Roles of cell adhesion molecules nectin and nectin-like molecule-5 in the regulation of cell movement and proliferation

In response to chemoattractants, migrating cells form protrusions, such as lamellipodia and filopodia, and structures, such as ruffles over lamellipodia, focal complexes and focal adhesions at leading edges. The formation of these leading edge structures is essential for directional cell movement. Nectin-like molecule-5 (Necl-5) interacts in cis with PDGF receptor and integrin alpha(v)beta(3), and enhances the activation of signalling molecules associated with these transmembrane proteins, which results in the formation of leading edge structures and enhancement of directional cell movement. When migrating cells come into contact with each other, cell-cell adhesion is initiated, resulting in reduced cell velocity. Necl-5 first interacts in trans with nectin-3. This interaction is transient and induces down-regulation of Necl-5 expression at the cell surface, resulting in reduced cell movement. Cell proliferation is also suppressed by the down-regulation of Necl-5, because the inhibitory effect of Necl-5 on Sprouty2, a negative regulator of the Ras signalling, is diminished. PDGF receptor and integrin alpha(v)beta(3), which have interacted with Necl-5, then form a complex with nectin, which initiates cell-cell adhesion and recruits cadherin to the nectin-based cell-cell adhesion sites to form stable adherens junctions. The formation of adherens junctions stops cell movement, in part through inactivation of integrin alpha(v)beta(3) caused by the trans-interaction of nectin. Thus, nectin and Necl-5 play key roles in the regulation of cell movement and proliferation.     

Effect of microRNA-143-3p-mediated CTNND1 on the biological function of lung cancer cells

Lung cancer poses a serious threat to human life with high incidence and miRNA is an important biomarkerin tumors. This study aimed to explore the effect of miR-143-3p on the biological function of lung cancer cells and theunderlying mechanism. Eighty-seven samples of lung cancer tissues and 81 samples of tumor-adjacent tissues frompatients undergoing radical lung cancer surgery in our hospital were collected. The lung cancer cells and lung fibroblastcells (HFL-1) were purchased, and then miR-143-3p-mimics, miR-NC, si-CTNND1, and NC were transfected intoA549 and PC-9 cells to establish cell models. MiR-143-3p and CTNND1 expression levels were measured by the qRTPCR,Bax, Bcl-2, and CTNND1 expression levels by the Western Blot (WB), and cell proliferation, invasion, andapoptosis by the MTT assay, Transwell assay, and flow cytometry. Dual luciferase report assay was used to determinethe relationship between miR-143-3p and CTNND1. In this study, miR-143-3p was lowly expressed in lung cancerand CTNND1 was highly expressed in lung cancer. The overexpression of miR-143-3p inhibited cell proliferationand invasion, promoted cell apoptosis, significantly increased Bax protein expression, and decreased Bcl-2 proteinexpression. The inhibition of CTNND1 led to opposite biological characteristic in cells. The dual luciferase reporterassay demonstrated that miR-143-3p was a target region of CTNND1. Such results suggest that miR-143-3p can inhibitthe proliferation and invasion of lung cancer cells by regulating the expression of CTNND1 and promote the apoptosisof lung cancer cells, sott is expected to be a potential target for lung cancer.     

牛乳腺上皮细胞体外培养体系研究进展

通过有效的培养方法,可实现牛乳腺上皮细胞的体外培养,并且所获细胞具有正常的生理特性及功能,从而建立牛乳腺上皮细胞的体外培养体系。本文论述了国内外近年来此领域的研究进展,综合比较了各种培养体系的建立方法,并对在培养体系建立的基础上进行的相关应用研究进行了总结。     

Scanning electron microscopy with an ionic liquid reveals the loss of mitotic protrusions of cells during the epithelial-mesenchymal transition

Epithelial-mesenchymal transition (EMT) is a key event in cancer metastasis and is characterized by increase in cell motility, increase in expression of mesenchymal cell markers, loss of proteins from cell-to-cell junction complexes, and changes in cell morphology. Here, the morphological effects of a representative EMT inducer, transforming growth factor (TGF)-β1, were investigated in human lung adenocarcinoma (A549) cells and pancreatic carcinoma (Panc-1) cells. TGF-β1 caused morphological changes characteristic of EMT, and immunostaining showed loss of E-cadherin from cell-to-cell junction complexes in addition to the upregulation of the mesenchymal marker vimentin. During scanning electron microscopy (SEM) with an ionic liquid, we observed EMT-specific morphological changes, including the formation of various cell protrusions. Interestingly, filopodia in mitotic cells were clearly observed by SEM, and the number of these filopodia in TFG-β1-treated mitotic cells was reduced significantly. We conclude that this reduction in such mitotic protrusions is a novel effect of TGF-β1 and may contribute to EMT.     

