| Objectives:Glioblastoma(GBM, World Health Organization grade IV glioma) is the most common malignant primary brain tumor in adults, and one of the most lethal forms of human cancer. Despite advances in treatments in the past decades, prognosis of the disease remains dismal, with 5-year survival rates averaging <3% in the United States and Europe. Recent studies have revealed that glioblastoma cancer stem cells(CSCs)constitute a reservoir of self-sustaining cells with the exclusive ability to self-renew and cause tumor outgrowth. The glioblastoma stem cells were first isolated from tumors by virtue of the expression of CD133, and were later identified to be highly tumorigenic,invassive and particularly resistant to chemoradiotherapy. However, the molecular mechanisms for maintenance of glioblastoma cancer stem cells remains largely unknown.There are experimental reports, in the maintenance of cancer stem cells in the dry, found that miRNA plays a role, miRNA is a class of 19 ~ 25 nucleotides of the non encoding single stranded small RNA molecules, in the regulation of gene transcription,generally through the target gene 3 ’end of the non translation region of the combination method to adjust the target gene transcription level.Studies have indicated that Hippo signaling pathway plays an important role in limiting the size of organs and inhibiting tumor. It is indicated that the Hippo signaling pathway is largely restricted to its two downstream effector factors YAP and TAZ. The activity of gene MST1-SAV1 directly affects the expression level of YAP/TAZ. Here, we detected the expression and activity of miR-130 b in the Hippo signal pathway, and detected the miR-130 b, CD133, SOX2, Nanog,MYC, BMI1 and, and to observe the effect of miR-130 b on glioma stem cells. These studies provide a new mechanism for the role of miR-130 b in the Hippo signaling pathway and the role of miR-130 b in the development of glioma cells, and provide a goodidea for the targeted therapy of glioma.methods: in order to detect the expression level of miR-130 b in glioma tissues and cells, we collected 8 cases of glioma tissues and 3 cases of normal tissues. NHA, U87 MG, LN444, LN18, U251 MG, SNB19 in normal cells.MiR-130 b detection kit(CL-has-miR-130b) was used to detect the samples of BioTNT. At the same time, we tested the effect of miR-130 b on the proliferation of glioma stem cells,and about 500 cells seeded on 6 well plates, and about 10 cells were inoculated on 24 well plate, with no serum DMEM / F12 culture for 13 days, and then added 2%B27 in the culture medium, 20 ng / ml, 20 g/ bFGF and 5 ml(BSA). After training, and then detect the size of the ball, calculate its multiple. We use WB detection the mir-130 b influence on the Hippo signaling pathway in yap and TAZ protein expression and nuclear protein P84 antibodies as reference(sigma USA). At the same time, to detect the Mst1 and SAV1 protein levels, with alpha tubulin antibody as internal control(sigma, USA). The effect of miR-130 b on the activity of TEAD was detected by luciferase assay. The TEAD plasmid was transfected into glioma cells in 48 holes, each group had 4 complex holes, and the 48 h was performed according to the instructions of LipofectamineTM2000. Mi RBASE software was used to predict the relationship between MST1/SAV1 and miR-130 b, and the software was used to predict and miR-130 b. The miR-130 b and MST1/SAV1 were used to detect the effect of MST1/SAV13 and MST1/SAV1.Result: Reference cancer gene GBM miRNA microarray data(TCGA), we found that miR-130 b levels were significantly upregulated in human glioma tissue(n = 496) compared with normal brain tissue(n = 10)(P <0.001). By RT-PCR to detect further validate the results. mi R-130 b levels in eight cases of GBM tissue compared with a significant increase in the three cases of normal brain tissues, and compared to the five kinds of glioblastoma cell lines, in normal human astrocytes(NHA) significantly reduced. mi R-130 b glioma glioma stem cell proliferation and the role into a ball, over-expression of miR-130 b and effectively promote the suspension culture cells produce about twice more than the ball. In contrast, mir-130 bcell formation inhibition sphere less than 2 times the normal sphere, our findings indicate that, miR-130 b promote glioblastoma balling effect. WB assay protein expression levels of that, overexpression of mi R-130 b makes YAP, increased expression of TAZ, and inhibit the expression of mi R-130 b can make the protein YAP, TAZ expression decreased,while the expression of nuclear factor P84 has no influence, And after over-expression of miR-130 b, MST1 and SAV1 lower protein levels, suggesting that miR-130 b directly affect the HIPPO signaling pathways related genes play a functional role. Luciferase further verified in the case of stable cell lines SNB19 miR-130 b and LN18, theinterference overexpression of YAP and TAZ, and the positive control cisplatin contrast,fluorescence is detected 1,0.25,0.28 and 1, 0.26,0.39, were statistically significant(P<0.001). Conclusion: YAP and TAZ are the TEAD a significant role, which reduces the activity of TEAD even stronger than cisplatin.MirBASE software forecast by the miR-130 b and MST1 / SAV1 regulatory sites have significantly reduced by the luciferase assay prompted miR-130 b and MST1 / SAV1-3’UTR measured fluorescence value,indicating the point of integration between them, weakening Expression of the fluorescence, whereas in the control group and the normal value of the fluorescence is no change in the suppression group, fluorescence enhancement, suggesting that inhibitors prevent binding to MST1 / SAV1-3’UTR These data illustrate miR- regulatory site 130 b and MST1 / SAV1-3’UTR combined presence, which is consistent with the theoretical prediction tool.All statistical analyses were carried out using SPSS statistical software(SPSS Inc., Chicago, IL, USA). The 2-tailed Student’s t-test was used to evaluate the significance of the differences between two groups of data in all pertinent experiments; a P value <0.05 was considered significant. |
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