in 50% successful cultures. The cultured IEC were characterized by gene expression of epithelial cell markers, cytokeratin and vimentin, and enterocyte markers like villin, zonula occluden (ZO1), fatty acid binding protein 2 (FABP2) and small intestinal peptidase (IP). Based on the morphology and gene expression profile, 10% FBS has been recommended for culturing primary buffalo IEC on collagen coated plates for 10days. However, 50% of the successful cultures could not show epithelial phenotype on 10% FBS culture conditions even on collagen coated plates. Interestingly, undifferentiated IEC showed an increasing expression of FABP2, IP and ZO1 transcripts compared to differentiated intestinal cells with 10% FBS on collagen plates. Therefore, future studies are needed to understand the role of FABP2, IP and ZO1 in differentiation of buffalo IEC.

LKB1 regulates epithelial-mesenchymal transition in Peutz-Jeghers hamartoma and intestinal epithelial cells

To investigate the molecular mechanism by which LKB1 regulates epithelial-mesenchymal transition (EMT) in Peutz-Jeghers hamartoma and intestinal epithelial cells. Immunohistochemistry was used to detect gene expression of LKB1, E-cadherin, and vimentin in 20 hamartoma tissues and 10 normal intestinal tissues, and collagen fiber deposition was analyzed using Masson trichrome staining. Normal intestinal epithelial NCM460 cells were transfected with LKB1 shRNA plasmid or negative control via lentiviral vectors, and the role of LKB1 in cell polarization and migration were determined using CCK8 and Transwell assays. Western blotting, quantitative real-time PCR (qPCR) and immunofluorescence were used to assess the alterations of EMT markers in the cells with LKB1 knockdown. Compared with normal intestinal tissues, hamartoma polyps showed significantly decreased LKB1 and E-cadherin expressions and increased vimentin expression with increased collagen fiber deposition. The cells with LKB1 knockdown exhibited enhanced cell proliferation and migration activities (P<0.01). Western blot analysis, qPCR and immunofluorescence all detected decreased E-cadherin and increased N-cadherin, vimentin, Snail, and Slug expressions in the cells with LKB1 knockdown. s LKB1 deficiency triggers EMT in intestinal epithelial cells and Peutz-Jeghers hamartoma, suggesting that EMT can serve as the therapeutic target for treatment of Peutz-Jeghers syndrome.

The association of vimentin and fibronectin gene expression with epithelial-mesenchymal transition and tumor malignancy in colorectal carcinoma.

Colorectal cancer is the most common malignancy of the gastrointestinal tract with very high mortality. One of the most distinguishing features for the establishment of an epithelial-mesenchymal transition phenotype is the alteration of mesenchymal markers expression and structural adhesion proteins. We evaluated the significance of vimentin and fibronectin gene expression in relation to invasion and metastasis in CRC patients. Tissue specimens were collected consecutively from forty-five colorectal carcinoma patients during surgeries. Tissues were divided into two separate parts for pathological and molecular assays. In order to histological staging, tissue sections were prepared from formalin-fixed paraffin-embedded blocks and stained with Hematoxylin and Eosin. To quantify gene expression, specimens were dissected and homogenized. Moreover, SW480, SW48, SW948, Caco-2, HT-29 and LS174T as human colon cancer cell lines were obtained and cultured, then molecular analyzing was performed. As results the expression of VIM gene increased in SW480, SW48 and SW948 while it decreased in Caco-2, HT-29 and LS174T. Moreover, FN was up-regulated in Caco-2, HT-29 and SW948, while it was down-regulated in SW480, SW48 and LS174T. In tissues, vimentin and fibronectin expression significantly increased in stromal cells, whereas vimentin decreased in colonic epithelial cells and fibronectin had no significant change. Vimentin and fibronectin expression were changed in tumor tissues. It was found an association between vimentin expression with age and tumor size; over-expression in older age and decreasing in larger tumor size. Furthermore, fibronectin over-expression is correlated to older age and high tumor stages; up-regulation with increasing age and high tumor stages.

Can mesenchymal stem cells pretreated with platelet-rich plasma modulate tissue remodeling in a rat with burned skin?

Our aim was to study the effect of platelet-rich plasma (PRP) on the proliferation of bone-marrow-derived mesenchymal stem cells (BM-MSCs) and to investigate their roles in the healing of experimental burn injury and the possible mechanism of action. Our work was divided into in-vitro and in-vivo studies. The in-vitro study included untreated MSCs and MSCs treated with PRP. Levels of TGF-β and cell proliferation were assessed. In the in-vivo study, 72 rats were distributed equally among 6 groups: control, burn, burn with MSCs, burn with PRP, burn with both MSCs and PRP, and burn with MSCs pretreated with PRP. On the 7th and 20th day after injury, the serum levels of transforming growth factor beta (TGF-β) and tumor necrosis factor alpha (TNF-α), as well as interleukin-10 (IL-10) levels in skin tissue were measured by ELISA; histopathology and gene expression of MMP-1, TIMP-2, Ang-1, Ang-2, and vimentin by real-time PCR were performed in all groups. In vitro: proliferation of MSCs and TGF-β increased in the PRP-treated group compared with the control group. In vivo: Ang-1, Ang-2, and vimentin were upregulated, whereas MMP-1 and TIMP-2 were downregulated. TGF-β and IL-10 were increased, whereas TNF-α was decreased in all treated groups with more significance in MSCs and PRP on day 20. Histopathology of burn skin was improved in all treated groups, particularly in MSCs pretreated with PRP 20 days post-burn.

Spheroid formation and modulation of tenocyte-specific gene expression under simulated microgravity.

For tendon tissue engineering, tenocyte-seeded scaffolds are a promising approach. Under conventional 2D culture however, tenocytes show rapid senescene and phenotype loss. We hypothesized that phenotype loss could be counteracted by simulated microgravity conditions. Human tenocytes were exposed to microgravity for 9 days on a Random Positioning Machine (RPM). Formation of 3D-structures (spheroids) was observed under light microscopy, gene expression was measured by real-time PCR. Cells under conventional 2D-culture served as control group. Simulated microgravity reached a value of as low as 0.003g. Spheroid formation was observed after 4 days, and spheroids showed stable existance to the end of the observation period. After 9 days, spheroids showed a significantly higher gene expression of collagen 1 (Col1A1) compared to adherent cells under microgravity (4.4x, p=0.04) and compared to the control group (5.6x, p=0.02). Gene expression of collagen 3 (COL3A1) was significantly increased in spheroids compared to the control group (2.3x, p=0.03). Gene expressions of the extracellular matrix genes Tenascin C und Fibronectin (TNC and FN) were increased in adherent cells under microgravity compared to the 1g-control group, not reaching statistical significance (p=0.1 and p=0.3). For the gene expression of vimentin, no significant alteration was observed both in the adherent cells and in the spheroids compared to the 1g control group. Gene expression of the tenocyte-specific transcription factor scleraxis (SCX) was significantly increased in spheroids compared to the control group (3.7x, p=0.03). Simulated microgravity could counteract tenocyte senescence in vitro and serve as a promising model for scaffold-free 3D cell culturing and tissue engineering. V (laboratory study).