| [Background]Glioma,which originates from the neuroepithelial ectoderm,is the most common primary malignant tumor of the central nervous system,accounting for about 40%-65%of intracranial tumors.Glioma is highly heterogeneous and invasive,characterized by uncontrolled cell proliferation,diffuse infiltration,anti-apoptosis and induced necrosis.Patients in China is increasing year by year and tends to be younger.The pathological type is more common in pleomorphic glioma.According to histopathological features,primary gliomas are classified into 4 gradesby WHO.The grade is associated with the degree of malignancy.Low-grade gliomas include? and ?grades,and high-grade gliomas include? and IVgrades.With the advancement of medical technology,the majority of glioma patients survived less than 12 months despite the combination of multiple treatments including surgical resection,radiation therapy,and drug chemotherapy.Becauseglioma often occurs in the deep intracranial and functional areasof brain,and infiltrates into the surrounding brain tissue,the unclear boundary with normal brain tissue makes it difficult to completely remove glioma.The recurrence of the tumor is the main reason for death of the patient.The multiple factors and steps have been implicated in thepathological process of gliomaformation and invasion.Therefore,it is an urgent problem to explore the molecular target of early diagnosis and treatment of glioma.MicroRNAs(miRNAs),a class of non-coding RNAs with biological regulatory functions in eukaryotes,are composed of aboutl8-25 nucleotides in length.MiRNAs do not affect the biological function by directly encoding protein,but play biological regulation functions bycompletely or incompletely pairing with the 3'-UTR of the targeted mRNAs to inhibit the transcription or promotethe degradation of the target genes.Mature miRNAs are assembled into ma-induced silencing complex from primary transcripts produced by a series of nuclease shearing processes.The complex degrades the target mRNA or represses translation of the target mRNA by base complementary pairing.By the early 1990s,miRNAs were discovered and reported successively.However,as the research progressed,the abnormal expression of miRNAs aredemonstrated to be involved in the occurrence and progression of various tumors and play important roles.MiRNAs act as oncogenes or tumor suppressors toregulate tumor formation and development by mediatingthe signaling pathways implicated by their target genes.Up-regulation or down-regulation of the corresponding miRNAs can induce apoptosis or inhibit proliferation,migration and invasion of tumor cells.Significant changes in the expression of various miRNAs in malignant tumor tissues also demonstrate that miRNAs are closely related to the occurrence and development of tumors.MiRNAs alter the biological behavior of tumor cells by regulatingthe corresponding proteins.For example,miR-107 overexpression inhibits cell proliferation in lung cell carcinoma;miR-15 and miR-16 induce apoptosis in chronic lymphocytic leukemia by targeting the apoptosis-inhibiting gene BCL2;miR-21 and miR-155 are up-regualted whilemiR-lOb and miR-125bare down-regulated in breast cancer,,suggesting that they act as oncogenes and tumor suppressorsin breast cancer,respectively;miR-150 blocksmetastasis and invasion of ovarian epithelial cancerby targeting ZEB1 expression;Main cellular and functional alterations of miRNAs involved in GBM pathogenesis.MiR-184 is located in the 25.1 segment of the q-arm of human chromosome 15,and its corresponding transcript has a molecular weight of 84 bp.MiR-184 is originally discovered in the mesoderm,ectodermal and pre-embryonic layers of drosophila embryosby Aboobaker.The mature miR-184 in the cell is mainly expressed asa single-stranded miR-184-3p.Studies have shown that miR-184 has different effects on a variety of cells,such as promoting the proliferation of tongue squamous cell carcinoma and hepatocellular carcinomacan,butinhibiting the proliferation of renal cell carcinoma and neuroblastoma cells.At present,few reportsaboutthe biological function of miR-184 in glioma are found,and its role in the progression of glioma has not been uniformly concluded.In this study,whether the differential expressionof miR-184 involved in glioma is associated with the pathological grade and pathological type of glioma and whether miR-184may become an important molecular marker of glioma will be investigated.MMPs are a family of endopeptidases that are dependent on zinc ions and have the ability to degrade extracellular matrices.They can promote the invasion and metastasis of tumor cells by degrading the basement membrane and enhancing the formation of tumor blood vessels.Whether MMP-2 and MMP9 are also differentially expressed in glioma compared with normal brain tissue,and whether it is closely correlated with miR-184 is also an experimental focus.In view of this,this study intends to analyze the expression of miR-184 in glioma and its influence on invasion and metastasis of glioma cells,which canprovide reference for clinical diagnosis and treatment.This study includes three parts:the first part:the relationship between different pathological grades of glioma and miR-184 expression;the second part:the effect of miR-184 on the proliferation,migration and invasion of glioma cell line U87;Part III:Expression of MMP-2 and MMP9 in invasive gliomas and theirrelationship with miR-184 expression.[Objective]1.To explore the differential expression of miR-184 in gliomas of different pathological grades,and to analyze the correlation between miR-184 expression difference and survival prognosis.2.In vitro experiments were performed to observe the changes in cell biological function of miR-184 mimics and miR-184 inhibitors(anti-miR-184)transfected into U87 glioma cells.3.The expression of metalloproteinases MMP-2 and MMP9 in gliomas and their relationship with miR-184 were analyzed.4.To explore the target genes associated with MRP-2 and MMP9 and their modes of action.