AKT regulates IL-1β-induced proliferation and activation of hepatic stellate cells

Background: Activated hepatic stellate cells (HSCs) are closely involved in the initiation, perpetuation, andresolution of liver fibrosis. Pro-inflammatory cytokine levels are positively correlated with the transition from liverinjury to fibrogenesis and contribute to HSC pathophysiology in liver fibrosis. Methods: In this study, we investigatedthe effect of the pro-inflammatory cytokine interleukin (IL)-1β on the proliferation and signaling pathways involvedin fibrogenesis in LX-2 cells, an HSC cell line, using western blotting and cell proliferation assays. Results: IL-1βincreased the proliferation rate and α-smooth muscle actin (SMA) expression of LX-2 cells in a dose-dependentmanner. Within 1 h after IL-1β treatment, c-Jun N-terminal kinase (JNK), p38, and nuclear factor-κB (NF-κB)signaling was activated in LX-2 cells. Subsequently, protein kinase B (AKT) phosphorylation and an increase in α-SMA expression were observed in LX-2 cells. Each inhibitor of JNK, p38, or NF-κB decreased cell proliferation, AKTphosphorylation, and α-SMA expression in IL-1β-treated LX-2 cells. Conclusion: These results indicate that JNK,p38, and NF-κB signals converge at AKT phosphorylation, leading to LX-2 activation by IL-1β. Therefore, the AKTsignaling pathway can be used as a target for alleviating liver fibrosis by the inflammatory cytokine IL-1β.     

Knockdown of lncRNA XIST prevents the epithelial-mesenchymal transition of TGF-β2-induced human lens epithelial cells via miR-124/Slug axis

Posterior capsular opacification (PCO) is linked to the pathological process of lens epithelial cells, which includes proliferation, migration, and epithelial-mesenchymal transition (EMT). Our goal was to investigate whether long noncoding RNA (lncRNA) XIST contributes to EMT via targeting miR-124/Slug axis in TGF-β2-induced SRA01/04 cells. EMT was induced by stimulating SRA01/04 cells with 10 ng/mL TGF-β2 for 24 h, and PCO microenvironment was simulated. The expressions levels of lncRNA XIST, miR-124, and Slug were measured by real-time polymerase chain reaction (RT-PCR) and western blot. The role and mechanism of lncRNA XIST in promoting EMT of TGF-β2-treated SRA01/04 cells were investigated by using cell transfection, cell counting kit-8 (CCK-8), immunofluorescence staining, transwell assay, wound-healing assay, RT-PCR, western blot and dual-luciferase reporter assay. The expression of Slug and lncRNA XIST was markedly increased, while miR-124 was downregulated in TGF-β2-treated SRA01/04 cells compared with the control group. Knockdown of lncRNA XIST reduced EMT, migration, and cell viability in TGF-β2-induced SRA01/04 cells; moreover, it adversely modulated miR-124 and adjusted the expression of Slug in SRA01/04 cells, while these effects were diminished by co-transfection with AMO-miR-124. Our data demonstrated that lncRNA XIST functioned as a competitive endogenous RNA (ceRNA) of miR-124 to modulate the expression level of Slug, thereby modulating EMT, migration, and cell viability in SRA01/04 cells. In conclusion, lncRNA XIST has a crucial role in promoting TGF-β2-induced EMT via modulating the miR-124/Slug axis in SRA01/04 cells, and it may provide a novel therapeutic option for PCO treatment.     

