| Research BackgroundMicroRNA and GliomaMicroRNA is a non-coding,endogenous,single-stranded molecule of about 20-25 nucleotide sequences.Lee et al.first discovered and reported lin-4 small molecule in 1993 and named it MicroRNA(miRNA).MicroRNAs follow the principle of complementary bases and bind to the 3 ’-utr(untranslated region)of the target mRNA,resulting in the hindered translation or degradation of the target mRNA,and thus participating in the post-transcriptional regulation of gene expression.Recently,numerous studies have shown that microRNAs are involved in post-transcriptional regulation and play a key role in both tumorigenesis and biological development.The occurrence of human malignant tumors is closely related to the abnormal expression of MicroRNA.Compared with normal brain tissues,a large number of microRNAs were detected in human Glioblastoma specimens by cDNA array analysis,and their expressions were different.Therefore,it was analyzed that some of the microRNAs with up-regulated expressions had the function of oncogenes,while some of the microRNAs with down-regulated expressions played the role of tumor suppressor genes.In-depth studies on its functions have shown that MicroRNA is involved in the regulation of almost all biological processes of Glioma,including the important role in cell differentiation,proliferation,invasion,metastasis,apoptosis,neovascularization and drug resistance of Glioma.Previous studies have shown an abnormally low expression of miR-132 in Glioma tumor tissue.Therefore,it is of great significance to investigate the expression and specific mechanism of miR-132 in brain Glioma in order to further understand the pathogenesis of brain Glioma.Glil and brain GliomaIn patients with brain Glioma,abnormal expression of active Glil could be detected in the pathological specimens removed by surgery.Moreover,the higher Glil is expressed,the higher the pathological grade,proliferation index and recurrence rate of Glioma are observed.At the same time,the higher expression of Glil had an important influence on the biological behaviors of Glioma cells,such as cell cycle and apoptosis,chemotherapy and radiotherapy tolerance.The abnormal activation of HH/Glil signaling pathway is considered as one of the important molecular mechanisms of brain Glioma formation.The three members of the GLI protein family,Glil,Gli2 and Gli3,played different roles.In the GLI family,Glil is the only one that activates target genes,and as a target gene of HH pathway and a strong positive feedback regulatory molecule,Glil is widely expressed in various tumor tissues.Its up-regulated level is considered as a marker of HH pathway activation,and has been recognized as the most effective target to target and suppress this pathway.So far,the mechanism of the abnormal activation of HH/Glil signaling pathway in brain Glioma has not been fully understood.Some scholars found that miR-214 acts on the SUFU in zebrafish,and indirectly affect the activity of HH/GLI1 signal pathways.miR-125b in the brain tumor may be direct effects on HH/GLI1 signaling pathways of key genes of miR-125b in the medulloblastoma cell expression level decreased,further functional analysis showed that act directly on the 3’ UTR of the SMO gene,lowered the activity of signaling pathways.The hypothesis of this project is based on the above domestic and foreign research progress and the previous research results of our research group.Since the Hedgehog/Glil signaling pathway is highly conserved in the evolutionary process,and Micro RNA can participate in post-transcriptional regulation,can miR-132 affect the proliferation and invasion of Glioma by regulating the activity of the Hedgehog/Glil signaling pathway?the current study aims to explore the effect of miR-132 via targeted inhibition on Glil expression on the proliferation and invasion of Gliomacells.This paper was divided into three parts.The expressions of miR-132 and Gli1 in Glioma tissues and Glioma cell lines at different levels were detected by RT-PCR and Western blot respectively.The cells overexpressed miR-132,and the proliferation and invasiveness of Glioma cell line U251 were determined by flow cytometry and Transwellassay.Luciferase reporter gene analysis was performed to verify whether Glil was a downstream target protein of miR-132.Glioma cell line U251 was treated with miR-132 mimic(miR-132mimic)or si-Gli1,and the expressions of Gli1,e-cadagin,vimentin and cyclin D1 were measured to explore the molecular mechanism of mir-132 targeted regulation of Glil expression on the colonization and invasion of glioma cells..PARTI The expression and clinical significance of miR-132 and Glil in Glioma tissuesObjectivesTo explore the expression of miR-132 and Glil in Glioma tissues and the correlation analysis and clinical significance of different pathological levels.MethodsThe tissue samples collected in this experiment were all from the tumor tissues ofGlioma patients who underwent tumor resection in the neurosurgery department of shandong provincial hospital affiliated to shandong university from January 2016 to December 2016.Altogether 51 samples were collected and studied with 28 from male patients and 23 from female patients,ranging from 40 to 72-years-old.There were 17,22 and 12 cases in stage Ⅱ Ⅲ,and Ⅳ,respectively.Twelve normal brain tissue samples due to traumatic intracranial decompression were used as the control group.Six of the patients were male and six were female,and the sample included patients between the ages of 38 and 69.There was no significant age or sex difference between the two groups.Each specimen was divided into two sections,one for rt-pcr and Western blot,and the other for pathological examination.Results1.RT-PCR showed that miR-132 was expressed in both normal brain tissues and Glioma tissues of patients with brain contusion*while the expression in Glioma tissues