| The gliomas is the most common type of primary tumor of central nervous system,and the invasion and recurrent rate of malignant gliomas are significantly higher than other tumors.Especially,the glioblastoma(GBM)showed a bad outcome and propensity to relapse,which was difficult to realize gross total resection and greatly affected the life and quality of patients,with a median life cycle around 1 year.Though undergoing the standard therapy combining surgery,radiotherapy and chemotherapy,the outcome for GBM was still pessimistic.Under the guiding of the current precision medicine,the traditional histologic subtyping gradually transmitted to the new diagnosis system based on the molecular genetic characteristics,the therapeutic strategy has turned to the precise targeting therapy by regulating the key molecular in the genesis and development of the tumors.microRNA is a kind of endogenous non-coding molecules composed of 18 to 25 nucleotides.The gene sequence of microRNA was located between general protein gene and even protein gene introns,and pri-microRNA(primary microRNA)was translated by RNA polymerase ? or polymerase ?.Pri-microRNA was cut into pre-microRNA(precursor microRNA)with around only 70 nucleotides in the nucleus by Drosha/DGCR8 protein complex.After running out of nuclear by output protein V,premicroRNA was further modified by RNA enzyme Dicer,finally become mature microRNA with only 18 to 25 nucleotides.They were inset in the RNA induce silence complex(RISC),when it combined 3 'untranslated of target genes region by the principle of base pairing,it induced the degradation of target genes m RNA or inhibit translation of the target genes m RNA.Each microRNA can regulate expression of around 200 target genes,can regulate the complicated process such as tumorgenesis,thus has significant antitumor potential.Many studies have shown that microRNA are differentially expressed in glioblastoma tumor tissues and normal control tissues,and the expression profiles of these microRNAs contribute to further classification of gliomas.Different patients have different levels of microRNA expression,different treatment responses,and different prognosis and outcomes.Therefore,microRNA can assist in grading,diagnosing and predicting the outcome of the clinical process of patients with glioblastoma.MicroRNA-497 is one of the members of microrna-15/16/195/424/497,a highly conservative microRNA located on chromosome 17p13.Previous studies have confirmed that microRNA-497 has been down-regulated in many malignancies,such as neuroblastoma,prostate cancer,non-small cell lung cancer,nasopharyngeal carcinoma,hepatocellular carcinoma,and breast cancer.The expression of microRNA-497 in human breast cancer was significantly lower than that of non-cancerous breast tissue,and the breast cancer patients with high expression of microRNA-497 had significantly better 5 years progression-free survival(PFS)and overall survival(OS)than those with low expression of microRNA-497.Single factor and multivariate analysis indicated that low expression icroRNA-497 was the independent influencing factor of poor prognosis in breast cancer patients.Giulia Regazzo et.al reported that expression level of microRNA-497 in serum of glioblastoma patients was significantly lower than patients with low-grade gliomas,indicating the expression of serum microRNA-497 can serve as a new diagnostic marker for clinical application.However,whether the role of microRNA – 497 in the role of gliomas tumor tissues are the same,whether microRNA – 497 was associated with the grade of tumor,patients' survival and prognostic outcome remains to be unknown,whether microRNA – 497 ca be used as a stratified index for disease or a therapeutic targets,needs further research.The present study will conduct the exploration from three aspects: clinical specimens,in vitro studies and in vivo research.Part I: The expression and clinical significance of microRNA-497 in glioma.Objective: To explore the expression of microRNA-497 in patients' glioma tissue,analyze the relationship between microRNA-497 and clinical pathological features of glioma,and further discuss the clinical significance of microRNA-497 in glioma.Methods: A total of 110 cases of glioma in the experimental group and 30 cases of non-tumor brain tissue in the control group were collected and sorted.The specimens were collected from 2005-2010,in department of neurosurgery of shanxi medical university first affiliated hospital,all specimens was obtained from patients with their families informed and signed informed consent for the experimental studies,and this study has been approved by the first affiliated hospital of shanxi medical university ethics committee.All of these patients has not been received radiotherapy,chemotherapy and other anti-tumor treatment before surgery,postoperative follow-up is fully completed,with data intact,and the average follow-up time was 30 months(ranging from 2 to 116 months).Rt-PCR was used to detect the expression of microRNA-497 for all experimental specimen,Cox proportional hazards models was used to evaluate the association of age,sex,tumor location,the recurrence rate,microRNA-497 expression level and the patients' overall survival.Results: By RT-PCR detection,microRNA-497 was found to be abundant in non-tumor tissues.Compared with glioma tissues,microRNA-497 had higher expression levels in non-tumor