The Bioactivity And Mechanism Of Inhibiting Human Brain Gliomas By Recombination Polypeptide Derived From The N-terminus Of Viral Macrophage Inflammatory Protein Ⅱ

Objective:To investigate suppression activity of the N-terminus recombination polypeptide of viral macrophage inflammatory protein Ⅱ and its molecular mechanism in human brain glioma cells and human brain glioma tumor.Methods:1. Immunofluorescence staining was used to test the expression of CXCR4 and GFAP in U251 and SHG-44 cells; 2. The effect of NT21MP on growth, migration ability and invasive activity in U251 and SHG-44 cells were detected by MTT assay, scratch assay and transwell chamber assay; 3. Cell cycle and apoptosis were determined by flow cytometry; 4. The real-time fluorescent quantitative PCR (qRT-PCR) was used to detect the expression of cell cycle associated mRNA (CyclinDl,CDK4) and apoptosis associated mRNA (Bakl,Bcl-2,Bax) with NT21MP; 5. The expression of cell cycle associated protein (CyclinDl,CDK4) and apoptosis associated protein (Bak1,Bcl-2,Bax,caspase3) were detected by western blotting; 6. The higher CXCR4 receptor expression human glioma cell was choosed by qRT-PCR; 7. The expression of CXCR4 was cuted by small RNA interference technology. The vitro activity tests were used to confirm whether NT21MP play a resistance role on human glioma cells by blocking the CXCR4 receptor signal. After the interference of CXCR4, the migration ability and invasive activity of glioma cell were evaluated by scratch assay and transwell chamber assay; Cell cycle and apoptosis was measured by flow cytometry; qRT-PCR and western blotting were used to detect cell cycle associated molecular (CyclinDl,CDK4) and apoptosis associated molecular (Bakl,Bcl-2,Bax, caspase3); 8. Established mice models of human brain glioma, the mice were put to death after four weeks, the tumor were stripped and weighed, and the tumor growth inhibitory rate of NT21MP was calculated; 9.The tumor tissue was analyzed by H&E staining.Results:1. CXCR4 expressed in human brain glioma cell lines U251 and SHG-44; 2. NT21MP inhibited the growth of brain glioma cells dramatically, which was obvious dosage-effect (P<0.05).The migration and invasion induced by SDF-1a/CXCR4 were reduced by NT21MP; 3. The brain glioma cell cycle was arrested in G1/S phase (P<0.05). Apoptosis occurred in brain glioma cells treated with NT21MP (P<0.05); 4. NT21MP increased the expression of Bakl, decreased Bcl-2/Bax ratio and the expression of CyclinD1, CDK4 in U251 and SHG-44 cells (P<0.05); 5. After CXCR4 interference in SHG-44 cells, the inhibit ratio of migration and invasion on SHG44 cells by NT21MP was decreased significantly, as well as the regulation ratio of cell cycle, cell apoptosis, cell cycle and apoptosis related molecules and protein (P<0.05); 6. The growth of brain glioma can be inhibited by NT21MP in vivo at the ratio of 60.7%(P<0.05); 7. H&E further validated that NT21MP inhibited the growth of the brain glioma (P<0.05).Conclusion:1. NT21MP can inhibit the proliferation, invasion, migration in U251 and SHG-44 cells and induce the differentiation from brain glioma cells to normal stellate cells in vitro by blocking the SDF-la/CXCR4 biological axis; 2. NT21MP blocked the cell cycle from G1 phase to S phase transition and promoted apoptosis of tumor cells by raising the expression of apoptosis related proteins (Bak1, Caspase3), down-regulating the ratio of Bcl-2/Bax and the expression of cycle regulation molecules (CyclinDl and CDK4) in U251 and SHG-44 cells.