MiR-223Promotes Growth And Invasion Of Glioma Cells By Down-regulating PAX6

Glioma is the most common primary malignant central nervous system. Because of its unique aggressive and difficult to treat, it is important to look for new therapeutic targets glioma. Recent studies have shown that tumor suppressor PAX6was involved in inhibition of glioma cell growth and invasion. But compared with adjacent normal tissue, the expression of PAX6in glioblastoma tissues were significantly decreased, suggesting that exploring the exact mechanism of the reduced expression of PAX6in gliomas could provide the new ideas for glioma’s treatmentmiRNAs belongs to a class of small, non-coding single-stranded RNAs. It can be the target gene mRNA3’-UTR, and consequently translation of target gene mRNA degradation or blocked, resulting in the post-transcriptional level of regulation protein. MiRNAs have a variety of biological effects. A lot of studies have shown that abnormal expression of miRNAs in cancer development and progression play an important role. Whether miRNAs works as tumor suppressor genes or oncogenes is depended on their target genes function. In different types of cancer cells, miR-223exhibit different role by targeting different genes. Glioma growth and metastasis with different miRNAs relevant, however, the role of miR-223in glioma biological function is still unclear. In addition, there is rare report about the relationship of miRNAs with PAX6in glioma. In this study, we invstigated firstly miR-223and PAX6expression levels in glioma cells (U251, U373) and tissue. And we analyzed the regulation of miR-223on PAX6using bioinformatics methods and the effects of different expression levels of miR-223, PAX6on U251and U373glioma cell proliferation, cell cycle and invasion, to explore the miR-223as a new target for the treatment of glioma feasibility.Objective:1. To detect the expression of miR-223and PAX6in glioma cells and tissues and its relationship2. Effect of miR-223on the expression of PAX6in glioma cells U251and U3733. To explore the effect of miR-223on the biological activity of glioma cells by regulating the expression of PAX6Methods:1. The expression of miR-223and PAX6in glioma tissues and its relationship(1) To detect the expression of PAX6in different pathological grade glioma tissues and normal brain tissue using microarray technology.(2) To detect the expression of miR-223and PAX6mRNA in glioma cell lines (A172, U251, U373, U138, XB1330) and normal human fetal brain glial cells (HFGC) using qRT-PCR.(3) To forecast the targeting relationship of miR-223with PAX6using bioinformatics software.(4) To identify the targeting relationship of miR-223with PAX6using dual luciferase reporter gene assay.2. Effect of miR-223on the expression of PAX6in glioma cells U251and U373(1) miR-SCR, anti-Con, miR-223mimics and anti-miR223were synthed by chemical synthesis methods, and were transfected into U251and U373cells to build miR-223acquired and lost models.(2) Construction PAX6overexpression and knockdown expression model inU251and U373cells.(3) To detect the effect of miR-223on the PAX6mRNA and protein expression levels using qRT-PCR and Western Blot respectively.3. Effect of miR-223on the biological activity of glioma cells by regulating the expression of PAX6(1) MTT test was used to detect the cell growth.(2) Flow cytometry was used to detect the cell cycle.(3) Cell invasion was tested bu transwell invasion assay.(4) Western Blot was used to detect invasion-related factors (MMP2, MMP9) and angiogenesis regulatory factors (VEGFA, HIF1α) expression levels.Results:1. The expression of miR-223and PAX6in glioma tissues and its relationship(1) Tissue microarray results showed that the PAX6protein expression in normal brain tissue was significantly higher than that in glioma tissues, and with the increase of the degree of brain glioma installments, PAX6protein expression was significantly reduced.(2) qRT-PCR results showed that, miR-223were expressed in glioma cell lines. Compared with normal human fetal brain glial cells (HFGC), the expression of miR-223in glioma cell lines was significantly increased, with the highest expression in the U138, XB1330cells (p<0.001), higher in A172cells (p<0.05) and moderate in U251, U373cells (p<0.01).(3) Bioinformatics software prediction results show that miR-223could regulate the expression of PAX6.(4) qRT-PCR results showed that, PAX6were expressed in glioma cell lines. Compared with normal human fetal brain glial cells (HFGC), the PAX6expression in glioma cell lines were significantly decreased with the lowest expression in the U138, XB1330cells lowest (p<0.001), moderate in U251, U373cells centered (p<0.01), and highter in A172cells (p<0.05). (5) Dual luciferase reporter gene assay showed that in group of co-transfected with miR-223mimics and PAX63’-UTR-psi-CHECKTM2recombinant plasmid, the luciferase activity was decreased significantly compared to the other groups. miR-223mimic significantly suppressed PAX63’UTR luciferase reporter gene activity, indicating that miR-223gene and the3’-UTR region PAX6targeting relationship existed; miR-223inhibitor by the recombinant PAX6-Mut3’UTR plasmid cotransfection found luciferase activity was no significant intensity differences of miR-223inhibitor is not affected, and PAX6-3’UTR suppressing effect is very obvious, and the inhibition can be reversed by miR-223inhibitor.2. Effect of miR-223on the expression of PAX6in glioma cells U251and U373(1) Successfully constructed Ientiviral shRNA-PAX6expression plasmid, and the mean titer was6.73×108TU/mL.