tissues.The study found that the expression levels of microrna-497 were also different in tissues of different grade glioma.microRNA-497 had significantly lower expression levels in the high-grade glioma tissues than in the low-grade glioma tissues.To analyze the survival condition of patients through the chi-square test,Kaplan Meier-inspection analysis,and Cox proportional hazards regression model was used for univariate and multivariate analysis(when the P value is less than 0.05 think results have significant differences),the study found that: the lower expression of microRNA-497 was significantly associated with high grade glioma(P < 0.001)and low Kamofsky performance score(P = 0.02),while has no association with age,sex,tumor location,and the recurrence rate for patients(P > 0.05).For high-grade glioma patients,microRNA-497 low expression group had a worse outcome than microRNA-497 high expression group((P < 0.001),which was absent for low-grade glioma patients.Conclusion: microRNA-497 had significantly lower expression levels in high-grade glioma tissues than in low-grade glioma tissues.For high-grade glioma patients,the prognosis of microRNA-497 low expression group was worse than that of the high expression group.Part II: In vitro study of regulatory role of microRNA-49 on angiogenesis and invasion of glioblastoma by targeting VEGFObjective: Using bioinformatics technology to query action target of microRNA-497,verify the regulation effect of microRNA-497 on VEGF.The effects and mechanisms of microRNA-497 on tumor angiogenesis and invasion in glioma cell lines were observed and discussed.Methods: Using bioinformatics prediction technology(http://www.targetscan.org)to predict the targeting relationship between microRNA-497 and VEGF-A,then the luciferase report system was used to detect regulation function of microRNA-497 on VEGF.U87 MG cells and HBMVEC cells were cultured to construct plasmid expressing microRNA-497 and the control plasmid.U87 MG cells were transfected with microRNA-497 expression plasmid and control plasmid,the Tube Formation experiment was used to test the influence of Metastable cell line(microRNA-497 mimics-U87 / microRNA-497 nc-U87)on HMBVEC cell,Transwell invasion experiment was used to analyze the influence of microRNA-497 on glioma cells invasion ability,and Western blot was used to test the expression of VEGF in microRNA-497 mimics-U87 and microRNA-497 nc cells.Results: Bioinformatics technology predicted microRNA-497 has targeted relationship with VEGF-A,dual luciferase detection show that the joint transfection microRNA-497 type combined with wild VEGFA 3 'UTR,inhibit the fluorescent report activity,while inhibitory effect is not obvious when combined with the mutant VEGFA 3'-UTR.By adopting co-culture of U87 MG with stable transfection of microRNA-497 overexpression and the brain microvascular endothelial cells(HBMVEC),it was found that over-expression of microRNA-497 can induce HBMVEC morphological differentiation,significantly reduce the number of sample tube cavity structure branch point,lumen formed structure is not complete,there are scattered around the cell.Western blot analysis showed that the expression level of VEGF in the microRNA-497 group U87 MG cells was significantly decreased(P < 0.05).Transwell experimental analysis showed that the invasion ability of U87 MG cells in the microRNA-497 group was significantly lower than that in the control group under the microscope(P < 0.05).Conclusion: microRNA-497 can targeted regulated VEGF,inhibit the expression of VEGF in U87 MG cells in vitro,significantly reduce the for mation of HBMVEC tube cavity,and significantly inhibit the invasion ability of U87 MG cells.Part III: In vivo study on inhibitory effect of microRNA-497 on proliferation of glioblastoma and the angiogenesis.Objective: Animal study have clearly demonstrated the effect of microRNA-497 on tumor formation in vivo,and also verified the changes of proliferation and angiogenesis in tumor cells,and the effect of microRNA-497 on tumor proliferation and angiogenesis.Methods: Take suspension of steady turned microRNA-497 mimics-U87 glioma cell line and microRNA-497 nc-U87 and normal cultured glioma U87 MG cells,which was injected into the right lower limb lateral subcutaneous tissue for nude mice.When tumor growed to the 25 th day,the e xecution of tumor mice,dissection of tumor tissue,weighing measurement were done.Immunohistochemical staining method was used to analyze the expression of ki-67,VEGF and CD34 in tumor tissue samples of different groups.Results: For the group with injection of microRNA-497 mimics-U87 cell,the tumor size was significantly reduced(P < 0.05)than the microRNA-497 nc-U87 cell group and U87 MG cells,while for the microRNA-497 nc-between U87 cell group and U87 MG cells,the size of the tumor had no significant difference.Immunohistochemical analysis showed that the expression levels of ki-67,VGEF and C D34 in microRNA-497 mimics-U87 group were significantly lower than that of microRNA-497 nc-u87 group and U87 MG group(P < 0.05),while there was no significant difference between m microRNA-497 nc-u87 group and U87 MG group.Conclusion: Overexpression of microRNA-497 can significantly inhibit the in vivo tumorgenesis of U87 MG cells in nude mice,and reduce the proliferation activity and angiogenesis of tumor cells. |
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