Sinusoidal endothelial cells of the liver: Fine structure and function in relation to age

Liver endothelial cells form a continuous lining of the liver capillaries, or sinusoids, separating parenchymal cells and fat-storing cells from sinusoidal blood. Liver sinusoidal endothelial cells differ in fine structure from endothelial cells lining larger blood vessels and from other capillary endothelia in that they lack a distinct basement membrane and also contain open pores, or fenestrae, in the thin cytoplasmic projections which constitute the sinusoidal wall. This distinctive morphology supports the protective role played by liver endothelium, the cells forming a general barrier against pathogenic agents and serving as a selective sieve for substances passing from the blood to parenchymal and fat-storing cells, and vice versa. Sinusoidal endothelial cells, furthermore, significantly participate in the metabolic and clearance functions of the liver. They have been shown to be involved in the endocytosis and metabolism of a wide range of macromolecules, including glycoproteins, lipoproteins, extracellular matrix components, and inert colloids, establishing endothelial cells as a vital link in the complex network of cellular interactions and cooperation in the liver. Fine structural studies in combination with the development of cell isolation and culture techniques from both experimental animal and human liver have greatly contributed to the elucidation of these endothelial cell functions. Morphological and biochemical investigations have both revealed little changes with age except for an accumulation of iron ferritin and a decrease in the activities of glucose-6-phosphatase, Mg-ATPase, and in glucagon-stimulated adenylcyclase. Future studies are likely to disclose more fully the role of sinusoidal endothelial cells in the regulation of liver hemodynamics, in liver metabolism and blood clearance, in the maintenance of hepatic structure, in the pathogenesis of various liver diseases, and in the aging process in the liver.     

Quantification of substrate and cellular strains in stretchable 3D cell cultures: an experimental and computational framework

The mechanical cell environment is a key regulator of biological processes . In living tissues, cells are embedded into the 3D extracellular matrix and permanently exposed to mechanical forces. Quantification of the cellular strain state in a 3D matrix is therefore the first step towards understanding how physical cues determine single cell and multicellular behaviour. The majority of cell assays are, however, based on 2D cell cultures that lack many essential features of the in vivo cellular environment. Furthermore, nondestructive measurement of substrate and cellular mechanics requires appropriate computational tools for microscopic image analysis and interpretation. Here, we present an experimental and computational framework for generation and quantification of the cellular strain state in 3D cell cultures using a combination of 3D substrate stretcher, multichannel microscopic imaging and computational image analysis. The 3D substrate stretcher enables deformation of living cells embedded in bead‐labelled 3D collagen hydrogels. Local substrate and cell deformations are determined by tracking displacement of fluorescent beads with subsequent finite element interpolation of cell strains over a tetrahedral tessellation. In this feasibility study, we debate diverse aspects of deformable 3D culture construction, quantification and evaluation, and present an example of its application for quantitative analysis of a cellular model system based on primary mouse hepatocytes undergoing transforming growth factor (TGF‐β) induced epithelial‐to‐mesenchymal transition.     

An orientation‐independent DIC microscope allows high resolution imaging of epithelial cell migration and wound healing in a cnidarian model

Epithelial cell dynamics can be difficult to study in intact animals or tissues. Here we use the medusa form of the hydrozoan Clytia hemisphaerica, which is covered with a monolayer of epithelial cells, to test the efficacy of an orientation‐independent differential interference contrast microscope for in vivo imaging of wound healing. Orientation‐independent differential interference contrast provides an unprecedented resolution phase image of epithelial cells closing a wound in a live, nontransgenic animal model. In particular, the orientation‐independent differential interference contrast microscope equipped with a 40x/0.75NA objective lens and using the illumination light with wavelength 546 nm demonstrated a resolution of 460 nm. The repair of individual cells, the adhesion of cells to close a gap, and the concomitant contraction of these cells during closure is clearly visualized.     

