Micro Rnalet-7d Inhibit Glioma Growth And Invasion

[Background and Objective]Gliomas are the most common types of brain tumors , glioblastoma accountfor about 60% of the total. Glioblastomas are one of the most malignancy andinfestation tumor . Although sophisticated regimens of conventional therapies arebeing carried out to treat patients with gliomas, the disease invariably leads todeath over months or years.So recent years , more and more researchers payattention to cure glioma and evaluate the prognosis with molecular biologymethod.MiRNA was a class of ~22bP small non-coding RNA which can regulategene expression at the Post-transcription level by perfect or imperfectcomplementarity to human the seed sequence in 3'UTR of its target gene. miRNAplays a very vital role in biological live process by regulating gene expression ,including growth , development, the genesis of diseases and so on. Recently ,more than one thousand of miRNA have been identified from many kinds oforganisms, however,only few miRNA target genes and their function have beenstudied clearly.The role of most miRNA in cells is still a riddle to berevealed.miRNA works by regulating gene expression.And as an endogenousmolecular which can regulate gene expression,miRNA has a great advantage intumor gene therapy.In the prophase of jobs,we study the miRNAs expression spectrum betweendifferent patho-grouping of glioma and normal brain tissue,by making use of themost complete miRNAs array and in the help of redundant human gliomaspecimen in our family . thereby we find the specifiy expression miRNAs in glioma,let-7d is one of this.Let-7d is one member of let-7 family,which has beenproved to be relation with proliferation and differentiation in leukemic cell.Thisreaserch emphasize on exploring the expression of let-7d in specimens of cerebralglioma and the influence on biological characteristics of human brain glioblastomaU87 cells.[Method[Method]Firstly, in situ hybridization (ISH) technique was applied to detect theexpression of let-7d in specimens of human cerebral glioma and normalbrain,buffy partical emerge in intracytoplasm stands for masculine, the morepositive staining,the higher expression of let-7d. Secondly, let-7doliogonucleotide was transfected to U87 cell by liposome before cultivating sometime, detecting the extinction luminance of each hole in 550nm wavelength toevaluate growth with MTT, to view cell apoptosis with hoechst ,to detect early cellapoptosis rate with AV-EGFP/PI method and observe invasion status of the cells ineach group.We then through analyzing the expression of Ras protein, to discusswhich downstream target gene was used by let-7d oliogonucleotide to educebiology effection.[Result]The expression level of let-7d miRNA in human brain glioma tissues waslower compared to normal brain tissues .Then through determine transfectionefficiency , we find that liposome mixd with let-7d oligonucleotide at 4:1 inquality could win the most high efficiency as well as the lowest cytotoxicity.TheU87 cells which were transfected with let-7d oligonucletide showed that growthwere slowed down, especially at 48h after tansfection; with hochest we observedtransfected cells chromatinic enrich ,fragmentation,nucleus exist bright bluefluorescence, the amount of early apoptotic cell obviously increased; Annexin V EGFP/PI showed that early apoptotic cell in experimental group account for14.22% compared with 5.85% in control group.Invation experiment showedexperimental group cell population in below-cave of Transwell cave obviouslyreduced. Finally, Western Blot was used to analysis the expression of Rasprotein,the expression result of experiment group was obviously lower thancontrol and blank group,which indicate that let-7d to produce a marked effectthrough regulate Ras.[Conclusion[Conclusion]miRNA let-7d may function as a Potential Growth Suppressor indevelopment and progression of human brain glioma,moreover,it probably educebiology effection through Ras Protein.let-7d can be gene therapeutic targetcandidate in human glioma. Antisense oligonucleotide transfetion is an effectiveapproach to inhibit over expressed miRNA and it has optimistic clinic trailprospects.