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Objective To investigate the effect of knocking-down Yes-associated protein (YAP)on the biologi-cal characteristics of SNB19 glioblastoma cell.Methods The expression of YAB in SNB19 was knockdown by YAB small interfering RNA (YABsiRNA).The downregulation of YAP expression was identified by Western blot analysis. The proliferative ability of cell was determined by methyl thiazoyl terazolium (MTT).The invasive ability of cell was examined by Transwell assay.Flow cytometry and Annexin V staining were used to detect the cell cycle and apoptosis respectively.The results were analyzed by the statistical software SPSS18.0.Results The expression of YAP in the cells transfected with YAPsiRNA was significantly reduced.The cell proliferation activity of SNB19 cells was inhibited, which decreased from (100.00 ±0.00)%to (52.32 ±3.10)%(F=33.00,P<0.01).The cell cycle was arrested in G0-G1 phase (F=8.76,P<0.01).The cell invasive ability was attenuated apparently,which decreased from (163.20 ±10.10)to (37.71 ±2.52)(F=282.05,P<0.01).The apoptosis ratio of the tumor cell which transfected with YAPsi-RNA was increased from (3.56 ±0.35)%to (18.99 ±0.66)%,(F=931.99,P<0.01).Conclusion Knocking-down YAP expression in glioma cells could inhibit the proliferative activity and invasive ability of SNB19 cell and could induce cell apoptosis.YAP could be served as a potential target for the gene therapy of glioma.
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Objective:This study aimed to explore the effect of Yes-associated protein (YAP) on the growth of the human glioma cell line LN229. Methods:YAPsiRNA was transfected into LN229 cells to knock down the YAP expression. The downregulation of the YAP expression was identified through Western Blot analysis. Colorimetric assay using methyl-thiazolyl-tetrazolium was applied to evaluate cell proliferation ability. Cell invasive activity was examined using Transwell assay. Flow cytometry and AnnexinV were used to detect cell cycle and apoptosis, respectively. The relevant molecules regulating proliferation, invasion, cell cycle progression, and apoptosis were examined through Western Blot analysis. Results:The YAP expression was downregulated after YAPsiRNA was trans-fected into LN229 glioma cells. Reduced YAP expression could arrest the cell cycle at G0/G1 phase, inhibit cell proliferation and inva-sion, and promote apoptosis. The expression of the proliferating cell nuclear antigen (Ki-67), matrix metallopeptidase-9 (MMP-9), cy-clin D1, and Bcl-2 were downregulated. Conclusion:The downregulation of YAP in LN229 cells suppresses cell proliferation and inva-sion, as well as promotes cell apoptosis. This study provides a novel evidence for further study on Hippo-YAP signal pathway in molec-ular pathology of glioma.
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Objective To study the inhibitory effect of knocking down miR-30a-5p on the U87 human glioma xenograft growth and its possible mechanism.Methods Nude mice bearing subcutaneous U87 human glioblastoma were established and separated into three groups (eight for each group) by randomized digital table method,including control group,scr-ODN treated group and AS-miR-30a-5p treated group.After relevant subcutaneous injection treatment,tumor size was measured every other day until the observation period ended.Researchers executed the animals after the treatment,stripped tumor tissues and extracted RNA and protein.Real-time PCR was conducted to detect the expression of miR-30a-5p.The histopathological characteristics and proliferation and apoptosis biological characters (including SEPT7,PCNA,cyclin D1,MMP-2,apoptosis related factor P53,bcl-2 and caspase3) were evaluated by HE and immunohistochemical staining,Westem blot analysis respectively,and the cell apoptosis was detected by TUNEL method.Results In AS-miR-30a-5p treated group,the tumor growth was delayed and the final tumor volume was smaller than that in the control and scr-ODN treated group (F =7.167,P ＜0.05),and the expression of miR-30a-5p was knocked down.The expression of PCNA,cyclin D1 were significantly downregulated while P53,SEPT7 and caspase3 up-regulated.Apoptotic index was increased significantly.Conclusion As-miR-30a-5p suppresses the growth of U87 human gliomas xenografts significantly.Malignant phenotype of tumors are reversed to a considerable degree.Therefore,miR-30a-5p can be a candidate for targeted therapy of human glioma.