Leptin promotes proliferation and invasion of osteosarcoma cells by upregulating the expression of SIRT1

Osteosarcoma (OS) is a primary high-grade malignant bone neoplasm, and the prognosis of OS remains poor due toearly metastasis. Leptin plays an essential role in tumorigenesis, but the role of leptin in the development of OS is still not fullyunderstood. In this study, we used a human osteosarcoma MG-63 cell line as an experimental model. MG-63 cells were treatedwith leptin, and cell proliferation, apoptosis, adhesion, invasion, and gene expression, were evaluated. The results showed thatleptin promoted proliferation, decreased adhesion, suppressed apoptosis, and promoted invasion, of MG-63 cells. Moreover,the expression of SIRT1 was upregulated in MG-63 cells exposed to leptin. Furthermore, MMP-2, 8, and 9 were significantlyupregulated by SIRT1, while SIRT1 knockdown inhibited the proliferation and invasion of MG-63 cells. In conclusion, ourresults suggest that leptin promotes OS cell proliferation and invasion by inducing the expression of SIRT1.     

Effect of structure and function of paraoxonase-1 (PON-1) on organophosphate pesticides metabolism

Paraoxonase-1 (PON1) is an important enzyme in various pathologies such as pesticide poisoning, diabetes,atherosclerosis, neuronal disorders, and cancer, due to its multifunctional activity since it acts on different metabolites.However, one of its main functions is the hydrolysis of organophosphate (OP) compounds from pesticides that causefatal poisoning at the level of the central nervous system (CNS). The objective of this review was to investigate whetherthe structure, genetics, and function of PON1 affect the metabolism of organophosphate pesticides or otherabnormalities. Information was selected from articles in the database PubMed–NCBI (https://www.ncbi.nlm.nih.gov/pubmed/) with a publication date between 2011 and 2019. The enzymatic activity of PON1 can be modified dependingon its chemical structure since there are different genetic polymorphisms that change PONI morphologies or the levelsof expression in the bloodstream. This leads to differences in susceptibilities to organophosphate pesticide poisoning.The results of this review reveal that phenotypic variants of PON1 have differences in affinities for OP substrates.     

Butyrate inhibits the bovine rumen epithelial cell proliferation via downregulation of positive regulators at G0/G1 phase checkpoint

Short-chain fatty acids (SCFAs) butyrate promote the postnatal rumen epithelial development and maturation in ruminants. However, molecular mechanisms of effects of butyrate on the bovine rumen epithelial cells (BRECs) proliferation remain elusive. Therefore, purpose of this study was to investigate the effects of butyrate on the expression of genes and proteins at G0/G1 and S phase of BRECs cycle. Our results showed that BRECs treated with butyrate inhibited (P < 0.05) the proliferation of BRECs, relatively to control. Flow cytometric assays revealed that butyrate triggers the BRECs cycle arrest at the G0/G1 phase. qRT-PCR analyses of mRNA level of genes involved in the G0/G1 phase of cell cycle showed that butyrate significantly upregulated (P < 0.001) the expression of mRNA encoding p21^Cip1 compared with control group, but it decreased (P < 0.05) the mRNA levels of cyclin D1 and CDK4 genes at G0/G1 phase checkpoint compared with control. Moreover, Western blot also revealed that butyrate downregulated the expression of cyclin D3, CDK6, p-Rb, and E2F1 proteins involved in the modulation of G0/G1 phase of cell cycle. In conclusion, our results demonstrated that butyrate inhibits the proliferation of BRECs via downregulation of positive regulators at G0/G1 phase checkpoint.     

Comparative study of the conditions required to image live human epithelial and fibroblast cells using atomic force microscopy

Successful imaging of living human cells using atomic force microscopy (AFM) is influenced by many variables including cell culture conditions, cell morphology, surface topography, scan parameters, and cantilever choice. In this study, these variables were investigated while imaging two morphologically distinct human cell lines, namely LL24 (fibroblasts) and NCI H727 (epithelial) cells. The cell types used in this study were found to require different parameter settings to produce images showing the greatest detail. In contact mode, optimal loading forces ranged between 2-2.8 x 10(-9) and 0.1-0.7 x 10(-9) (N) for LL24 and NCI H727 cells respectively. In tapping (AC) mode, images of LL24 cells were obtained using cantilevers with a spring constant of at least 0.32 N/m, while NCI H727 cells required a greater spring constant of at least 0.58 N/m. To obtain tapping mode images, cantilevers needed to be tuned to resonate at higher frequencies than their resonance frequencies to obtain images. For NCI H727 cells, contact mode imaging produced the clearest images. For LL24 cells, contact and tapping mode AFM produced images of comparable quality. Overall, this study shows that cells with different morphologies and surface topography require different scanning approaches and optimal conditions must be determined empirically to achieve images of high quality.     

