Preliminary Studies Of Relationship Between Mirna And Notch Pathway In The Regulation Of Glioma

Glioma is a neuroectodermal tumor, which accounts for32%of braintumors. About80%of glioma are malignant and can be divided into four grades:grade Ⅰ, grade II, grade III and grade IV. As the grades increasing, themalignant degree of glioma rises. It is difficult to suppress the occurrence anddevelopment of glioma completely due to limited knowledge of its pathogenesisand etiology. Meanwhile, as glioma has strong capacity of migration andinvasion to the surrounding tissues, results in the high risk of recurrence inglioma patients. The current level of health care can only prolong the survivaltime of patients, rather than achieving radical results.Although surgery, drugsand chemotherapy at early stage do have certain effects, the average survivaltime is only14months after treatment. It is vital to select efficient targets andprevent glioma recurrence in treatment.MicroRNA is a family of small non-coding RNAs with length around22nucleic acids. Despite its recent discovery in the1990, it has became a rising star in the basic medical research field. In addition, MicroRNAs participate inthe occurrence and development of various types of tumors including the glioma.Previous research indicated that MiRNA might play pivotal roles in thediagnosis and treatment of glioma. Nevertheless, further studies would berequired to fully understand the mechanisms and distinguished functions ofdifferent MiRNAs in glioma.Notch signaling is highly conserved in evolution, which has different levelsof expression in vertebrates and invertebrates. It has been confirmed in Manystudies that Notch signaling plays an important regulatory role in thedevelopment, invasion and vascular generation of glioma. Meanwhile, as manyStudies reported, MicroRNAs is involved in the Notch signaling pathway.Although there are some reports of miRNA regulating Notch signaling, it is stillunknown whether Notch signaling can regulate miRNA expression.Our study group had selected and identified a series of moleculars locatedat the down-stream of Notch signaling and carried some research experimentspreviously. With the review of former papers, this study focused some relatedexperiments including cell culture, miRNA transfection, reporter assay, cellbiology observation and clinical specimen research, which aiming to find thecharacteristics and functions of those three miRNAs on glioma.1、 A preliminary observation of the Notch signaling potentialdownstream miRNA (miR-342-5p) direct regulation of several downstreammolecules.Construct the reporter gene vector of ph2, ph3, Araf, PFN2and GAS2which were potential downstream molecules of the miR-342-5p; detect theeffection of miR-342-5p to the above reporter genes’ expression by reportergene assay and found that the3’-UTR of these genes were not directly regulatedby miR-342-5p, suggesting that miR-342-5p has other target genes or it does not regulate the downstream molecules through the3’-UTR.2、Preliminary observation of effection of miR-9on glioma cells.Cell culture, transfection, cell biology observation were used to detect theeffection of miR-9to glioma cells. Preliminary results indicate that miR-9couldregulate glioma cell neurite outgrowth, while cell migration seems to be nosignificant difference.3、Preliminary observation of effection of miR-188-5p on glioma cells.Cultured glioma cell lines and synthetic of miR-188-5p mimics andinhibitor were used to observe effection of miR-188-5p to the migration ofglioma cells. We confirmed that miR-188-5p could inhibit glioma cell migrationby the scratch test. Further researches demonstrate that miR-188-5p can inhibitglioma cell invasion through transwell experiments. In addition, to detectwhether miR-188-5p affect cell proliferation, we stained glioma cellproliferation-associated antigen expression through immunofluorescence, whichdemonstrate that miR-188-5p had no impact on cell proliferation.We further explored the mechanism of the inhibition of miR-188-5p toglioma cell migration and invasion. As MMPs family molecules, especiallyMMP2have great important effects on the cell migration and invasion, and wefound that MMP2, MMP13were target genes of miR-188-5p in pre-study of ourlaboratory, we observed the MMPS molecular expression in a variety of gliomacell lines. We confirmed that all the glioma cell lines we detected had expressionof miR-188-5p and MMPs molecules by WB experiment and initiallydemonstrated that miR-188-5p can inhibit the expression of MMPs molecules.On the basis of the above work, we carried out a certain extent in vivoobservation. Protein and RNA were extracted from glioma tumor tissue andnormal tissue adjacent to the tumor. First we confirmed the expression levels ofmiR-188-5p in tumor tissue was lower than that in the normal organization by qRT-PCR; we followed confirmed that expression of MMPs in tumor tissue washigher than normal tissue by WB. This is entirely consistent with the cellularlevel to verify the results. In addition, the preliminary work and literature havedemonstrate that Notch signaling pathway related molecules plays an importantregulatory role in the occurrence and development of glioma, therefore wedetect the expression of the related molecules of Notch signaling pathway in thetumor tissue, which indicate that the expression level of Notch downstreammolecules Hes1、Hes5in tumor tissue were higher than in normal tissue.Conclusion:Through this research, we successfully constructed the reporter genevector of ph2, ph3, Araf, PFN2and GAS2which were potential downstreammolecules of the miR-342-5p and found that these genes is not directly regulatedby miR-342-5p through3’-UTR; we found that miR-9could regulate glioma cellneurite outgrowth, while had little effect on cell migration; we found thatmiR-188-5p can inhibit glioma cell migration and invasion, and had nosignificant effect on cell proliferation; we initial demonstrate that the MMPsfamily are downstream target molecules of miR-188-5p, and may be involved inthe regulation of glioma cells through the Notch signaling pathway. Theseresults have great significance for understanding the role and mechanism ofNotch signaling pathway and miRNA in glioma cells.