The Mechanism And Functional Study Of MiR-9in Human Glioblastoma Cells

【Background】Gliomas, the most common and deadly primary human brain tumors. Which could beclassified as grade I to grade IV on the basis of histopathological and clinical criteria bythe World Health Organization (WHO). Grade I gliomas occur more in children than inadults, are curable with complete surgical resection and rarely progress to higher-gradelesions. WHO grade II or III gliomas are invasive, evolve into higher-grade gliomas andhave a poor outcome. Glioblastoma (GBM), the most common and malignant gliomas, hasa very badly prognosis and develop rapidly without evidence of a less malignant precursorlesion(primary glioblastoma), or less commonly through progression from a lower gradetumour (secondary glioblastoma).MicoRNAs (miRNAs) are small noncoding RNAs that regulate gene expression bytargeting the mRNAs of a large number of human genes. Interesting, most miRNAs arehighly conserved through evolution, suggesting that they might play crucial roles inmultiple key biological processed. MicroRNAs can be subdivided into two classesaccording to their tumorigenic functions, oncogenic miRNAs and tumour-suppressor miRNAs. To date, it is worth noticing that while great efforts have been made inuncovering differentially expressed miRNAs between malignant and normal tissues orcells, investigating their function in cancer, and then looking for the new targets whichwere regulated by the miRNAs. MicroRNAs are frequently deregulated in cancer, alsoinclude gliomas. And miRNAs deregulation has been associated with gliomapathobiology.Aberrant miR-9levels have been reported in many types of cancer, suggesting thatmiR-9play important role in tumor formation and progression. And is highly conserved inanimals and plants. However, the miR-9research is still not very thoroughly in glioma.What is vital target genes regulated by miR-9to affect the progress in glioma? Why ismiR-9high expression in glioma? If we can clarify the raise mechanism of miR-9, it willbe as a therapeutic targets, and a new strategy for clinical diagnosis and treatment forglioma.【Aims】To explore the miR-9expression in the glioma clinical samples and cell lines. Toinvestigate the miR-9biological effects in glioma malignant progress. To identify relatedtarget genes of miR-9in glioma. To find the new transcription factors and epigeneticregulation in miR-9expression of glioma, providing the new theoretical basis for theprevention, early diagnosis and treatment of glioma.【Methods】1.Using qRT-PCR detected the miR-9expression level in clinical tissue sample andglioma cell lines；2.Transient transfection miR-9mimic/inhibitor raised/down-expressed theexpression of miR-9, MTT, flow cytometry, cell scratches, transwell, angiogenesis andadhesion experiment detecting miR-9affect biological function of glioma cells in vitro;3.Using bioinformatics analysis software to predict potential target genes of miR-9, and usingmolecular cloning technology connect target gene3’UTR fragment to PGL3report plasmid;4.The luciferase report gene identify the directly regulating target genes of miR-9, and usingqRT PCR, Western blot and ELISA experiment validated on mRNA and protein levels;5.Bioinformatics website forecast the new transcription factors and H3K27histone methylation that regulate the miR-9expression levels, using qRT-PCR detecting transcription factorsexpression and H3K27histone methylation status;6. Transient transfection siRNA/up-expressed plasmid to lower/highly expression the transcription factors and histonedemethylases, qRT-PCR analysis the expression levels of miR-9.【Results】1.qRT-PCR shows miR-9is up-expression in glioma tissue samples and glioma cell lines,compared with normal brain tissue and glia cell HEB. miR-9is highly expressed in gliomacell lines, relative lower expression in A172cell, and higher in U251cell. Compared with thenormal intestinal epithelial cell, miR-9is high expression in colorectal cancer cell lines; andmiR-9is lower expression in kidney cancer cell lines compared with normal renal epithelialcell. All results show miR-9expression present organization specificity;2. Transienttransfected miR-9mimic in A172cell could effectively increase the expression of miR-9invitro. MTT result show miR-9enhance cell proliferation, FCM analysis that over-expressedmiR-9increase cell in S phase, G1phase decrease, miR-9obviously increase the number ofmigration and invasion glioma cell, and miR-9conditioned medium enhanced formationvessels ability of HUVEC. While transfected inhibitor miR-9in U251cell increase the cellproliferation ability, G1-to-S arrest, S phase decreased and G1phase increased, the number ofmigration and invasion glioma cell is significantly reduced, and condition medium cultureHUVEC reduced the tube formation ability;3. miR-9mimic could significantly improve themiR-9expression in HUVEC, transwell experiment shows over-expressed miR-9group ofHUVEC are more number of cells migrate through the artificial membrane, over-expressedmiR-9could form more tube compared with NC group. And up-expressed miR-9enhance theHUVEC cell adhesion ability;4.Bioinformatics predictions suggest that THBS2, COL18A1,PTCH1and PHD3may be potential miR-9downstream target genes, which affect thebiological function of glioma cells, include proliferation, migration/invasion, andangiogenesis;5. Using qRT-PCR, Western blot, ELISA and luciferase report gene experimentsfurther confirmed THBS2, COL18A1, PTCH1and PHD3are direct regulated downstreamtarget genes of miR-9;6. After up-expressed C-MYC in A172cell, qRT-PCR result showexpression of Has-miR-9is increased, while siRNA C-MYC in U251found miR-9is decreased, especially differentially expressed is miR-9-2;7. Over-expressed OCT4in A172cell, qRT-PCR demonstrate expression of miR-9is increased, however, down-regulated OCT4in U251found miR-9is decreased, interesting differentially expressed is miR-9-1/3;8.Transfected over-expressed KDM6A/B plasmid found that miR-9expression is increasing,whereas known-down KDM6A/B expression in U251reduced miR-9expression, andespecially is miR-9-2.【Conclusions】1. MiR-9is highly expression in glioma tissue samples and glioma cell lines, and presentthe tissue specificity.2. MiR-9promote the glioma cell proliferation, cell cycle G1-to-S arrest,cell migration and invasion, and angiogenesis;3. MiR-9enhanced endothelial cell migration,invasion and angiogenesis, increase the endothelial cell adhesion ability;4. THBS2,COLA8A1, PTCH1and PHD3is direct downstream regulated target genes of miR-9;5.Transcription factors C-MYC and OCT4regulate the miR-9expression;6. H3K27methylation status could regulate the expression levels of miR-9.