Mirna In Human Glioma Associated Preliminary Study Of Gene Therapy

Research background:MicroRNAs (miRNAs) are a class of small non-coding RNAs and consist of20-25 ribonucleotides. The mature miRNAs with full or partial complementarity tothe target mRNAs direct the cleavage of target mRNAs or act as repressors oftranslation, involved in modulating the development, differentiation, proliferationand apoptosis of cells. Recent studies have shown a distinct connection betweenmiRNAs and the development of cancer, some of which are specificly expressed inglioblastoma.Research objection:More than 400 human microRNAs have been discovered while their functionsremain to be undiscovered. Especially, little was reported about the functions ofglioma-related microRNAs. This topic seeks to carry out the glioma-related analysisof microRNA expression profiling and its functional studies. We try to explore thepossibly specific microRNAs which may be involved in the pathophysiologicalprocess of glioma, and provide a new target for the future use of specific microRNAsilence technology for glioma disease treatment.Research Methods:. Screen: Collect different pathological diagnosis of malignant glioma, as well asthe surgical specimens of normal brain tissue from decompression of severe traumatic brain injury patients, and then screen the glioma-specific expression ofmiRNAs using miRNAs microarray methods and recent research at home andabroad.. VerificationVerification: We verify the specificity of selected miRNAs in human gliomatissues and normal brain tissues via application of real-time fluorescencequantitative PCR technology, and further compared the relevance of theexpression of specific miRNAs associated with different grades of glioma.. Function Study: Selected human glioma cell lines, U87, TJ905 and U251 cells,were cultured in 10% newborn calf serum DMEM (Gibco Inc.) medium, andplaced in 37℃, 5% CO2 incubator, taking logarithmic phase for cell experiments.Specific miRNAs oligonucleotide synthesized by Shanghai GenePharmaCompany and specific miRNAs expression vectors constructed by WuhanGenesil Company, were transfected into U87, TJ905 and U251 glioma cells. Andthe expression of specific miRNAs in human tissues and cells were detected byreal-time fluorescence quantitative PCR. The effects of the proliferation,transformation, invasion, and apoptosis of upward or downward specificmiRNAs on transfected glioma cells were evaluated via MTT assay, Transwellassay, Flow cytometric method and Soft agar colony assay.Research Results:. MiR-21 high expressed in human brain different-grade gliomas and U87 cell linecompared with normal brain tissue. Inhibiting miR-21 could effectively lead toU87 cells proliferation suppression, apoptosis induction, invasion reduction,Caspase-3 activity elevation and Caspase-9 activation, but did not affect PTENand Caspase-8 expression. Mechanism for its induction of apoptosis maybeachieve through the Caspase-9, 3 ways. . MiR-181a and miR-181b were strongly down-regulated in all glioma samplesand glioma cell lines, U87,TJ905 and U251 cells compared with normal braintissue. MiR-181a expression showed downward trend with the increased level ofglioma, and its expression was negatively correlated with tumor grades; miR-181b expression stabilized in more than WHO-III of glioma, and showed nostatistical significance between WHO-II and WHO-III of glioma; MiR-181cexpression were down-regulated in WHO-IV of glioma, but showed nosignificant differences in both WHO-II and WHO-III of glioma.. Up-regulation of miR-181a and miR-181b could effectively lead to glioma cellsgrowth suppression, transformation inhibition, apoptosis increase, and invasionreduction.ResearcResearch Conclusions:. MiR-21 showed high expression in glioma tissues and U87 cells. Down- regulatedexpression of miR-21 in U87 cells could effectively inhibit U87 cell proliferation,induce apoptosis, and reduce the invasion ability. The abnormally high expressionof miR-21 in glioma may function as an antiapoptotic factor.. MiR-181a, miR-181b and miR-181c show different low level of expression inglioma and cell lines, among which miR-181a expression negatively correlateswith tumor grades in glioma. The aberrantly down-regulated miR-181a and miR-181b maybe function as tumor suppressors in human glioma.