In Vitro Invasion Of C6Glioma Cells Transfected With Cx43cDNA

Background:Glioma is the most common primary tumor in central nervous system. One ofthe most important hallmarks of malignant glioma is their invasive behavior whichmakes it a poor prognosis and recurrence. Thus, glioma invasive research has becomea hot topic. Invasion of tumor cells into normal tissue is thought to be amulti-factorial process, mainly consisting of adhesion, ECM degradation andmigration. Matrix metalloproteinases degrade ECM, creating space for invadingglioma cells. Cx43is the most abundant connexin in the central nervous system andexpresses mainly in astrocytes. The observation is consistent with low Cx43expressionwithin the core of the glioblastoma and high expression in the peri-tumor. It has beendemonstrated that silencing of Cx43suppressed invasion of rat hepatocellularcarcinoma cells, however, the relationship between Cx43and rat C6cells invasionremains elusive. So, this study is to explore the relationship between overexpressionof Cx43in C6cells which was mediated by liposome and its invasion, which couldprovide new ideas and strageties for clinical precaution and treatment of glioma.Objective:It is to increase expression of Cx43by transfecting pCMV-Cx43cDNA plasmid intoC6cells. It is to study the connection between overexpression of Cx43and invasionof C6cells in vitro and to explore its possible mechanism.Method:1. pCMV-Cx43cDNA plasmid was transfected into C6cells by liposome. C6cellsoverexpressed Cx43. Overexpression of Cx43in C6cells was cloned stably by usingG418.2. Cx43mRNA and Cx43protein expression were detected by RT-PCR andWestern-blot, respectively.3. Invasion of C6cells was checked by Transwell and MTT method.4. The activity of MMP-2and MMP-9in cell medium were detected by zymography. 5. MMP-9protein expression was detected by Western blot.Results:1. pCMV-Cx43cDNA plasmid was successfully transfected into C6cells. Cell linesoverexpressing Cx43(C6-Cx43) or empty vector (C6-non) were selected stably byusing G418.2. RT-PCR: the expression of Cx43mRNA in C6cells, C6-Cx43cells and C6-noncells were as follows:1.15±0.05,1.86±0.07,1.08±0.05. The expression ofCx43mRNA in C6-Cx43cells increased significantly, compared with C6-non cellsand C6cells (P＜0.05); Western blot: the expression of Cx43protein in C6cells,C6-Cx43cells and C6-non cells were as follows:0.36±0.06,1.25±0.1,0.31±0.07.The expression of Cx43protein in C6-Cx43cells was significantly increased,compared with C6-non cells and C6cells (P＜0.05).3. The results of invasion test showed that absorbance value of C6-Cx43cells wasgreatly increased, compared with C6-non cells and C6cells (P＜0.05),whichindicated that the ability of invasion of C6-Cx43cells is stronger than that of C6-noncells and C6cells.4. zymography assay showed that the activity of MMP-9in cell medium of C6-Cx43cells was greatly enhanced, compared with that of C6and C6-non cells (P＜0.05).5. Western blot: the expression of MMP-9protein in C6cells, C6-Cx43cells andC6-non cells were as follows:0.29±0.03,0.43±0.02,0.27±0.01. The expression ofMMP-9protein in C6-Cx43cells was significantly increased, compared with that ofC6-non cells and C6cells (P＜0.05).Conclusion:1. pCMV-Cx43cDNA plasmid is successfully transfected into C6cells. Cx43mRNAand Cx43protein expression of C6cells are increased significantly.2. Overexpression of Cx43of C6cells increases glioma cells invasion, which maybecorrelates with the increasing of MMP-9activity and the upregulation of MMP-9protein expression.