| Glioma is the most common malignant tumor in the central brain system, accountsfor about45%-60%of intracranial tumors. The invasive growth is one of the keybiologic features of glioma, which limits the glioma topical treatments such as thefeasibility and effectiveness of surgical and radiotherapy. Therefore, new approaches toimprovement of the efficacy of anti-glioma treatments are urgently needed. Thus, newtherapeutic targets and tools should be developed based on a better understanding of themolecular pathogenesis of glioma. B7-H3is an important negative molecule of B7family, involving in tumor immune escape. Previous studies have showed that theabnormal expression of B7-H3in many tumor tissues including glioma, was directlyrelated to the biological characteristics of the tumor cells. However, the mechanismregulating B7-H3expression on tumor cells and the downstream signal of B7-H3characterizing the tumor cells remain unclear. MicroRNAs, a kind ofnon-protein-coding of single small RNAs (about22nt), could regulate gene translationat the post-transcriptional level and play a important role in the biological characteristicsof glioma cells, including cell proliferation, invasion and metastasis, differentiation,tumor formation and the generation of stem cells. Studies have found that miR-29a maytarget B7-H33’-UTR sequence, and modulates the expression of B7-H3. If there anyother microRNA could modulate the expression of B7-H3, and the mechanism ofmicroRNAregulation of B7-H3in glioma cells invasion remain to be further studied.In view of these, this study used the means of biology information method anddual-luciferase reporter system testing to find new microRNAs regulating B7-H3expression on glioma, and miR-339-5p was found in this meaning. The expression ofB7-H3and miR-339-5p were further analyzed in glioma tissues with different gradesof clinical pathogenesis. Furthermore, the in vitro experiment by transfectingmiR-339-5p to the U87cell lines was taken to explore the role of miR-339-5p onglioma cell invasion and the mechanism of miR-339-5p regulation B7-H3, and thus provide a theoretical basis to the further explore the molecular mechanisms and clinicaltreatment of glioma.Part1: miR-339-5p regulation the expression of B7-H3and theclinical significance of miR-339-5p expression on glioma tissuesObjective: To search the mircroRNAs which could regulate the expression ofB7-H3, and detect the expression of the mircroRNAs and B7-H3in glioma and theirrelated clinical significances.Methods: First, we pridicted the mircroRNAs which may target B7-H33’-UTRsequence through biology information method, as well as analyzed the mircroRNA chipdata of glioma in situ tumor model to find mircroRNAs that may regulate B7-H3andglioma invasion. And then we tested the regulation of the choosen mircroRNAs on theexpression of B7-H3by dual-luciferase reporter system. At last, we detected expressionof B7-H3, miR-29a and miR-339-5p in glioma tissues of different clinical pathogensisvia Real-time fluorescence quantitative (RT-PCR) method.Results:(1) Online prediction (URL http://www.microrna.org) showed that thereare20different mircroRNA binding sites in B7-H33’-UTR sequences.Together with theresults of analyzing the mircroRNAchip data of glioma in situ tumor mode, We foundthree specific microRNAs: miR-339-5p, miR-185-5p, miR-539-5p. Thedual-luciferase report system at last identified one of the three, miR-339-5p, coulddirectly target B7-H33’ UTR sequence and inhibite B7-H3gene expression, with asimilar effect by the reported miR-29a.(2) Real time quantitative PCR experimentshowed that the expression of miR-29and miR-339-5p negatively correlated with theclinical pathogensis of glioma tissues, and also B7-H3gene expression showed acorrelation with the clinical pathogensis of glioma tissues.Conclusion: miR-339-5p could down-regulate B7-H3expression, with a similarinhibition effect by miR-29a. B7-H3highly expressed in grade IV tumor tissues, alsoknown as high-grade astrocytoma or glioblastoma (GBM), and the miR-339-5p had anopposite expression pattern, which remind us that miR-339-5p may regulate theexpression of B7-H3in glioma. Objective: Study effect of miR-339-5p inhibit B7-H3molecules in glioma;Observation miR-339-5p reduce B7-H3in glioma and the influence of the biologicalcharacteristics and mechanism.Methods:Transfection miR-339-5p into glioma cell lines U87cells by genetransfection technique, detected B7-H3molecule and mRNA expression levels aftertransfection by flow cytometry and real-time PCR method; study miR-339-5p effect onglioma cell invasion and proliferation through cell invasion assay and CCK-8assay;simultaneously detected expression of CXC chemokine receptor (CXC chemokinereceptor), CXCR1, CXCR2, CXCR3, CXCR4and CXCR7in U87by flow cytometryafter transfection.Results:(1) according to flow cytometry and real-time PCR, transfection ofmiR-339-5p can inhibit B7-H3molecule and the expression of B7-H3mRNA in gliomacell;(2) cell invasion assay results show that transfected miR-339-5p can reduce theinvasion and metastasis of glioma cells, miR-339-5p inhibitors can neutralize thisdownward capability; but the cell proliferation assay showed that transfection ofmiR-339-5p glioma cell does not affect the value;(3) transfected with miR-339-5pexpression of brain glioma cells are significantly decreased CXCR4, other chemokinereceptor expression did not change.Conclusion: miR-339-5p could regulate B7-H3gene expression, the effect wassimilar with miR-29a which has been reported. B7-H3was high expression in gliomacell lines and high grade glioma tissues, while the miR-339-5p express the oppositetrend, show that miR-339-5p may regulate the expression of B7-H3in glioma.MiR-339-5p can inhibite the expression of B7-H3in glioma, and can inhibit theglioma cells invasion, but does not affect its proliferation. MiR-339-5p can reduce theexpression of CXCR4, and CXCR4by miRNAs online software to predict does notexist miR-339-5p binding sites, show that miR-339-5p inhibiting tumor cell invasion may through the cut after the B7-H3through CXCR4signaling pathways. |
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