Study On Expression Of S100A4 In Glioma And Its Role In The Migration And Invasion Of Glioma Cells

Gliomas are the most common primary tumors in the central nervous system(CNS). The current standard therapy includes maximal safe resection followed by radiotherapy in combination with chemotherapy. Because of the current optimal therapeutic strategies, to some extent,quality of life was improved and survival was prolonged, but malignant gliomas still could not be cured. Malignant gliomas are highly invasive and characterized by widespread infiltration throughout the brain.Although such patients accept the standard therapy, the median survival is only 14 months. Molecular targeted therapy related to tumor progression may provide potential cure of gliomas. In recent years, many researches are focused on studying biological mechanism of gliomas as well as finding new molecular targets, aiming to inhibit growth, proliferation, migration and invasion of gliomas through these targets.S100A4 belongs to the S100 protein family, which is Ca2+-binding and low molecular mass proteins(10 to 12 KDa). It generally exists as homo- or heterodimers within cells and is localized in the nucleus, cytoplasm or both. Like other S100 proteins, S100A4 possesses no enzymatic activity, instead, it exerts the effects by interacting with and modulating the activity of other proteins. Some studies have showed S100A4 has diverse biological functions and is associated with development and progression in some of human tumors, which indicates S100A4 might be one of molecular targets for glioma treatment.Malignant glioma cells move into surrounding brain parenchyma is a complex,four-step process: ①detachment of invasive cells from the tumor mass, ②attachment to extracellular matrix( ECM), ③degradation of the ECM, ④change morphology,and migration. Aimed at effectively control of the glioma recurrence and invasion into normal brains, one of novel therapeutic strategies is to inhibit the migration and invasion of malignant glioma cells. In this study, we firstly examined S100A4 expression in glioma tissues and glioma cell lines compared with control brain tissues and GES-1 cell line respectively. Experiments in vitro were then designed to investigate the role of S100A4 in above four-step process of migration and potentialmechanism of S100A4 in promoting the migration and invasion by using S100A4over-expressed glioma cell lines. Two parts were included:1. The expression of S100A4 in human glioma tissues and glioma cell lines.First, a total of 43 glioma specimens with different pathological grades and 11 control brain tissues were collected from surgeries for immunohistochemical staining to determine the expression of S100A4 in human gliomas and analyzed the correlations between S100A4 expression and the malignacy of gliomas. Then, S100A4 mRNA and protein expression was examined by real-time PCR and Western blot respectively in four malignant glioma cell lines and GES-1 cell line which was served as control.The results of immunohistochemical staining revealed that S100A4 was negatively or just weakly expressed in control brain tissues, while strongly expressed in the nucleus,cytoplasm or both in glioma tissues. The expression level of S100A4 was positively correlated with the pathological grades in glioma tissues. Compared with GES-1 cell line, S100A4 was highly expressed in U87, LN229, U251 and SNB19 glioma cell lines. The S100A4 expression determined by Real-time PCR as well as Western blot was in accordance with immunochemistry.2. The role of S100A4 in migration and invasion of glioma cells. Small interfering RNA(siRNA)of S100A4 was transfected into SNB19 and U251 glioma cells to knock down the expression of S100A4. Differently treated Glioma cells were assigned into three groups: control group, siRNA-negative control treated group(siRNA-NC) and siRNA-S100A4 group. Both RT-PCR and western blot assays were used to detect the S100A4 expression at mRNA and protein level respectively. The migration and invasion of glioma cells were determined by wound healing assay and transwell invasion assay respectively. The expression of E-cadherin, N-cadherin,Vimintin, MMP-9 and MMP-2 was detected by Western blot. The lamellipodia and focal adhesion of glioma cells were observed by immunofluorescence(IF). The distribution of S100A4 and non-musle myosin heavy chain IIA(NM IIA) in glioma cells were also examined by IF. The combination of S100A4 and NMIIA in glioma cells was detected by Co-immunoprecipitation(Co-IP). Results: Compared with control group and siRNA-NC group, S100A4 expression was inhibited insiRNA-S100A4 group both at mRNA and protein level. After S100A4 expression was knocked down, the ability of migration and invasion of glioma cells was markedly impeded. Knockdown of S100A4 also resulted in restoration of E-cadherin expression and suppressed expression of N-cadherin, Vimintin and matrix metalloproteinases(MMPs) both in glioma SNB19 and U251 cells. S100A4 combined and mainly colocalized with NMIIA at the leading edge of glioma cells.The lamellipodia became smaller or unextended, and focal adhesion became larger in siRNA-S100A4 treated group compared with siRNA-NC group and control group.Conclusions:1. S100A4 expressed negatively or just weakly positively in control brain tissues,but expressed positively in gliomas, and the expression of S100A4 in high grade gliomas was higher than in low grade gliomas.2. S100A4 was highly expressed in malignant glioma cell lines, and the ability of migration and invasion was declined significantly after the glioma cells were treated with siRNA specific to S100A4.3. S100A4 plays an important role in the whole process of invasion and migration of glioma cells.4. S100A4 might be a potential candidate for anti-glioma therapy to prevent the invasion and migration of glioma cells.