Studies Of Function And Expression Of MicroRNA-7-5P In Microvascular Endothelial Cells Of Glioblastoma

BackgroundThe occurrence and development of malignant tumors are complex process that have multiple genes and a variety of signaling pathways participate in, and a variety of related developmental genes play an extremely important role in this process. As one of the important factors of tumor development, the expression and regulation of microRNAs is becoming more and more attention and research subject. MicroRNAs are a class of small molecules RNA with length18-22nt and non-coding, which have gene regulatory functions. MiRNAs disorders are often associated with the development and progression of cancer. MiRNAs target mRNAs by completely or not completely complementary binding, causing degradation of target mRNAs, or inhibit translation, which exerts its biological effects.Glioblastoma (Glioblastoma, WHO IV grade), easily called glioblastoma multiforme tumor (Glioblastoma multiforme, GBM), are the most common and most malignant primary tumors in the central nervous system tumors. The treatment of glioblastoma, is troubled by the problem of neurosurgery, the main treatment is to maximize the clinical surgical resection and postoperative supplemented by means of radiotherapy and chemotherapy. Although some aspects of treatment have made new progress in recent years, but because of its high degree of malignancy, easy to relapse after surgery, the prognosis remains poor. Therefore, to explore new glioblastoma pathogenesis and find new and more effective treatments is still a major research focus at present.Microvascular proliferation is a unique pathological features of GBM, however, the function of the proliferative of microvascular endothelial cells is poorly understood in the currentAlmost all studies and chemotherapy drugs are targeted to GBM cells, and act on the anti-angiogenic effects of chemotherapy drugs are not expected to occur.Because GBM microvessel only to a very small part in the tumor, previous studies that by extracting the entire tumor total RNA or genomic DNA to find GBM microvascular specific gene is clearly inappropriate.This study will extract non-tumor brain tissue microvascular and GBM microvessels, compared the differences in miRNA expression profiling in GBM microvascular and brain microvascular with micro-chip technology, apply of modern bioinformatics tools to analyze and integrate of miRNA expression profiling,and study certain factors that can enhance or inhibit the proliferation of GBM microvessels on miRNA, mRNA and protein levels, in order to find a more effective inhibition of GBM vascular proliferation potential targets.Objective1. Extracting and purificating of microvascular from GBM and normal brain tissue to detect differences miRNAs between them;2. Screened significant differences miRNA, to study its effects on vascular endothelial cells biological behavior;3. To study the function and mechanism of the filter out significant differences miRNA and provide experimental evidence for clear whether its can be used as a new target for the treatment of GBM.Methods1. Applying dextran sedimentation density gradient centrifugation, extract and purify of GBM microvascular and non-tumor normal brain tissue capillaries. Look microvascular as the specimen, and extract the total RNA from it.2. By miRNA microarray technology, screening differences expression microRNAs in GBM microvascular and brain tissue microvascular, using quantitative real-time PCR to further validate the gene chip result, verify its expression in the microvasculature organization and human umbilical vein endothelial cells (HUV-EC-C). 3. By bioinformatics tools(TagetScan5.1, MicroCosm Targets Version5) prediction the target genes of differences miRNA, and perform KEGG pathway analysis for the specific target genes. Select OK the significant differences microRNA and their target genes that related to the regulation in the biological behavior of endothelial cells.4. Construct the lentiviral vectors with over express the desired gene and its negative control vector; perform packaging and viral titer in the293T cells;5. Using scratch test, MTT assay and flow cytometry, to observe the effect of target gene expression in vitro migration, proliferation and cell cycle on vascular endothelial cells.6. Cloned the predicted target genes3’-UTR into the XbaI site GV272and construct luciferase reporter gene, with the miRNA lentivirus and negative control lentivirus co-transfected293T cells, by analyzing the expression of luciferase to preliminary screening target genes of these highly expressed miRNA in endothelial cells. Using Western Blot to further confirmed the regulation of miRNAs to their target gene in cells and tissues.7. All results are expressed