Study On Mechanism For P21/53Promoting Invasion Of Human Glioma Stem Cells By Inhibiting TIPM-2Expression

Background and Objective:Malignant brain glioma is the most common primary central nervous system ofhuman malignant intracranial tumors.Tumor cells to normal brain tissue invasivegrowth is an important biological features of malignant glioma, this to normal braintissue invasion characteristics makes surgery is difficult to cure the tumor, and thepoor efficacy of radiotherapy and chemotherapy, postoperative recurrence rate is high,and poor prognosis.Invasive growth and malignant glioma surgery, resistant treatmentresistance mechanism is unclear.Solid tumors in histologic heterogeneity of garment, its composition includingtumor cells, interstitial inflammatory infiltration and tumor vascular structures.Nowthere are a lot of research suggests that the tumor cells, there is a small part of thecells, they with stem cell properties, which we call the tumor stem cells, these cells inthe tumor play an important role in the occurrence and development.Reports theexperiment, if transplanted tumor cells into immunodeficiency mice model, onlyCSCs can promote the birth and development of tumor.From these cells, the lowgrade or part cause phenotypic differentiation tumor cells can be induced.In recentyears, in leukemia, breast cancer, glioma, prostate cancer, colon cancer, andpancreatic cancer research process, the mechanism studies of CSCs tumordevelopment is more and more get attention.The concept of cancer stem cells may let’s understanding of tumor biology and design new treatments have a profoundimpact on tumor growth.In recent years a large number of studies have shown that cancer stem cells,cancer stem cells, CSCs) in the tumor origin and developmentPlay an importantrole.From the past a lot of the literature and our recent studies have found that there isa small proportion of malignant glioma glioma stem cells (glioma stem cells, GSCs),these glioma stem cells form tumors, multi-directional differentiation potential, aswell as the ability of self-renewal.Also found that glioma stem cells are highlyaggressive, but the specific molecular mechanism is not yet clear.Transcriptional regulation factor p53as a tumor suppressor genes can induce cellgrowth retardation, apoptosis, cell differentiation and DNArepair.But the p53mutantcould make the wild-type p53function inactivation of tumor suppressor gene, and thefunction of oncogenes.P53protein is associated with the cell cycle division, itsexpression in almost all sorts of nucleated cells, but at a lower level.Now, have madeit clear that the p53is negative regulation of cell growth cycle factors, and regulatingcell cycle, DNArepair, cell differentiation, apoptosis, and other important biologicalfunctions, in the process of inhibit cancer cell growth plays an important role.Is amember of the family of C1P p21genes, it is located at the downstream of the p53gene cell cycle dependent kinase inhibitors.P21and p53constitute the G1cell cyclecheckpoint, because without after DNAdamage repair cannot pass, reduces thedamaged DNAreplication and accumulation, and thus play a role of tumorsuppressor.Studies have shown that p21and tumor differentiation, infiltration depth,proliferation and metastasis, prognosis of value.At present, a study shows the P53/P21have the functions of oncogenes or tumor suppressor genes.However, the role ofP53/P21in GSCs attack is still not very clear.Now found that metalloproteinases tissue inhibiting factor2(TIMP-2), matrixmetalloproteinase9(MMP9) and matrix metalloproteinase2(MMP2) pathway inmalignant tumor invasive play an important role.Because they are of the ECMcomponents have specific enzyme activity, so MMP2and MMP9can degrade almostall types of ECM.MMP2and MMP9in the activation and reconstruction and poor prognosis of the tumor biological characteristics.Past literature reported furtherproved by lowering the expression of MMP2and MMP9, can inhibit tumorcells.TIMP-2as natural inhibitor of MMP2and MMP9, expressed in many tumorsincluding glioma is lower, but the reason it is not clear.We through the computerforecast reports, documents and bioinformatics analysis, we found that the TIMP-2May be one of potential target genes of P53/P21.In this study, we first from human glioma cell line U87glioma specimens andthe original generation in the isolation and identification of CD133+glioma cells, toidentify the CD133+glioma cells with GSCs properties;Then, we tested the TIMP-2MMP2and MMP9in glioma cells and the expression in GSCs;Finally, we studied theP53/P21of TIMP-2regulation function and prove that is the important mechanismof the P53/P21promote GSCs attack.Materials and Methods:1from human glioma cell line U87glioma specimens and the original generation ofisolated CD133+glioma cells.2. Immunofluorescence staining from neural stem cell markers, nerve cell formation,multi-directional differentiation potential, sorting out the GSCs three aspectsidentification.3. The GSCs Transwell invasion in vitro experiment testing and the comparison ofGCs aggressivity.4. Using Transwell invasion in vitro experiments to detect P53/P21impact the GSCsand GCs aggressivity.5. The qRT-PCR and western bloting technology to detect TIMP-2and itsdownstream target genes of MMP2, MMP9expression in GSCs and GCs.6. The qRT-PCR and western bloting technology testing after transfection P53/P21inhibitors GSCs TIMP-2, the expression of MMP2and MMP9in change.Results:1. Success from human glioma cell line U87glioma specimens and the originalgeneration to separate and identify express CD133+markers of glioma cells, these cells have the ability to form a nerve cells in the ball.By using immunofluorescencestaining multi-directional differentiation potential, according to the experimentalmethods the CD133+cells after induction of the ball can differentiate into cellsexpressed GFAP, beta tubulin III.Through the above results, we isolated CD133+cells with stem cell characteristics.2. P53/P21in GSCs attack ability.Through the experiment data show that GSCs invasive significantly higher than thatof GCs (P <0.05).After transfection P53/P21inhibitors, the invasive ability of GSCssignificantly decreased (P <0.05).3. Through the experiment data show that in both mRNAlevel and protein level,TIMP-2expression in GSCs to significantly lower GCs (P <0.05), on the contrary,MMP2, MMP9mRNAlevel and protein level expression was significantly lower thanin the GCs GSCs (P <0.05).4. P53/P21expression of TIMP-2pathway.After the transfection P53/P21inhibitors in GSCs, MMP2, MMP9mRNAandprotein levels were significantly decreased (P <0.05), and TIMP-2significantlyincreased the expression of mRNAand protein levels (P <0.05).Conclusions:his experiment adopts the flow separation technique from human glioblastoma cellline U87and brain specimens of primary glioblastoma in separation and identify ofglioma stem cells "CD133+glioma cells, prove that CD133+highly invasive gliomacells, found through the high expression of P53/P21promoted the GSCs invasive, itspossible mechanism is at least by cutting TIMP-2, raised MMP2and MMPexpression.This experiment for the first time reveals the P53/P21in human gliomastem cells play an important role in invasion, to know the invasive malignant gliomagrowth mechanism, to explore new treatment to provide new evidence.