HMGB1 promotes the proliferation and invasion of oral squamous cell carcinoma via activating epithelial-mesenchymal transformation

This study aimed to investigate the role of high mobility group box-1 (HMGB1) expression in oral squamouscell carcinoma; HMGB1 promoted the proliferation and invasion of oral squamous cell carcinoma via activatingepithelial-mesenchymal transformation (EMT). In this study, RNA transfection was used to silence the expression ofHMGB1 in oral squamous cell carcinoma cells. CCK-8, cell clone formation and trans-well assays were used to detectthe proliferation and invasion of cells before and after HMGB1 silencing. qRT-PCR and Western blot were used todetect changes in EMT marker protein expression before and after transfection. HMGB1 was significantly higher inOSCC tissues than in adjacent tissues, and of the cell lines examined, HMGB1 was highest in SCC-9 cells. Additionally,HMGB1 silencing decreased SCC-9 cell proliferation and viability. Down-regulation of HMBG1 expression inhibitednot only the proliferation but also the invasion of SCC-9 cells. The expression of N-cadherin, Snail, and Slug, but notE-cadherin, were promoted after silencing HMGB1. The expression of HMGB1 in OSCC tissue and cell lines washigher, and HMGB1 silencing decreased SCC-9 cell proliferation and invasion, suggesting that HMGB1 has positiveeffects on OSCC development. Down-regulation of HMBG1 expression regulates EMT markers, suggesting thatHMBG1 promotes OSCC cell proliferation and invasion is likely to be associated with EMT activation.     

Signal analysis of total internal reflection fluorescent speckle microscopy (TIR-FSM) and wide-field epi-fluorescence FSM of the actin cytoskeleton and focal adhesions in living cells

Fluorescent speckle microscopy (FSM) uses low levels of fluorescent proteins to create fluorescent speckles on cytoskeletal polymers in high‐resolution fluorescence images of living cells. The dynamics of speckles over time encode subunit turnover and motion of the cytoskeletal polymers. We sought to improve on current FSM technology by first expanding it to study the dynamics of a non‐polymeric macromolecular assembly, using focal adhesions as a test case, and second, to exploit for FSM the high contrast afforded by total internal reflection fluorescence microscopy (TIR‐FM). Here, we first demonstrate that low levels of expression of a green fluorescent protein (GFP) conjugate of the focal adhesion protein, vinculin, results in clusters of fluorescent vinculin speckles on the ventral cell surface, which by immunofluorescence labelling of total vinculin correspond to sparse labelling of dense focal adhesion structures. This demonstrates that the FSM principle can be applied to study focal adhesions. We then use both GFP‐vinculin expression and microinjected fluorescently labelled purified actin to compare quantitatively the speckle signal in FSM images of focal adhesions and the actin cytoskeleton in living cells by TIR‐FM and wide‐field epifluorescence microscopy. We use quantitative FSM image analysis software to define two new parameters for analysing FSM signal features that we can extract automatically: speckle modulation and speckle detectability. Our analysis shows that TIR‐FSM affords major improvements in these parameters compared with wide‐field epifluorescence FSM. Finally, we find that use of a crippled eukaryotic expression promoter for driving low‐level GFP‐fusion protein expression is a useful tool for FSM imaging. When used in time‐lapse mode, TIR‐FSM of actin and GFP‐conjugated focal adhesion proteins will allow quantification of molecular dynamics within interesting macromolecular assemblies at the ventral surface of living cells.     

Centrifugation of bacterial cells on slide surface improves the spatial distribution, cell recovery and reduces the time of enumeration for fluorescence in situ hybridization

Fluorescence in situ hybridization (FISH), which is used for the enumeration of bacteria in various ecosystems including the human intestinal tract, has several limitations. One of the major problems encountered is the uneven distribution of bacterial cells on the slide surface, which increases the coefficient of variation between repetitions and thereby increase the time required for enumeration. In order to improve the spatial distribution, we designed a centrifugation device, which allows the direct centrifugation of bacterial cells onto the slide surface. Another problem is the loss of bacterial cells during the hybridization procedure. This leads to an underestimation of the true cell numbers in the sample. To overcome this problem we tested the use of silanized or chrome gelatine coated slides. Our study indicates that the use of the centrifugation device in conjunction with chrome gelatine coated slides highly improves the quality of enumeration data obtained by manual and automated microscopic counting and shortens the time of analysis.     