[Materials and Methods]1.Collection of glioma tissue and normal brain tissue specimens,acquisition of U87 glioma cell line:A total of 40 patients with glioma diagnosed in our hospital from January 2015 to January 2016 were collected,and 10 patients were included.Normal brain tissue,the sex and age of the two groups are comparable.Human glioma cell line U87 cells were purchased from the Cell Bank of the Chinese Academy of Sciences.2.RT-PCR and immunohistochemistry experiments:Analysis of the differential expression of miR-184 in normal brain tissue and glioma.At the same time,the median survival time of the 75%quantile array and the median survival time of the miR-184 expression positive intensity of the 25%quantile array were compared with the miR-184 expression positive intensity.3.Resuscitation,culture,passage and transfection of U87 cell line by U87 cell line,culture,passage and transfection.MTT assay combined with colony formation assay to detect the effect of miR-184 on glioma cell proliferation;cell scratch assay to detect the effect of miR-184 on glioma cell migration;Transwell invasion assay to detect miR-184 on glioma cells The impact of the invasion.4.Glioma has strong invasiveness.It may be related to metalloproteinases(MMPs).Immunohistochemical staining of glioma brain tissue and normal brain tissue and MMP2 and U87 glioma cell culture.Amplification of MMP9 mRNA,analysis of differences in the content of MMP2 and MMP9 in tissues and differences in gene expression.Finally,Western Blot was used to detect the difference in protein expression between MMP2 and MMP9 in miR-184-molded?miR-184 inhibitor transfection groups.The results showed that miR-184 was positively correlated with MMP2,and TargetSca target gene database was used to predict that miR-184 regulates MMP2 target gene as matrix metalloproteinase-2 inhibitor(TIMP-2).use TIMP-2 expression was detected by RT-PCR and Western Blot.At the same time,luciferase double reporter assay was used to verify that TIMP-2 is a target gene for miR-184 to alter MMP2.[results]1.miR-184 was detected in both glioma and normal brain tissues,and the difference was statistically significant(P<0.05).The positive expression level of miR-184 was positively correlated with glioma grade.The higher the malignant degree of glioma,the stronger the positive expression of miR-184.2.The median survival time of the 75%quantile array of miR-184 positive expression was significantly shorter than that of miR-184 expression positive intensity 25%quantile array[(8.0±0.4)ratio(12.0±0.1)month,?2=12.480,P<0.001].3.miR-184 transfection was positively correlated with proliferation,colony formation,invasion and migration of tumor cells.4.Compared with MMP-9,there was no significant difference in expression between glioma and normal tissues.MMP-2 expression level in glioma was higher than that in normal tissues.RT-PCR and Western blot showed that miR-184-mimetic transfection significantly promoted the expression of MMP2,while miR-184 inhibitor transfection inhibited MMP2 expression.The expression level of MMP9 was not significantly different among the three groups.5.The TargetSca target gene database suggests that the potential target gene for miR-184 to promote MMP2 expression is TIMP-2.The expression of TIMP-2 was significantly decreased by U87 transfection of miR-184 by real-time fluorescent quantitative PCR.The expression of TIMP-2 protein in the miR-184 group was significantly lower than the control groups,and the expression of TIMP-2 was negatively correlated with the target gene TIMP-2.Luciferase reporter gene experiments further demonstrated that miR-184 can directly act on TIMP-2 to affect MMP2 expression.[Conclusions]1.Compared with normal brain tissue,miR-184 is up-regulated in glioma and positively correlated with glioma pathological grade,suggesting that miR-184 can be used as a potential marker for pathological grading of glioma.The median survival time of the 75%quantile array of miR-184 positive expression was significantly shorter than the 25%quantification of the positive expression of miR-184,indicating that the expression level of miR-184 is related to the median survival of glioma patients.Negative correlation.2.Overexpression of miR-184 can positively promote the proliferation,cloning,invasion and migration of glioma.It suggests that it may be positively associated with enzymes that degrade the basement membrane.3.The expression levels of miR-184 and MMP-2 were positively correlated in U87 glioma cells.Prompt related target genes4.TIMP-2 is the target gene locus for miR-184 to regulate MMP2;In summary,glioma is the most common primary central nervous system malignant tumor,accounting for 40%-65%of intracranial malignant tumors.It is highly heterogeneous and invasive with a very poor prognosis.Exploring the entry point of molecular targets for early diagnosis and treatment of glioma is an urgent problem to be solved.miRNAs are assembled from a nuclease by a series of nucleases and assembled into an RNA-induced silencing complex(RISC)to recognize target mRNAs by base-pair pairing and direct silencing complexes.Degrading target mRNA or repressing translation of target mRNA.Mature miR-184 is mainly expressed in single-stranded miR-184-3P,and miR-184 promotes or inhibits the progression of various tumors.At present,there are relatively few studies on the biological function and regulation mechanism of miR-184 in glioma.Therefore,whether the expression of miR-184 in glioma is related to pathological grade and pathological type,and whether it is associated with glioma Regarding the high invasiveness,whether it can become an important molecular marker of glioma,these have not been determined so far,MMPs are considered to play an important role in the invasion and metastasis of malignant tumors.The association of miR-184 with MMPs has not been addressed in previous studies.The effect of miR-184 on the differential expression of miR-184 in different pathological grades of glioma and the difference in miR-184 expression were correlated with survival prognosis.It was found that the degree of miR-184 expression was positively correlated with glioma grade.miR-184 is positively correlated with proliferation and invasion of glioma cells.In addition,MMP2 was significantly elevated during glioma invasion and was negatively regulated by TIMP-2,a target molecule downstream of miR-184.The expression of miR-184 in gliomas and its effect on invasion and metastasis of glioma cells may provide new targets for clinical diagnosis and treatment.It should be noted that this study can not fully simulate or in vivo the cell survival environment,and can not assess the impact of complex cell surrounding environment.The next step is to increase the live animal experiment and observe the miR-184 changes in the biological activity of tumor cells in vivo. |
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