as mean plus or minus standard deviation. Comparisons between groups were performed with the t test. The relationship between miR-7-5p and RAF1expression was explored by Pearson’s method. P<0.05was considered to be statistically significant. Statistical analyses were performed with SPSS software (version16.0).Results1. Applying dextran sedimentation density gradient centrifugation the microvascular organization was successfully extracted and purified from GBM and normal brain tissue, using trypan blue stain them, and then observed them under the microscope, we can find the normal brain tissue microvascular complete and clear each branch obvious, and however the GBM capillaries present broken, lumps and branch clutter.2. By microarray analysis showed that miR-7-5p significantly reduced in GBM microvessels relative to normal brain tissue microvascular (P<0.01), and reduce2.61 times relative to the normal non-tumor brain tissue microvascular. In all five cases of GBM microvascular and brain microvascular, the results of qPCR verify and the gene chip analyses have exactly the same.3. Bioinformatics prediction results show, the3’-UTR of RAF1that participate in the biological behavior of endothelial cells and miR-7-5p5’end "seed sequence" have better matching sites. Determination of luciferase activity shows that the RAF1is a direct target of the miR-7-5p downstream (P<0.01).4. MTT assay, cell cycle experiments showed that miR-7-5p induced endothelial cell cycle arrest in the G1phase, and inhibited the proliferation of endothelial cell in vitro. Scratch test showed that miR-7-5p has no effect on vascular endothelial cells in migration in vitro.5. Western blot analysis showed that the infection of LV-miR-7-5p leaded to a decrease of RAF1protein expression in HUV-EC-C cells. Actin was used as internal (P<0.01); In the GBM and normal brain tissue capillaries, the expression of miR-7-5p and RAF1shows negatively correlated. Pearson’s correlation analysis with statistical significance. Correlation coefficient was-0.786.(P<0.01).ConclusionsDextran sedimentation density gradient centrifugation methods can be successfully extracted microvascular from GBM normal brain tissue, by trypan blue staining, and microscopy found a significant difference between GBM and normal brain microvascular in the form, this is in line with the objective law. The study began with the GBM and normal brain tissue microvascular. Therefore, there have a good reference value for the pinpoint valuable microRNAs and their targets gene that play a vital role for the occurrence of GBM microvascular, and may provide a more meaningful experimental basis for the treatment of GBM. In the current study of the gene chips, we revealed the miR-7-5p significantly downregulated in GBM microvascular, and verified the result by qPCR in the GBM microvascular tissue and HUV-EC-C cell lines. Moreover, we successfully packaged the lentivirus of over express miR-7-5p (LV-miR-7-5p) and its negative control lentivirus (LV-miR-NC), and performed the MTT assay, cell cycle test and scratch test in vitro. Experimental results showed that the overexpression of miR-7-5p can significantly inhibit the proliferation of the HUV-EC-C cells by induce the cell cycle arrest in G1phase, but for the migration of vascular endothelial cells had no effect. These results strongly supported the miR-7-5p may be an inhibitor of GBM angiogenesis.To explore the mechanism of miR-7-5p inhibition GBM microvascular endothelial cell growth, we have identified the potential target genes of miR-7-5p.Bioinformatics analysis shows that the RAF1may the be potential target of miR-7-5p.Moreover, RAF1plays an important role in the development and occur for many types of cancer. Therefore, we apply the luciferase activity assay to identify the role of miR-7-5p in expression of the RAF1.The luciferase activity assay shows that miR-7-5p can directly targeting the3’-UTR of RAF1. Western blot results showed that the overexpression of miR-7-5p can significantly reduced the expression of RAF1in HUV-EC-C cells. We have carried out western blot experiments in five-groups of GBM microvascular and normal brain tissue microvascular, the result shows that the RAF1upregulated in GBM tissue capillaries, however downregulated in the brain microvessels, and there have a negative correlation appeared between the miR-7-5p and the RAF1.These data confirm the miR-7-5p can downregulate the expression of RAF1, which also shows RAF1may be a contributing factor in angiogenesis of GBM. This study suggests that miR-7-5p may play a role of tumor suppressor genes in the formation and development of microvessels in GBM through the regulation of RAF1which downstream target genes, and play an important effect in the regulation of proliferation of endothelial cells.