Effects of substrate texture and curvature on the morphology of cultured cells

The ultrastructural characteristics of several growth matrices were examined using two cell types chosen for their distinct growth habits. Chinese hamster ovary cells and Balb-c 3T3 mouse fibroblasts were grown on flat substrates (glass, tissue culture plastic, Millipore filters) as well as spherical (glass, tissue culture plastic, cross-linked dextran) substrates. Cells were plated maintaining equal densities and growth surface area. Once the majority of the cells reached confluency, the cell's morphology on each matrix was examined using scanning electron microscopy. Digital analysis was performed on cell attachment area to compare the effect of each matrix on cell spreading. Variation in cell shape was dramatic between matrices, being most noticeable between a textured surface (filter, dextran bead) and that of a smooth (glass) surface. Even within smooth surfaces, some variation was observed. There was also an effect of matrix curvature on cell attachment area, the greatest being in the 3T3-c Balb cells, causing an overall decrease in the area of attachment between cell and matrix. The changes seen could also be related to the particular cell type used. Hamster ovary cells tended to be cylindrical and showed little effect between matrices, whereas the mouse fibroblasts, which are more flattened, showed the matrix effect to a greater degree. This study demonstrates the necessity of being aware of substrate-induced cell changes in tissue culture, where some variation in cell shape may be due to the surface on which the cells are grown as opposed to the experimental procedure.     

AFM studied the effect of celastrol on β1 integrin‐mediated HUVEC adhesion and migration

Integrin‐mediated human umbilical vein endothelial cells (HUVECs) adhesion to the extracellular matrix plays a fundamental role in tumor‐induced angiogenesis. Celastrol, a traditional Chinese medicine plant, has possessed anticancer and suppressed angiogenesis activities. Here, the mechanism underling the antiangiogenesis capacity of celastrol was investigated by exploring the effect of celastrol on β1(CD29) integrin‐mediated cell adhesion and migration. Flow cytometry results showed that the HUVECs highly expressed CD29 and cell adhesion assay indicated that celastrol specifically inhibited the adhesion of HUVECs to fibronectin (FN) without affecting nonspecific adhesion to poly‐L‐lysine (PLL). After cell FN adhesion being inhibited, the cell surface nanoscale structure and adhesion force were detected by atomic force microscope (AFM). High‐resolution imaging revealed that cell morphology and ultrastructure changed a lot after being treated with celastrol. The membrane average roughness (Ra) and the major forces were decreased from 31.34 ± 4.56 nm, 519.60 ± 82.86 pN of 0 μg/ml celastrol to 18.47 ± 6.53 nm, 417.79 ± 53.35 pN of 4.0 μg/ml celastrol, 10.54 ± 2.85 nm, 258.95 ± 38.98 pN of 8.0 μg/ml celastrol, respectively. Accompanying with the decrease of adhesion force, the actin cytoskeleton in the cells was obviously disturbed by the celastrol. All of these changes influenced the migration of HUVECs from the wound‐healing migration assay. Taken together, our results suggest that celastrol can be as an inhibitor of HUVEC adhesion to FN. This work provides a novel approach to inhibition of tumor angiogenesis and tumor growth. SCANNING 35:316‐326, 2013. © 2012 Wiley Periodicals, Inc.     

Effect of the downstream transition region of a flow measurement flume of rectangular compound cross section on flow properties

In this study, the effect of the downstream expansion region of a flow measurement flume of rectangular compound cross section on some of the flow properties; such as the discharge coefficient, C_d, the approach velocity coefficient, C_v and the modular limit, ML were investigated. For this reason, extensive laboratory tests were conducted with nine models of different downstream transitions. The aforementioned hydraulic quantities were then related with the relevant parameters to obtain sets of curves from which one could decide which kind of downstream transition type would produce the highest modular limit. It was found that model type A yielded the highest modular limit with a downstream slope of about 1/7.