| Neuroglioma is a common primary intracranial tumor in the Department of Neurosurgery,and it is also one of the tumors for arduous treatment.Glioma has the characteristics of high malignant degree,radical aggressive,easy recurrence and so on.But natural,glioma is a polygenic disorders disease,it makes cancer cell escape the natural steps of cell death through overexpression of protooncogene and with the mutation and deletion of anti-oncogenes.Come so far,the main method for glioma clinically treatment are operation,radioactive therapy and medical chemotherapy.Despite the above methods have made great progress,but the prognosis of patients is still pessimistic.With the advance of basis research of glioma,an abundant researches revealed the biological mechanisms of glioma,including the discovery of cancer stem cells,termed glioma stem cells(GSC).Some of these researches include the delineation of molecular heterogeneity both between tumors from different patients as well as within tumors from the same patients.Therefore,study on its molecular pathogenesis at the level of molecular in tumor cell,and find breakthroughs from tumorigenesis and development,that is the key to unearth the more effective,more precise and faster anti-cancer therapies.miRNAs are one member of the non-coding RNA family,its contains 18-25 bases and negatively regulate protein-coding gene expression through post-transcriptionally.Researches showed that miRNAs played an integral role in neuroglioma occurrence and development.They regulate various of signal pathways associate with tumorigenesis,and effect tumor growth and progression,cell aggression and response to treatment.miR-320 a is located in chromosome 8p21.3 and aberrantly low-expressed in cancers of liver,breast and colorectum.Studies indicated that miR-320 a had many types of cell bio-effects,such as regulating cell proliferation,invasion and migration.In tumor,increasing evidences demonstrated that miR-320 a have many targets,but its effects and functions in glioma still needs to deeply explore.Aquaporin 4(AQP-4)is one member of the aquaporin family which is special abundant in brain tissues and with various functions in different structures.Generally,AQP-4 is defined as a pure water molecules transport protein that participates in cerebral edema,cell water regulation and cerebrospinal fluid formation in brain tissue.But actually,the functions of AQP-4 are more than the above,it could regulate the dissociation and recombination of cell membrane to promote cell motility,such as astrocyte.It worth noting that matrix metalloproteinase 9(MMP-9)has the same function with AQP-4 in cell invasion and migration.Therefore,we proposed a hypothesis: whether miR-320 a would regulate the glioma cell aggressiveness,and what the function of AQP-4 and MMP-9 in this process.ObjectiveIn this study,we used glioma cell lines U87 and U251 treated with miR-320 a and miRNA negative control(miR-NC)mimics to explore the cell invasion and migration.Based on bioinformatics analysis,explore the action mechanism of miR-320 a,and the functions of AQP-4 and MMP-9 in cell aggressiveness.By the experimental results,hope that we could provide more theoretical basis and drug targets for clinical treatment of glioma.MethodChinese Glioma Genome Atlas(CGGA)data analysis were used to distinguishing the difference of miR-320 a expression in low and high grade of glioma and the difference of overall survival between high and low expression of miR-320a;Targets of miR-320 a in AQP-4 mRNA 3’-UTR were analyzed by miRbase,Targetscan and microRNA.org,then synthesized the sequence and constructed the plasmid,dual luciferase report system was used to validated the results;The expression of AQP-4 in human glioma and tumor pathological were observed by histochemical and immunohistochemical stain;Human glioma cells U87 and U251 were cultured in vitro and then transfected with miR-320 a and miRNA negative control mimics respectively,after 48 hours going further experiments;The transfection efficiency was identified by qPCR;The cell dynamic were determined by Transwell assay;AQP-4 and MMP-9 mRNA expression were tested by qPCR;The expression of AQP-4 and MMP-9 proteins were detected by western blot;Immunofluorescence(IF)was employed to determine the location and expression of AQP-4 in U87 and U251 cells;Down-regulation of AQP-4 gene by RNA interference,qPCR and western blot were used to detect the expression of AQP4 after 48 hours;The invasion and migration abilities of cells were determined by Transwell assay.Results1.CGGA data analysis showed that miR-320 a is significantly decreased in WHO grade Ⅲ and Ⅳ glioma samples compared with WHO grade Ⅱ(p<0.01).The overall survival of high expression miR-320 a were distinctly overmatches vs low expression patients(p<0.01).2.Bioinformatic analysis found three target regions of miR-320 a in AQP-4 mRNA 3’-UTR.The dual luciferase report system identified the region of 848-870 with in 2000 bps was the effective target region of miR-320 a.3.The glioma(WHO grade Ⅱ and Ⅳ)and normal brain tissues were examined by hematoxylin-eosin(HE)and immunohistochemical(IHC)staining.Compared with normal brain tissue,the HE staining showed that with the increases of tumor malignancy: tumor cell pleomorphism clearly,form tumor giant cell,nuclei were darker and more irregularly sized,and observed mitotic nuclei.And the IHC staining indicated that the expression of AQP-4 was markedly increased and polar distribution could be observed in malignant glioma,and was usually proportional to WHO pathological grading.4.In vitro,the miR-320 a was significantly upregulated compared with miR-NC group(p<0.01)after transfection,and there was no difference between miR-NC and normal control group.Transwell assay showed that the overexpression of miR-320 a could inhibit the cell invasion and migration obviously compared with the miR-NC group.5.The experiments of qPCR and western blot identified that AQP-4 were down-regulated by overexpression of miR-320 a,but the expression of MMP-9 did not changed.6.Results of WB and qPCR proved the down-regulated the expression of AQP-4 gene had effectively inhibit the expression of AQP-4,and transwell assay showed that the Down-regulated the expression of AQP-4 could inhibit the cell invasion and migration.7.The results of IF showed that AQP-4 located in cell membrane was down-regulated by either overexpression of miR-320 a or AQP-4 gene interference.Conclusion1.miR-320 a is aberrant down-regulated in human gliomas and associated with the clinical prognosis.2.The invasive and migratory ability of glioma cells can be suppressed by down-expression of AQP-4.3.miR-320 a can directly target 3’-UTR of AQP-4 suppress the protein expression,and inhibit the invasion and migration ability of glioma cells. |
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