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The miR-17-92 cluster,consisting of six individual microRNAs,including miR-17,miR-20a,miR-18a,miR-19a,miR-19b and miR-92a-1,is a kind of typical oncogenic polycistron microRNA.MiR-19 (containing miR-19a and miR-19b),as the key oncogenic component in miR-17-92 cluster,is overexpressed in human cancers including lymphomas,leukaemia,lung cancer,breast cancer and multiple myeloma,and has been one of research focus in recent years.It is found that miR-19 promotes tumor growth,invasion and metastasis through negative regulation of target genes such as PTEN,PP2A,Bim,SOCS1,and is correlated strongly with PI3K-AKT-mTOR pathway.MiR-19 plays an important role in tumor genesis and development.
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ObjectiveTo confirm the effect of miR-93 inhibitor in glioma cell growth and invasion.MethodsMalignant glioma cells were transfected with miR-93 inhibitor by lipofectamin to downregulate their overexpression of miR-93.Real time-PCR was taken to measure miR-93 expression after transfection.The cell cycle kinetics and cell growth rate were detected by flowcytometry and MTT assay,the cell proliferative ability was evaluated by soft agar assay,and the invasive ability was detected by transwell assay.ResultsThe highlevel expression of miR-93 was downregulated effectively in glioma cells after transfecting the miR-93 inhibitor.Meanwhile,the cell cycle progress was delayed,S phase cells were reduced,the speed of growth was slowed,cloning formation ability was receded,the number of cells through the matrigel was reduced,and invasive ability was significantly repressed.ConclusionDownregulation of miR-93 expression could inhibit the proliferative ability and invasive ability of glioma cells.
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objective To identify SEPT7as one of the target genes of miR-19a and miR-19b.MethodsmiR-19a inhibitor and miR-19b inhibitor mediated by lipofectamine2000,were transfected to SNB19 glioma cells for knocking down miR-19a/19b overexpression.Real time PCR was conducted to detect the expression of miR-19a/miR-19b in transfected cells.The expression of SEPT7was determined by Western blot analysis.RT-PCR was used to detect the mRNA expression of SEPT7; and Luciferase reporter assay was used to identify the direct regulation of miR-19a/19b on SEPT7.ResultsIn SNB19 glioma cells transfected with miR-19a/19b inhibitor,the expression of miR-19a/miR-19b was significantly reduced,whereas the protein expression of SEPT7 was upreguhtted; no significant change of SEPT7 mRNA level was found and luciferase activity became stronger as compared to control cells.ConclusionSEPT7 is the target negatively regulated by miR-19a and miR-19b.
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Objective Increasing evidence suggest that aberrant activation of PI3K/Akt is involved in many human cancers, and that inhibition of the PI3K/Akt pathway might be a promising strategy for cancer therapy. The study is to evaluate the effects of combined therapy of PI3K inhibitor (LY294002) and Ad-PTEN in athymic mice xenogeneic transplant model of human glioma and to reveal the possible mechanisms involved.Methods Twenty-four athymic mice were randomly divided into 4 groups (DMSO、Ad-vector plus DMSO、LY294002 alone and Ad-PTEN plus LY294002), and were treated, respectively. Athymic mice xenogeneic transplant model was established by inoculation (sc) with LN229 glioma cells. Body mass (BM) and diameter of tumor mass were measured. Furthermore, The protein expressions of PTEN、p-Akt、CyclinD1、Caspase-3、MMP-2、p-FAK in tumor tissues were analyzed with immunohistochemistry.Results The tumor-inhibiting rate of was significantly higher in Ad-PTEN plus LY294002 than in the LY294002 alone (92.46 vs 65.59%)( P <0.05).The protein expressions of PTEN and Caspase-3 were significantly higher, while PCNA、CyclinD1、bcl-2 and MMP-2、p-FAK was significantly lower in Ad-PTEN plus LY294002 group than in the other three groups ( P <0.05).Conclusions LY294002 plus Ad-PTEN achieve better outcome than either alone in treating glioma possibly through enhancement of the inhibitory action of PI3K/Akt pathway and Ad-PTEN pathway.