| Human brain glioma is the most common primary brain tumor of the central nervous system,accounting for 40% to 43% of intracranial tumors.The clinical treatment of glioma is very poor at home and abroad.In the past 30 years,there was no significant improvement in the high recurrence rate and low cure rate of malignant glioma.Glioma tumor cells have the characteristics of aggressive growth,rapid proliferation and easy to relapse.The median survival time of high-grade gliomas was only 14-16 months,and the 5-year survival time of it is less than 50%.Glioma is a kind of diseases with abnormal expression of many genes,including the over expression of oncogenes,the mutation or deletion of tumor suppressor genes.Indeed,it includes abnormalities of multiple signal pathways.Surgical treatment,postoperative concurrent chemoradiotherapy and late adjuvant chemotherapy were less effective,and postoperative concurrent chemoradiotherapy and late adjuvant chemotherapy may have toxic and side effect on the central nervous system.Thus,the treatment of glioma is still difficult in the department of neurosurgery.Therefore,it has become a hot topic in the basic research of glioma,to fundamentally correct the gene therapy related to the occurrence and development of glioma.MicroRNAs(miRNAs)is a class of endogenous non coding RNA,which have about 19-25 nucleotides(NTS).Through the specific,completely or incompletely,binding of the 3 ’UTR non transcribed region of the target gene,the mature miRNAs regulate the expression level of the gene by post-transcriptional.MiRNAs usually regulate multiple or a group of target genes with similar functions,which makes miRNAs "natural" superior to the single gene therapy that often used in current gene therapy.It had been shown that miRNAs could regulate tumorigenesis and development of glioma,by modulating the signal pathways involved in the target genes,and promote the malignant biological behavior of tumors.MiRNAs plays a decisive role in the occurrence,development and prognosis of glioma cells.Many different types of miRNAs are expressed in glioma cells,which can induce apoptosis or inhibit proliferation of glioma cells,by up-regulating or down-regulating the corresponding miRNAs,or affect the migration and invasion of glioma cells.Therefore,the study of the expression of miRNAs in glioma cells may provide a new strategy for the diagnosis and treatment of glioma.MiR-451 is located on chromosome 17q11.2.In recent years,abnormal expression of miR-451 has been reported in breast cancer,ovarian cancer,lung cancer,gastric cancer and colorectal cancer and glioma.Recent studies have shown that miR-451 is closely related to the development and progression of glioma.MiR-451 can inhibit the invasion and migration of glioma and play a role in tumor suppression,but the specific mechanism is still unclear.The preliminary study of this experiment shows that miR-451 plays an important role in inhibiting the invasion and migration of glioma cells,but which signal pathway plays a role in inhibiting the invasion and migration of glioma has not been described.Through bioinformatics analysis,we found that IKK-βwas one of the miR-451 target genes.The activation of IKK is a critical step in the activation of NF-кB classical pathway.Research shows that NF-кB has high expression in early malignant glioma.NF-кB signaling pathway has become an important target for the treatment of malignant glioma.In this study,we used miR-451 mimics to act on malignant glioma cell lines,and,by luciferase experiment,IKK-β was confirmed to be the target gene of miR-451.Through in vivo and in vitro experiments,the molecular mechanism of miR-451,by targeting IKK-βto regulate NF-кB/Snail signaling pathway,were confirmed.Therefore,it can inhibit the invasion and migration of glioma.So as to provide a new clue for miR-451 targeted therapy of glioma,and provide a new strategy for clinical treatment of glioma.The research is divided into three parts:In the first part,the relationship between miR-451 and IKK-β gene in glioma was studied by luciferase assay.We prepared the luciferase reporter plasmids,which contain IKK-βbinding sites and mutation sites.The Binding sites of IKK-β and the sequences of binding site mutations were cloned into the pGL3 reporter plasmids,which could sequence the correct orientation and sequence of it.LN229、U251 and U87 glioma cells were co-transfected with pGL3-IKK-β3’UTR-Wild/Mut and miR-451 mimics.The results showed that IKK-βcould bind the 3’UTR region of miR-451.By up-regulating the level of miR-451,it could decrease the activity of the wild-type IKK-β-luciferase.It shows that IKK-βis a direct target of miR-451 in glioma cells.In the second part,in vitro,we studied the negative effect of miR-451 on the invasion and migration of glioma,by targeting IKK-β,regulating NF-кB/Snail signaling pathway.I used real-time quantitative PCR to determine the effect of miR-451 on mRNA expression of IKK-β in LN229,U251 and U87 cell lines and miR-451 overexpressing LN229,U251 and U87 cell lines.The migration ability of LN229,U251 and U87 cells,of which,the IKK-β was knocked down by miR-451,were detected by the scratch test and Transwell test.The results showed that,through targeting IKK-β,miR-451 inhibited the activity of NF-кB/Snail signaling pathway;the protein encoded by IKK-β,NF-кB(p50,p65)and Snail gene decreased markedly;the ability of invasion and migration of LN229,U251 and U87 cells was significantly decreased after the knockdown of IKK-β.In the third part,in vivo,the role of miR-451 in the inhibition of glioma invasion and migration,through targeting IKK-β,regulating NF-кB/Snail signaling pathway,was investigated.In vivo,we prepared intracranial models of heterologous glioma in nude mice.We used stereotaxic injection to establish the intracranial glioma model.Nude mice were randomly divided into black control group,lentiviral miRNA negative control group(LV-miRNA-NC)and lentiviral miRNA-451 group(LV-miRNA-451)in vivo imaging.The survival rate of each group was recorded and the survival curves were analyzed by Kaplan-Meier method.HE and immunohistochemical staining were performed in the brain tissue of nude mice.Compared with Black control group and LV-miRNA-NC group,the LV-miRNA-451 group showed low fluorescence intensity and a longer survival time.The expression of IKK-β,NF-кB(p50,p65)and Snai in IKK-β/NF-кB/Snail pathway in LV-miRNA-451 group was decreased by immunohistochemistry.These results demonstrate that,through targeting IKK-β,regulating NF-кB/Snail signaling pathway in glioma,miR-451 can inhibit the invasion and migration of glioma.Conclusion: 1.IKK-β is the target gene of miR-451 in glioma.2.After transfecting LV-miRNA-451 into glioma cells,with lentivirus as carrier,miR-451 could be expressed steadily in glioma cells.3.The results showed that,by targeting IKK-β,regulating NF-кB/Snail signaling pathway,miR-451 could inhabit the invasion and migration of human glioma cells.4.MiR-451 is a tumor suppressor gene.It is a potential target for gene therapy of glioma. |
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