HAb18G/CD147 Regulates Vinculin-mediated Focal Adhesion Formation And Cytoskeleton Reorganization In HCC

Tumor cytoskeleton has important influence on invasion and migration. The formation of skeleton is regulated by focal adhesion, which is marked by vinculin. Focal adhesion is composed by a dozen proteins, such as integrin, FAK, talin, paxillin, vitronectin, actin and so on. HAb18G/CD147(CD147) is a single-pass transmembrane glycoprotein, belonging to the immunoglobulin superfamily. C D147 is also known as a tumor associated marker of poor prognosis, which have broadly expression in many tumors. Previous studies showed that CD147 is involved in the dynamic adjustment of the focal adhesion aggregation and dissociation, and the marker protein of vinculin by direct interaction and the regulation of vinculin expression level, affect the adhesion plaque area and intensity, resulting in the reorganization of cytoskeleton.Our previous research found that C D147 is the marker of maligant type tumor, stimulating fibroblast cell to produce elevated levels of MMPs and promoting the invasion and metastasis potentials. It’s not clear that the role of C D147 in cytoskeleton arrangement. In order to study the correlation between C D147 high expression and cytoskeleton reorganization, and the influence of C D147 on the ability of tumor cell adhesion, we adopt a set of methodssuch as Western blot, construction of fluorescent reporter gene plasmids, Confocal Laser Scanning Microscopy, Fluorescence Photobleaching Recovery(FRAP) and Fluorescence Resonance Energy Transfer(FRET) to detect biological behaviors of the HCC cells. Part 1: CD147 regulates the expression of vinculin and their interactionIn this study, we used four HCC cell lines: SMMC-7721, T7721(CD147 is overexpressed), K7721(CD147 is knocked out) and R7721(CD147 is reexpressed in K7721 ccells). Firstly, we examine the expression levels of these four cell lines. Using the si RN A interfere we revealed the correlation between CD147 and vinculin. The immunofluorescence and co- immunoprecipitation are good methods to detectthe interaction of these two proteins. We also constructed plasmids of p EGFP-CD147 and Dsred2-N1-vinculin, and observed the protein conformation change by confocal microscopy.The experimental results show that C D147 exhibited a one-way regulation for vinculin. In K7721 cells, vinculin expression was markedly up-regulated, while in the high expression of CD147 in SMMC-7721, R7721, T7721 cells, vinculin expression was very low. Immunoprecipitation showed that CD147 and vinculin have interaction in SMMC-7721 cell lysate. The FRET phenomena occur in focal adhesion position. This effect occurs in less than 10 nm range. Part 2: The observation of focal adhesion and cytoskeleton reorganization in different cell linesVinculin is the most stable protein in focal adhesion. Many report showed that vinculin directively interacts with actin,thus regulates the reorganization of cytoskeleton. These are four different expression in four different expression of CD147. We observed the vinculin localization in focal adhesion and analyzed the parameters of focal adhesion such as area, intensity, circularity and diameter in four cell lines which have different expression levels of CD147. Using the phalloidin staining, we observed the cytoskeleton reorganization.Focal adhesion is vital structure in cancer cell migration. In the process of assemble/disassemble of focal adhesion as a directional motion mode, cytoskeleton is stretched. Droved by myosin proteins, cells continue to form the FA front and end point where FA constantly is dissociated. In the process of continuous dynamic focal adhesion assembly and dissociation, cells directively move towards the adhesion plaque. CD147 may regulate cell movement by influencing the dynamic process of focal adhesion assembly and dissociation. Since the formation of FA is a dynamic process, we adopted the in vitro method of fluorescence photobleaching recovery by transfecting p EYFP-vinculin plasmid into the four cell lines and using the 488 nm laser to bleach the focal adhesion of formation in the cell membrane. We measured the recovery time, processed the data and calculated by the mathematical model equation.The average intensity of focal adhesion of SMMC-7721 cells are stronger than the other three kinds of cells. In the K7721 cells, derangement of actin meshwork, loose and no obvious microfilament bundles. There are more small branches located in the edge of the cell. The area and intensity of R7721 cells are less than the SMMC-7721 cells. The adhesion plaque of T7721 cells distributed widely, even arrived at the central position of cel s. Part 3: The influence of CD147 on cell adhesion, spreading and morphologyFocal adhesion is an important mode of cell adhesion to extracellular matrix. Different components of extracellular matrix lead to different modes of adhesion and different types of adhesion force. In the cell adhesion and spreading experiments we used Matrigel and Fibronectin respectively to precoat the cell culture dishes. Adhesion potentials of the four cell lines were measuredand. In the cell spreading experiment, we used Matrigel as the extracellular matrix. Cells were resuspended in serum- free medium and cultured in Matrigel-coated culture dishes. At intervals of 15 minutes, Cells were observed with the Differential Interference Contrast Microscope(DIC). We found that cell spreading area and unfolded form are completely different which may affect the the adhesion of cells. Cell spreading can be considered as the first step of cell adhesion, suggesting that there is a correlation between the cell spreading area and the speed of focal adhesion formation.K7721 cells spread process is slow because of CD147 knockdown. T7721 cells are obvious filopodia for spreading, and SMMC-7721 is lamellipodia. Focal adhesion observation showed that focal adhesion of T7721 cell distributed in the whole bottom o f cell, and there was no focal adhesion distribution in cell center position of SMMC-7721 and R7721 cells.