(2) Successfully constructed PAX6-pcDNA3.1eukaryotic expression plasmid.(3) miR-223expression in U251and U373cells:qRT-PCR results showed that after transfected with miR-SCR, miR-223mimics, anti-Con and anti-miR223, the expression of miR-223was significantly increased in U251and U373glioma cells transfected with miR-223mimics. And this effect could be reversed transfected with anti-miR223. (4) PAX6in U251and U373cells:qRT-PCR and Western Blot results are shown after transfected with PAX6-pcDNA3.1eukaryotic plasmid, PAX6mRNA and protein expression were increased,after infected with lentiviral vector shRNA-PAX6, PAX6mRNA and protein expression were decreased,indicating that the success of transfection or infection model.(5) Effect of miR-223on the PAX6mRNA and protein expression: qRT-PCR results showed that in glioma U251and U373cells after transfected with miR-223mimics, PAX6expression was decreased and transfected with miR-223inhibitor, which could be reversed the PAX6expression. Western Blot results showed that after transfected with miR-223mimics PAX6protein expression was significantly decreased; this effect can be reversed by anti-miR223.3. Effect of miR-223on the biological activity of glioma cells by regulating the expression of PAX6(1) MTT cell proliferation results:U251, U373cells transfected with the expression PAX6plasmid48h, cell proliferation decreases significantly to72h down most significant; transfected with shRNA-PAX6Plasmid48h, cells significantly increased proliferative capacity have begun to72h increased the most significant; while miR-223overexpression,48h cells were significantly increased proliferation occurs; while inhibiting miR-223expression,48h decreased cell proliferation occurs; (2) The results of cell cycle by flow cytometry in U251, U373cells overexpressing PAX6lead to significant increase in the number of cells in G1phase, S phase cells decreased, ie cell cycle arrest in G1phase, inhibiting G1to S phase conversion; inhibit PAX6after,G1phase cells decreased, S phase cells increased significantly, which promotes cell cycle G1to S phase transformation; overexpression of miR-223, G1phase cells decreased, S phase cells was significantly increase, which promotes cell cycle G1to S phase transformation, anti-miR223after, G1phase cells increased, S phase cells was significantly reduced, which inhibit the G1to S phase of the transformation;(3) the expression levels of different PAX6Transwell invasion assay results when PAX6overexpression of two glioma U251, U373cells in Matrigel gel hydrolysis and through the basement membrane Transwell chamber significantly reduced the number of cells; while expressing silence PAX6Both glioma U251, U373cells and through hydrolysis Matrigel Transwell chamber glue the number of cells increased compared with the control group, the difference was statistically significant (p <0.05).(4) Different miR-223expression levels Transwell invasion assay results miR-223overexpression, through the basement membrane Transwell chamber number of cells increased significantly compared with the control group, inhibition of miR-223expression, the two kinds of brain glial hydrolysis of tumor cells through Matrigel Transwell chamber glue and the number of cells was significantly reduced, the difference was statistically significant (p<0.05).(5) U251and U373cells each factor Western Blot test results miR-223overexpression MMP2, MMP9, HIF1α, VEGFA protein expression was significantly increased, transfected miR-223inhibitor, MMP2, MMP9, HIF1α, VEGFA protein decreased; PAX6overexpression MMP2, MMP9, HIF1α, VEGFA protein expression was significantly decreased after the expression of interference PAX6, MMP2, MMP9, HIF1α, VEGFA protein expression appeared to increase, showing overexpression of miR-223with the same effect.Conclusion:1. In glioma cells, high expression of miR-223and low expression of PAX6showed the opposite relationship. PAX6protein levels in glioma tissue and tumor staging also has the opposite relationship.2. PAX6mRNA and protein expression levels were regulated by miR-223expression levels and PAX6is a directly target gene of miR-223.3. The changing of expression of PAX6results to affect cell viability, cell cycle, invasion force; it may suppress the expression of invasion-associted factor MMP2, MMP9and angiogenesis regulatory factor HIF1α, VEGFA protein expression leves.4. miR-223affects cell viability, cell cycle and invasion by targeting the expression PAX6.