was significantly decreased.With more advanced TNM staging and pathological grading,its expression decreased further.The difference was significant(p<0.05).2.RT-PCR showed that Glil mRNA was expressed in both normal brain tissue and Glioma tissue of patients with brain contusion.Compared with normal brain tissue of patients with brain contusion without tumor lesions,Glil mRNA expression in Glioma tissue was significantly increased.Glil expression increased with more advanced pathological grade.The difference was significant(p<0.05).3.Compared with the control group,western blot results also showed a significant increase in Glil protein expression in Glioma tissues,and the level was correlated with pathological grade.Conclusions1.MiR-132 and Glil mRNA were expressed in normal brain tissues and Glioma tissues of patients with brain contusion,but miR-132 was down-regulated and Glil mRNA was up-regulated in Glioma tissues.2.Glil protein expression was significantly increased in Glioma tissues,and the level was correlated with pathological grade3.The expression of miR-132 and Glil in Glioma is related to the degree of malignancy of Glioma.PART ⅡEffect of miR-132 overexpression on invasion and proliferation of Glioma cells.ObjectivesTo investigate the effect of overexpression of miR-132 on biological behaviours of Glioma cells.MethodsHuman brain Glioma cell line U251 was cultured in vitro and divided into two groups.Then the exogenous synthesized miR-132 mimic(miR-132mimic)were transfected into human brain Glioma cell line U251 through liposomes with the negative control group(NC).The cells overexpressed miR-132 to form an overexpressed cell line,and then the effect of miR-132 on the proliferation and invasion of U251 cells was determined by flow cytometry and Transwell,and the role of miR-132 in the proliferation and invasion of Glioma cells was studied.Results1.Flow cytometry showed that transfection with miR-132 mimic reduced proliferation efficacy of U251 cells by 34.5%.2.Transwell assay showed that compared withmiR-NC transfected cells?the invasion ability of U251 cells was significantly decreased after miR-132 overexpression(p<0.05).Conclusions1.Overexpression of miR-132 can reduce the proliferation efficacy of Glioma cells.2.The invasion ability of Glioma cells was significantly reduced after miR-132 overexpression.PART ⅢMiR-132 decreases the proliferation and invasion of Glioma cells by inhibition of GlilObjectivesTo explore the mechanism by which miR-132 attenuates Glioma cell proliferation and invasion by inhibiting Glil.MethodsExperimentalgroup Ⅰ:miR-NC and pmiR-Glil-mut mutant plasmid;Experimental group;2:miR-NC and pmiR-Glil-wt wild-type plasmid;Experimental group;3:miR-132mimic and pmiR-Glil-mut mutant plasmid.Experimental group;4:miR-132mimic and pmiR-Glil-wt wild-type plasmid.were transfected into HEK293T cells.Double luciferase reporter gene analysis was used to verify whether Glil was a downstream target gene of miR-132.Glioma cell line U251 was treated with miR-132 mimic(miR-132mimic)or si-Glil,and the expressions of Glil,e-calcinin,vimentin and cyclin D1 were measured.Results1.Online gene prediction of miR-132 targeting and inhibition of Glil expression revealed complementary binding sites between miR-132 and 3’-utr of Glil mRNA.The dual luciferase reporter assay showed that the transfection of miR-132 mimic significantly inhibited the relative luciferase activity in HEK293T cells,The difference was statistically significant(p<0.05),indicating the targeted regulation between miR-132 and Glil mRNA.2.Overexpression of miR-132 targeted and inhibited Glil expression,and inhibited proliferation or invasion of U251 cells.Compared with themiR-NC transfection group,the mRNA expressions of miR-132 and e-cadherin were significantly increased after the simulated transfection of miR-132.However,the mRNA levels of Glil,Vimentin and Cyclin D1 were significantly decreased.The difference was statistically significant(p<0.05).Western blot results showed a significant increase in e-cadherin and a decrease in Glil,Vimentin and Cyclin D1 levels.3.SiRNA interference of Glil significantly inhibited the proliferation and invasion of U251 cells.Compared with the si-nc transfection group,the down-regulation of Glil significantly increased the expression of e-cadherin mRNA,while the mRNA levels of Glil,Vimentin and Cyclin D1 were significantly decreased.The difference was statistically significant(p<0.05).Western blot shows similar results.Compared with si-NCtransfected cells,flow cytometry showed that the transfection of si-Glil reduced the proliferation efficacy of U251 cells by 40.6%and significantly inhibited the invasion efficacy of U251 cells,compared with si-nctransfected cells.The difference was statistically significant(p<0.05).Conclusions1.Complementary binding sites exist between miR-132 and 3’-utr of Glil mRNA.Targeted regulation exists between miR-132 and Glil mRNA.2.Transfection with miR-132 mimic or si-Glil significantly inhibited the expression of Glil,Vimentin or Cyclin D1 in U251 cells,up-regulated the expression of e-cadherin,and inhibited cell proliferation and invasion.3.The overexpression of miR-132 can inhibit the proliferation or invasion of Glioma cells by targeting the inhibition of Glil expression.4.In conclusion,both miR-132 and Glil are expressed in glioma tissues,and the expression level is related to the degree of malignancy of glioma.Overexpression of miR-132 can reduce the proliferation and invasion ability of glioma cells.In conclusion,combined with this study,this study proposed for the first time that the overexpression of miR-132 inhibits the proliferation or invasion of glioma cells by targeting the inhibition of Glil expression,so miR-132 is also a tumor suppressor gene.It may provide a new target for the treatment of glioma. |
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